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Extremely Bright, Near-IR Emitting Spontaneously Blinking Fluorophores Enable Ratiometric Multicolor Nanoscopy in Live Cells
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  • Jonathan Tyson
    Jonathan Tyson
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
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  • Kevin Hu
    Kevin Hu
    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
    Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06511, United States
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  • Shuai Zheng
    Shuai Zheng
    Department of Chemistry, University of California, Berkeley, California 94720, United States
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  • Phylicia Kidd
    Phylicia Kidd
    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
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  • Neville Dadina
    Neville Dadina
    Department of Chemistry, University of California, Berkeley, California 94720, United States
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  • Ling Chu
    Ling Chu
    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
    Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
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  • Derek Toomre
    Derek Toomre
    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
    Nanobiology Institute, Yale University, West Haven, Connecticut 06516, United States
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  • Joerg Bewersdorf*
    Joerg Bewersdorf
    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
    Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06511, United States
    Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, Connecticut 06510, United States
    Nanobiology Institute, Yale University, West Haven, Connecticut 06516, United States
    *(J.B.) Email: [email protected]
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  • Alanna Schepartz*
    Alanna Schepartz
    Department of Chemistry, University of California, Berkeley, California 94720, United States
    Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, United States
    California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720, United States
    Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
    Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, United States
    *(A.S.) Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-2127-3932
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Cite this: ACS Cent. Sci. 2021, 7, 8, 1419–1426
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https://doi.org/10.1021/acscentsci.1c00670
Published July 23, 2021

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Abstract

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New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multicolor, live-cell, single-molecule localization microscopy (SMLM). Here we report the design, synthesis, and biological evaluation of Yale676sb, a photostable, near-IR-emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.

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Synopsis

The bright and near-IR-emitting spontaneously blinking fluorophore Yale676sb is paired with HMSiR to enable two-color live-cell single-molecule localization microscopy with no chemical additives and a single 642 nm laser.

Introduction

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Single-molecule localization microscopy (SMLM) (1−4) is a powerful technique for visualizing intracellular architecture at the nanoscale (5) and across large fields of view. (6) The technique is characterized by the detection and localization of fluorescent markers that cycle rapidly between emissive (ON) and non-emissive (OFF) states. For optimal results, the sample and imaging conditions must maintain the majority of fluorescent markers in the OFF state, such that the neighboring molecules in the emissive ON state can be treated as sparse single emitters. (7−11) Organic fluorophores are favored over fluorescent proteins for SMLM because they are generally brighter and more photostable and because their photophysical properties can be fine-tuned using chemistry. (7,12−17,17−21) The challenge is that many SMLM-compatible organic fluorophores require the addition of exogenous nucleophiles, redox modulators, and/or oxygen depletion systems to switch efficiently between ON and OFF states. These additives can be cytotoxic and damage or alter biological samples. (7,12,13,15,17) An additional challenge is that many established SMLM-compatible fluorophores are cell-impermeant (7,12,13,20,22,23) and/or require cytotoxic high-power and/or short-wavelength lasers. (7,12,16,18,22,24,25)
The spontaneously blinking fluorophore (SBF) hydroxymethyl Si-rhodamine (HMSiR) (15) (Figure 1a) reported by Urano and co-workers overcomes many of these limitations. It is cell-permeant and photostable and is believed to cycle rapidly between ON and OFF states by virtue of a pH-dependent spirocyclization reaction that occurs in the absence of chemical additives (15) (Figure 1a). For HMSiR, the midpoint of this pH-dependent equilibrium (referred to as pKcycl) occurs at approximately pH 6.0. Thus, at pH 7.4 roughly 98% of the HMSiR molecules in solution occupy the OFF state, which enables facile detection and localization of the sparse subset of molecules that are emissive (ON). (15) HMSiR’s cell permeability, photostability, and ability to blink in the absence of chemical additives has enabled multiple minimally invasive single-color SMLM experiments, including those that visualize organelle membrane dynamics in live cells for extended times, (26) others that resolve the morphology of dopaminergic neurons in an intact Drosophila melanogaster adult brain, (27) and still others that enable turn-on visualization of intracellular protein targets. (28)

Figure 1

Figure 1. (a) Structure and pH-dependent equilibrium of the spontaneously blinking fluorophore HMSiR. (15) (b–d) Structures of previously reported fluorophores considered as potential HMSiR partners for multicolor live-cell SMLM (16,18,21,29) (e) Structure of the spontaneously blinking fluorophore reported herein, Yale676sb.

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Despite these advances, there remains a need for new SBFs that effectively partner with HMSiR to enable multicolor live-cell SMLM experiments without the need for chemical additives or photoactivation. (13) Although two green-emitting SBFs whose emission spectra are separable from HMSiR have been reported (Figure 1b), (18,19) including one (HEtetTFER) that can be paired with HMSiR for two-color SMLM in fixed cells, (18) their use demands high-intensity lasers that excite at 488 and 561 nm, respectively. These light sources can induce substantial cytotoxicity as phototoxicity is especially pronounced in the blue and green spectrum. (14) Two other previously reported SBFs are excitable in the far-red/near-IR (Figure 1c), (20,21) but they are spectrally indistinguishable from HMSiR and therefore not suitable for two-color experiments. Although both the fluorescent protein mEos3.2 and CP550 (Figure 1d), a carbopyronin fluorophore that reacts irreversibly with intracellular glutathione, (20) have been paired with HMSiR for two-color, live-cell SMLM, (15,20) these experiments require an additional ∼560 nm laser, which is inferior to red-light excitation for live-cell microscopy. (10,14) Furthermore, sequential multicolor imaging with multiple lasers is slow and the images are prone to sample motion artifacts. Finally, the spontaneously blinking carborhodamine, HMCR550, which was designed using quantum calculations, has an excitation maximum at 560 nm and would likewise require multiple lasers to pair with HMSiR (650 nm excitation) for a two-color live-cell SMLM experiment.
Here we report the rational design of a new near-IR-emitting SBF that pairs effectively with HMSiR to enable simplified two-color SMLM experiments in live cells (Figure 1d). Yale676sb emits at 694 nm, the longest wavelength of any reported SBF, and possesses, to our knowledge, a higher quantum yield (0.59) than any previously reported nanoscopy-compatible Si-rhodamine (SiR) fluorophore. Yale676sb and HMSiR can be excited simultaneously with a single 642 nm laser and imaged ratiometrically for simultaneous multicolor SMLM of two distinct intracellular organelles (ER + mitochondria) in live cells.

Results

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New Spontaneously Blinking Fluorophores: Design Considerations

Three distinct chemical and photophysical properties are needed to ensure compatibility with HMSiR for ratiometric two-color, live-cell SMLM. The first is an emission maximum > 690 nm to ensure adequate separation from HMSiR (emission maximum = 670 nm) via ratiometric imaging. (5,30,31) The second is a pKcycl value between 5.3 and 6.0 to ensure the sparsity of emissive/ON molecules. (15,21) The third requirement is a high quantum yield; although a quantum yield > 0.2 can yield respectable SMLM images, higher values are always more desirable. (13,22) The challenge is that the quantum yields of rhodamine-based fluorophores typically decrease as the absorption and emission maxima increase (Supporting Information (SI) Figure S1). As a result, molecules that absorb and emit at higher, less cytotoxic wavelengths that are compatible with live cells are relatively dim. This correlation is reflected in the relatively low quantum yield of HMSiR (0.31) when compared to those of the green-light-emitting SBFs HMJF526 (0.87) (16) and HEtetTFER (0.76). (18) We therefore sought a design approach that would yield fluorophores possessing both long-wavelength emission and high quantum yield.

HMSiRindol, HMSiRjulol, and HMSiRTHQ

Previous work has demonstrated that introduction of heterocyclic indoline, (32,33) julolidine, (34) or tetrahydroquinoline (32) moieties into the core of a SiR chromophore can shift the excitation and emission maxima by up to 50 nm relative to SiR itself (Figure 2a). To evaluate whether these effects would be preserved in the context of a HMSiR core, we synthesized HMSiR, as well as the heterocyclic derivatives HMSiRindol, HMSiRjulol, and HMSiRTHQ (Figure 2b and Supporting Information Schemes S1–S4) according to a recently reported general method for Si-rhodamine fluorophore synthesis. (35) We then characterized the photophysical properties and aqueous spirocyclization equilibrium (pKcycl) of each new fluorophore (Figure 2b–d).

Figure 2

Figure 2. (a) Structures and photophysical properties (ƛabs, ƛem, and Φ) of previously reported SiR fluorophores with red-shifted absorption and emission spectra and HMSiR analogs HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ. Normalized (b) absorption and (c) emission spectra of HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ in 0.2 M sodium phosphate (pH = 4.5 for HMSiR, pH = 2.0 for HMSiRindol, HMSiRjulol and HMSiRTHQ). (d) pH-dependent change in absorbance of 2 μM HMSIR (650 nm), HMSiRindol (697 nm), HMSiRjulol (684 nm), and HMSiRTHQ (678 nm) as a function of pH in 0.2 M sodium phosphate buffer at room temperature. The absorbance of each fluorophore was monitored at the wavelength of maximal absorbance in panel b.

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Each of the new fluorophores displayed absorption (Figure 2b) and emission (Figure 2c) maxima that were red-shifted by at least 23 nm relative to HMSiR, with the emission maxima increasing in the order HMSiR < HMSiRTHQ < HMSiRjulol < HMSiRindol. As expected, the absorption and emission maxima of the HMSiR series were nearly identical to those of the analogous SiR variants reported previously. (32−34) The pKcycl of each new HMSiR analog was determined from a plot of the pH dependence of the absorption of each fluorophore at the absorption maximum of the open/ON form (Figure 2d and SI Figure S2); the pKcycl is the pH at which the concentration of the open/ON state equals that of the closed/OFF state. (15) The pKcycl values of HMSiRindol and HMSiRTHQ were 6.4 and 6.9, respectively, both significantly higher than the value for HMSiR (6.0). The pKcycl value of HMSiRjulol (pKcycl = 9.0) was shifted even more dramatically, presumably because the additional electron-donating alkyl groups disfavor cyclization. A related previously reported rhodamine analog with julolidine groups also displayed a high pKcycl. value. (15) The absorbance vs pH curves for HMSiRindol, HMSiRjulol, and HMSiRTHQ are sigmoidal, whereas that of HMSiR is bell-shaped due to cyclization of the protonated fluorophore at low pH; this protonation is disfavored when the exocyclic amine is constrained by a five- or six-membered ring. (15,36)
The final criterion needed to ensure compatibility with HMSiR for ratiometric two-color, live-cell SMLM is a high quantum yield. The quantum yields measured for HMSiRindol, HMSiRjulol, and HMSiRTHQ also paralleled the values for the analogous SiR variants; the quantum yield of HMSiRindol, like SiR700, was low (0.13), whereas those of HMSiRjulol and HMSiRTHQ (0.43 and 0.38, respectively) were comparable to that of HMSiR (0.31) (SI Figure S3).
These data indicate that neither HMSiRTHQ, HMSiRjulol nor HMSiRindol possess the characteristics necessary to partner with HMSiR for two-color SMS nanoscopy. Although all three fluorophores exhibit emission maxima that are shifted by at least 23 nm from that of HMSiR, and HMSiRjulol and HMSiRTHQ display acceptable quantum yields (0.43 and 0.38), none feature pKcycl values low enough to prevent significant multiemitter artifacts at physiological pH. In each case, chemical modifications are needed to increase the electrophilicity of the xanthene core, favor spirocyclization, and decrease pKcycl. Ideally, these modifications should also increase quantum yield to increase brightness and resolution, but as outlined below, this goal is complicated by the complex interplay between quantum yield, emission maximum, and pKcycl.

Interplay between Quantum Yield, Emission Maximum, and pKcycl

The quantum yields of rhodamine fluorophores are limited by a nonradiative decay process known as twisted intramolecular charge transfer (TICT). (37−39) TICT involves the excited-state transfer of an electron from the exocyclic nitrogen of the fluorophore to the neighboring carbon π system with concomitant twisting of the Caryl–N bond; the charge-separated state subsequently decays to the ground state without emission of a photon. Processes that decrease the propensity for Caryl–N bond rotation increase quantum yield. For example, the quantum yields of rhodamine B and tetramethyl rhodamine (TMR) are higher in viscous solvents (37) and at low temperature where Caryl–N bond rotation is inhibited. (37,40) Indeed, the modestly increased quantum yields of HMSiRjulol (0.43) and HMSiRTHQ (0.38) relative to HMSiR (0.31) can be ascribed to restricted Caryl–N bond rotation, (34) although these effects appear to be less dramatic in the SiR series than with conventional rhodamines: rhodamine 101, the rhodamine analog of HMSiRjulol, displays a near-perfect quantum yield of 0.99. (40)
TICT is also inhibited in fluorophores in which the ionization potential (IP) of the exocyclic nitrogen is increased by electron-withdrawing groups (EWGs). (18,38,41) Addition of EWGs to a fluorophore core also decreases pKcycl by lowering the energy of the fluorophore’s lowest unoccupied molecular orbital (LUMO). (15,18) However, the addition of EWGs typically induces moderate to large decreases in excitation and emission wavelength maxima. For example, an EWG-containing fluorophore reported by Lv et al. possesses an exceptional quantum yield (0.66) but is blue-shifted by ∼20 nm relative to HMSiR (ƛabs/ƛem = 631 nm/654 nm). (41) We reasoned that combining the effects of restricted aryl-N bond rotation with an EWG would simultaneously reduce pKcycl and increase quantum yield by inhibiting TICT. If these changes were introduced into the HMSiRTHQ scaffold, even a moderate decrease in excitation and emission maxima would not jeopardize the emission shift needed to remain orthogonal to HMSiR. HMSiRTHQ was preferred as a starting point because its pKcycl (6.9) and quantum yield (0.38) are both close to those of HMSiR, in contrast to HMSiRindol, whose quantum yield is low (0.13), or HMSiRjulol, whose pKcycl is very high (9.0).

Design of the Bright, Near-IR-Emitting SBF, Yale676sb

To test this hypothesis, we synthesized Yale676sb, a variant of HMSiRTHQ in which two N-methyl groups were replaced symmetrically by monofluorinated N-ethyl groups (Figure 3 and SI Scheme S5). As predicted, Yale676sb was characterized by a 10-fold more favorable spirocyclization equilibrium than HMSiRTHQ (pKcycl = 5.9 vs 6.9) and a greatly improved quantum yield (0.59 vs 0.38) (SI Figure S3). Interestingly, Yale676sb exhibited absorption and emission ƛmax that are both virtually identical to those of HMSiRTHQ. Addition of a stronger difluorinated N-ethyl group to generate Cal664sb resulted in a further increase in quantum yield to 0.74 (SI Figure S3) but, in this case, led to an emission ƛmax that was too close to that of HMSiR (667 nm vs 677 nm) to support two-color ratiometric imaging. The photophysical properties associated with Yale676sb suggest that it should be an ideal partner for HMSiR: an emission maximum > 690 nm, a pKcycl value between 5.3 and 6.0, and a high quantum yield. The quantum yield of Yale676sb (0.59) is, to our knowledge, higher than any Si-rhodamine derivative prepared and utilized for fluorescence nanoscopy.

Figure 3

Figure 3. Structures and photophysical properties (ƛabs, ƛem, and Φ) of (a) HMSiR and HMSiR2FlEt, and (b) HMSiRTHQ, Yale676sb, and Cal664sb. Normalized (c) absorption and (d) emission spectra of HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ in 0.2 M sodium phosphate (pH = 4.5 for HMSiR; pH = 2.0 for HMSiRindol, HMSiRjulol, and HMSiRTHQ). (e) pH-dependent spirocyclization equilibria. Normalized absorption of open form of 2 μM HMSiR, HMSiR2-FlEt, HMSiRTHQ, Yale676sb, and Cal664sb as a function of pH in 0.2 M sodium phosphate buffer at room temperature.

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To deconvolute the effects of aryl-N bond rotation and the monofluoro electron-withdrawing group, we also prepared HMSiR2-FlEt, which carries the same monofluorinated N-ethyl groups but allows aryl-N bond rotation (SI Scheme S7). HMSiR2-FlEt was characterized by a minimal change in absorption and emission ƛmax relative to HMSiR; however, it displayed a 10-fold more favorable spirocyclization equilibrium than HMSiR (pKcycl = 5.0 vs 6.0), a value too low for efficient blinking at physiological pH of 7.4. (21) Its improvement in quantum yield was more modest relative to Yale676sb (0.51 vs 0.59). These comparisons emphasize the benefits of combining restricted aryl-N bond rotation with an EWG.

Evaluation of the Single-Molecule Properties of Yale676sb

To ensure that the bulk photophysical parameters of Yale676sb would translate into efficient single-molecule parameters, we evaluated its properties under SMLM imaging conditions. We quantified the “ON time” to determine the dye’s compatibility with HMSiR by imaging single dye molecules immobilized on glass coverslips (SI Figure S4). By monitoring individual molecules, we were able to determine the ON time to evaluate the compatibility of Yale676sb and HMSiR. Because both dyes are imaged on the same camera using ratiometric imaging, similar ON times allow a single camera integration time to be effective for acquiring data from both fluorophores. (42) From these data, we determined that Yale676sb has an ON time of 4.5 ms at pH 7.4, which is close to the ∼10 ms ON time reported for HMSiR, and in theory should allow even faster imaging. (15) This short ON time, combined with the high quantum yield also makes the Yale676sb/HMSiR combination suitable for high-speed imaging, with camera frame rates as high as 400 frames per second (fps). With an OFF time of 3.8 s, we expect an ON fraction or duty cycle of 0.0012.

Single-Color Live-Cell SMLM with Yale676sb

We next tested whether Yale676sb would support single-color, live-cell SMLM imaging. U2-OS cells that were engineered to overexpress the endoplasmic reticulum (ER)-localized protein Halo-Sec61β (43) were treated with 300 nM Yale676sb-CA (SI Scheme S8) for 30 min, washed, and immersed in a standard live-cell imaging solution using a custom-built SMLM instrument (see the SI discussion of methods). Figure 4a shows a representative super-resolution image (out of n = 16 images) acquired over 5 s. These images revealed multiple tubules in the cell periphery that were ∼99 ± 15 nm (mean ± s.d.) wide, a value comparable to ER morphology metrics acquired using both STED and 4Pi-SMS. (44) A time series illustrates changes in ER morphology that occur over the course of 10 s (Figure 4B). On average, we detected ∼800 photons per blink, corresponding to a localization precision distribution with a peak at ∼20 nm (Figure 4c,d).

Figure 4

Figure 4. (a) Super-resolution image of the ER in U2-OS cells using Yale676sb. Average reconstructed signal as a function of position along the seven line profiles indicated by yellow lines is shown. Insets: (b) dynamic ER network remodeling; histograms illustrating range in no. of photons (c) and localization precision (d) associated with single molecules in panel a. (e) Two-color super-resolution image of the ER and mitochondria in U2-OS cells using Yale676sb (magenta) in conjunction with HMSiR (green). Insets: (f, g) super-resolved mitochondrial and ER networks in close proximity. Scale bars: 5 μm for panels a and e; 1 μm for panels b and f. All reconstructions using 5 s of acquired frames.

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Ratiometric Two-Color Live-Cell SMLM with Yale676sb and HMSiR

Next we sought to evaluate whether Yale676sb would support live-cell multicolor imaging in combination with HMSiR. U2-OS cells were transiently transfected with Halo-Sec61β (to reveal the ER) and SNAP-OMP25 (to reveal the outer mitochondrial membrane), treated with Yale676sb-CA and HMSiR-BG, and imaged using the identical SMLM setup. As predicted from the absorption and emission spectra of Yale676sb and HMSiR, both dyes could be excited with the same 642 nm laser and ratiometrically separated from two simultaneously acquired images detecting the emission wavelength ranges of 650–680 and 680–750 nm, respectively (SI Figure S5). Figure 4e shows a two-color super-resolution image, accumulated over 5 s, revealing the intertwined mitochondrial and ER networks of the cell. We detected comparable average photon numbers per frame for the two dyes (∼500 and 590 photons for Yale676sb and HMSiR, respectively), especially given that the filters and excitation wavelength were optimized for HMSiR.

Conclusions

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In summary, here we report a new spontaneously blinking Si-rhodamine, Yale676sb, that can be used alongside HMSiR to enable two-color ratiometric SMLM in living cells in physiological media. This new experiment was facilitated by three unique photophysical metrics associated with Yale676sb: (1) an exceptionally high quantum yield for a silicon rhodamine derivative (0.59); (2) an unusually long emission maximum (694 nm); and (3) a pKcycl value (5.9) that is nearly identical to that of HMSiR (6.0).
The unique photophysical metrics associated with Yale676sb result from the simultaneous introduction of both heterocyclic rings as well as electron-withdrawing dialkyl amino groups (DAGs) into the silicon rhodamine core. When either of these structural features is introduced in isolation, at least one of the three critical photophysical metrics required for two-color SMLM becomes nonoptimal. Silicon rhodamine dyes with only heterocycle-containing dialkyl amino groups (such as HMSiRindol, HMSiRTHQ, and HMSiRjulol) display long-wavelength emission (689–716 nm) but resist spirocyclization. As a result, their pKcycl values (6.4–9.0) are too high to ensure adequate distribution of single-molecule emitters (Figure 2). By contrast, silicon rhodamine dyes with only electron-withdrawing substituents, such as HMSiR2-FlEt, display a high quantum yield, but their spirocyclization equilibrium is too favorable, and their pKcycl values are too low (Figure 3). By combining these two substitution patterns in Yale676sb, the competing effects on pKcycl are balanced, while the red shift from the heterocycle-containing DAG is maintained (Figure 3). Moreover, because both the rotational restriction from the heterocycle-containing DAGs and the electron-withdrawing capacity of the 2-fluoroethyl substituent inhibit twisted intramolecular charge transfer, the quantum yield increase from the latter is not only maintained, but enhanced (0.51 vs 0.59).
As expected, switching from a 2-fluoroethyl to a more electron-withdrawing 2,2-difluoroethyl substituent at the nitrogen in Cal664sb further increases the quantum yield, though at the expense of both pKcycl and emission wavelength (Figure 3). This pattern would likely continue with increasingly electron-withdrawing substituents. Despite these blue shifts, Cal664sb displays a comparable quantum yield to a previously reported and exceptionally bright Si-rhodamine fluorophore (compound 9 in ref (41)), but with a >30 nm longer emission maximum. (41)
Finally, we note that while the quantum yield increase relative to HMSiR observed with HMSiR2-FlEt is not as dramatic as that observed with Yale676sb, it is comparable to that observed from more commonly used azetidinyl substituents. (16,35,38,45,46) Being that the former requires only one position at each aniline nitrogen to be substituted, whereas the latter requires two, use of the 2-fluoroethyl substituent may serve as an alternative method for increasing quantum yield of rhodamine derivatives, especially those with more complex DAGs. This approach and others described herein may serve as versatile methods for the preparation of even more greatly enhanced fluorescent labels.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00670.

  • Description of all synthetic and imaging procedures and characterization of all fluorophores (PDF)

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Author Information

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  • Corresponding Authors
    • Joerg Bewersdorf - Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States;  Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06511, United States;  Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, Connecticut 06510, United States;  Nanobiology Institute, Yale University, West Haven, Connecticut 06516, United States;  Email: [email protected]
    • Alanna Schepartz - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, United States;  California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720, United States;  Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States;  Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, United States;  Orcidhttps://orcid.org/0000-0003-2127-3932;  Email: [email protected]
  • Authors
    • Jonathan Tyson - Department of Chemistry, University of California, Berkeley, California 94720, United States;  Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
    • Kevin Hu - Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States;  Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06511, United States
    • Shuai Zheng - Department of Chemistry, University of California, Berkeley, California 94720, United States
    • Phylicia Kidd - Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States
    • Neville Dadina - Department of Chemistry, University of California, Berkeley, California 94720, United States
    • Ling Chu - Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States;  Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
    • Derek Toomre - Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, United States;  Nanobiology Institute, Yale University, West Haven, Connecticut 06516, United States
  • Author Contributions

    J.T. and K.H. contributed equally to this work.

  • Notes
    The authors declare the following competing financial interest(s): J.B. discloses significant financial interest in Bruker Corp. and Hamamatsu Photonics.

Acknowledgments

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This work was supported by the NIH (Grant Nos. 1R01GM131372 and 1R35GM134963 to A.S., R01GM118486 to J.B. and D.T., 1T32EB019941 to K.H., and P30DK045735 to the Yale Diabetes Research Center) and the Wellcome Trust (Grant No. 203285/B/16/Z). Work at the Molecular Foundry to determine quantum yields was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

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    Imaging Intracellular Fluorescent Proteins at Nanometer Resolution
    Betzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.
    Science (Washington, DC, United States) (2006), 313 (5793), 1642-1645CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The authors introduce a method for optically imaging intracellular proteins at nanometer spatial resoln. Numerous sparse subsets of photoactivatable fluorescent protein mols. were activated, localized (to ∼2 to 25 nm), and then bleached. The aggregate position information from all subsets was then assembled into a superresoln. image. The authors used this method - termed photoactivated localization microscopy - to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, the authors imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
  2. 2
    Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3 (10), 793– 796,  DOI: 10.1038/nmeth929
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    2
    Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
    Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei
    Nature Methods (2006), 3 (10), 793-796CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The authors have developed a high-resoln. fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be detd. with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. The authors demonstrated an imaging resoln. of 20 nm. This technique can, in principle, reach mol.-scale resoln.
  3. 3
    Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 2006, 91 (11), 4258– 4272,  DOI: 10.1529/biophysj.106.091116
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    Ultra-high resolution imaging by fluorescence photoactivation localization microscopy
    Hess, Samuel T.; Girirajan, Thanu P. K.; Mason, Michael D.
    Biophysical Journal (2006), 91 (11), 4258-4272CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Biol. structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resoln. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large nos. of fluorescent mols. on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small nos. of them at a time, at low excitation intensity. To control the no. of visible fluorophores in the field of view and ensure that optically active mols. are sepd. by much more than the width of the point spread function, photoactivatable fluorescent mols. are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable mols., the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive mols. are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the mols. are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small no. of mols. are visible at a given time, their positions can be detd. precisely; with only ∼100 detected photons per mol., the localization precision can be as much as 10-fold better than the resoln., depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are obsd. by FPALM of PA-GFP on glass. FPALM images are compared with images of the same mols. by wide-field fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with at. force microscopy and show that the full width at half-max. of features ∼86±4 nm is significantly better than the expected diffraction-limited optical resoln. The no. of fluorescent mols. and their brightness distribution have also been detd. using FPALM. This new method suggests a means to address a significant no. of biol. questions that had previously been limited by microscope resoln.
  4. 4
    Heilemann, M.; van de Linde, S.; Schüttpelz, M.; Kasper, R.; Seefeldt, B.; Mukherjee, A.; Tinnefeld, P.; Sauer, M. Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes. Angew. Chem., Int. Ed. 2008, 47 (33), 6172– 6176,  DOI: 10.1002/anie.200802376
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    4
    Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes
    Heilemann, Mike; van de Linde, Sebastian; Schuttpelz, Mark; Kasper, Robert; Seefeldt, Britta; Mukherjee, Anindita; Tinnefeld, Philip; Sauer, Markus
    Angewandte Chemie, International Edition (2008), 47 (33), 6172-6176CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review.
  5. 5
    Zhang, Y.; Schroeder, L. K.; Lessard, M. D.; Kidd, P.; Chung, J.; Song, Y.; Benedetti, L.; Li, Y.; Ries, J.; Grimm, J. B.; Lavis, L. D.; De Camilli, P.; Rothman, J. E.; Baddeley, D.; Bewersdorf, J. Nanoscale Subcellular Architecture Revealed by Multicolor Three-Dimensional Salvaged Fluorescence Imaging. Nat. Methods 2020, 17 (2), 225– 231,  DOI: 10.1038/s41592-019-0676-4
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    5
    Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging
    Zhang, Yongdeng; Schroeder, Lena K.; Lessard, Mark D.; Kidd, Phylicia; Chung, Jeeyun; Song, Yuanbin; Benedetti, Lorena; Li, Yiming; Ries, Jonas; Grimm, Jonathan B.; Lavis, Luke D.; De Camilli, Pietro; Rothman, James E.; Baddeley, David; Bewersdorf, Joerg
    Nature Methods (2020), 17 (2), 225-231CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Combining the mol. specificity of fluorescent probes with three-dimensional imaging at nanoscale resoln. is crit. for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-mol. switching super-resoln. microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi app. and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.
  6. 6
    Stehr, F.; Stein, J.; Schueder, F.; Schwille, P.; Jungmann, R. Flat-Top TIRF Illumination Boosts DNA-PAINT Imaging and Quantification. Nat. Commun. 2019, 10 (1), 1268,  DOI: 10.1038/s41467-019-09064-6
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    6
    Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification
    Stehr Florian; Stein Johannes; Schueder Florian; Schwille Petra; Jungmann Ralf; Schueder Florian; Jungmann Ralf
    Nature communications (2019), 10 (1), 1268 ISSN:.
    Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation.
  7. 7
    Wang, L.; Frei, M. S.; Salim, A.; Johnsson, K. Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy. J. Am. Chem. Soc. 2019, 141 (7), 2770– 2781,  DOI: 10.1021/jacs.8b11134
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    Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy
    Wang, Lu; Frei, Michelle S.; Salim, Aleksandar; Johnsson, Kai
    Journal of the American Chemical Society (2019), 141 (7), 2770-2781CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. Super-resoln. fluorescence microscopy is a powerful tool to visualize biomols. and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resoln. Different phys. approaches to super-resoln. microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomols. In this perspective, the role of small-mol. fluorescent probes for live-cell super-resoln. microscopy and the challenges that need to be overcome for their generation are discussed. Recent trends in the development of labeling strategies are reviewed together with the required chem. and spectroscopic properties of the probes. Finally, selected examples of the use of small-mol. fluorescent probes in live-cell super-resoln. microscopy are given.
  8. 8
    Huang, B.; Bates, M.; Zhuang, X. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem. 2009, 78 (1), 993– 1016,  DOI: 10.1146/annurev.biochem.77.061906.092014
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    8
    Super-resolution fluorescence microscopy
    Huang, Bo; Bates, Mark; Zhuang, Xiaowei
    Annual Review of Biochemistry (2009), 78 (), 993-1016CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review. Achieving a spatial resoln. that is not limited by the diffraction of light, recent developments of super-resoln. fluorescence microscopy techniques allow the observation of many biol. structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resoln. fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biol. structures and processes.
  9. 9
    Toomre, D.; Bewersdorf, J. A New Wave of Cellular Imaging. Annu. Rev. Cell Dev. Biol. 2010, 26 (1), 285– 314,  DOI: 10.1146/annurev-cellbio-100109-104048
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    9
    A new wave of cellular imaging
    Toomre, Derek; Bewersdorf, Joerg
    Annual Review of Cell and Developmental Biology (2010), 26 (), 285-314CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)
    A review. Fluorescence imaging methods that push or break the diffraction limit of resoln. (approx. 200 nm) have grown explosively. These super-resoln. nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resoln., speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity-down to single mols. with tens of nanometers resoln. Nanoscopes have revealed transient intermediate states of organelles and mols. in living cells and have led to new discoveries but also biol. controversies. We highlight common unifying principles behind nanoscopy such as the conversion of a subset of probes between states (ground or excited) and the use of scanning (ordered or stochastic). We emphasize major advances, biol. applications, and promising new developments.
  10. 10
    Schermelleh, L.; Ferrand, A.; Huser, T.; Eggeling, C.; Sauer, M.; Biehlmaier, O.; Drummen, G. P. C. Super-Resolution Microscopy Demystified. Nat. Cell Biol. 2019, 21 (1), 72– 84,  DOI: 10.1038/s41556-018-0251-8
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    10
    Super-resolution microscopy demystified
    Schermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C.
    Nature Cell Biology (2019), 21 (1), 72-84CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
    Super-resoln. microscopy (SRM) bypasses the diffraction limit, a phys. barrier that restricts the optical resoln. to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
  11. 11
    Schermelleh, L.; Heintzmann, R.; Leonhardt, H. A Guide to Super-Resolution Fluorescence Microscopy. J. Cell Biol. 2010, 190 (2), 165– 175,  DOI: 10.1083/jcb.201002018
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    11
    A guide to super-resolution fluorescence microscopy
    Schermelleh, Lothar; Heintzmann, Rainer; Leonhardt, Heinrich
    Journal of Cell Biology (2010), 190 (2), 165-175CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    A review. For centuries, cell biol. has been based on light microscopy and at the same time been limited by its optical resoln. However, several new technologies have been developed recently that bypass this limit. These new super-resoln. technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single mols. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.
  12. 12
    Jradi, F. M.; Lavis, L. D. Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy. ACS Chem. Biol. 2019, 14, 1077– 1090,  DOI: 10.1021/acschembio.9b00197
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    12
    Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy
    Jradi, Fadi M.; Lavis, Luke D.
    ACS Chemical Biology (2019), 14 (6), 1077-1090CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    A review. The development of single-mol. localization microscopy (SMLM) has sparked a revolution in biol. imaging, allowing 'super-resoln.' fluorescence microscopy below the diffraction limit of light. The last decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-mol. fluorescent probes for this technique. In this review, written by chemists for chemists, the authors detail the history of single-mol. localization microscopy and collate the collection of probes with demonstrated utility in SMLM. The authors hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biol. samples with exquisite detail.
  13. 13
    Li, H.; Vaughan, J. C. Switchable Fluorophores for Single-Molecule Localization Microscopy. Chem. Rev. 2018, 118 (18), 9412– 9454,  DOI: 10.1021/acs.chemrev.7b00767
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    13
    Switchable Fluorophores for Single-Molecule Localization Microscopy
    Li, Honglin; Vaughan, Joshua C.
    Chemical Reviews (Washington, DC, United States) (2018), 118 (18), 9412-9454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The past decade has witnessed an explosion in the use of super-resoln. fluorescence microscopy methods in biol. and other fields. Single-mol. localization microscopy (SMLM) is one of the most widespread of these methods and owes its success in large part to the ability to control the on-off state of fluorophores through various chem., photochem., or binding-unbinding mechanisms. The authors provide here a comprehensive overview of switchable fluorophores in SMLM including a detailed review of all major classes of SMLM fluorophores, and the authors also address strategies for labeling specimens, considerations for multichannel and live-cell imaging, potential pitfalls, and areas for future development.
  14. 14
    Fernández-Suárez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9 (12), 929– 943,  DOI: 10.1038/nrm2531
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    14
    Fluorescent probes for super-resolution imaging in living cells
    Fernandez-Suarez, Marta; Ting, Alice Y.
    Nature Reviews Molecular Cell Biology (2008), 9 (12), 929-943CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resoln. imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resoln. Current research is focused on further improving spatial resoln. in an effort to reach the goal of video-rate imaging of live cells with mol. (1-5 nm) resoln. Here, the authors describe the contributions of fluorescent probes to far-field super-resoln. imaging, focusing on fluorescent proteins and org. small-mol. fluorophores. The authors describe the features of existing super-resoln. fluorophores and highlight areas of importance for future research and development.
  15. 15
    Uno, S.; Kamiya, M.; Yoshihara, T.; Sugawara, K.; Okabe, K.; Tarhan, M. C.; Fujita, H.; Funatsu, T.; Okada, Y.; Tobita, S.; Urano, Y. A Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Live-Cell Super-Resolution Imaging. Nat. Chem. 2014, 6 (8), 681– 689,  DOI: 10.1038/nchem.2002
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    15
    A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging
    Uno, Shin-nosuke; Kamiya, Mako; Yoshihara, Toshitada; Sugawara, Ko; Okabe, Kohki; Tarhan, Mehmet C.; Fujita, Hiroyuki; Funatsu, Takashi; Okada, Yasushi; Tobita, Seiji; Urano, Yasuteru
    Nature Chemistry (2014), 6 (8), 681-689CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Single-mol. localization microscopy is used to construct super-resoln. images, but generally requires prior intense laser irradn. and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodol. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramol. spirocyclization reaction. Optimization of the intramol. nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equil. between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-mol. localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resoln. imaging of microtubules in live cells for up to 1 h.
  16. 16
    Zheng, Q.; Ayala, A. X.; Chung, I.; Weigel, A. V.; Ranjan, A.; Falco, N.; Grimm, J. B.; Tkachuk, A. N.; Wu, C.; Lippincott-Schwartz, J.; Singer, R. H.; Lavis, L. D. Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging. ACS Cent. Sci. 2019, 5 (9), 1602– 1613,  DOI: 10.1021/acscentsci.9b00676
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    16
    Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging
    Zheng, Qinsi; Ayala, Anthony X.; Chung, Inhee; Weigel, Aubrey V.; Ranjan, Anand; Falco, Natalie; Grimm, Jonathan B.; Tkachuk, Ariana N.; Wu, Carl; Lippincott-Schwartz, Jennifer; Singer, Robert H.; Lavis, Luke D.
    ACS Central Science (2019), 5 (9), 1602-1613CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Rhodamine dyes exist in equil. between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equil. toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compds.-ligands that shift to the fluorescent zwitterion upon binding a biomol. target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, the authors develop a quant. framework for the development of new fluorogenic dyes, detg. that the lactone-zwitterion equil. const. (KL-Z) is sufficient to predict fluorogenicity. This rubric emerged from the anal. of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging expts. The authors then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resoln. immunofluorescence. JF526 constitutes a new label for advanced microscopy expts. and the quant. framework will enable the rational design of other fluorogenic probes for bioimaging.
  17. 17
    Tachibana, R.; Kamiya, M.; Suzuki, S.; Morokuma, K.; Nanjo, A.; Urano, Y. Molecular Design Strategy of Fluorogenic Probes Based on Quantum Chemical Prediction of Intramolecular Spirocyclization. Commun. Chem. 2020, 3 (1), 82,  DOI: 10.1038/s42004-020-0326-x
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    17
    Molecular design strategy of fluorogenic probes based on quantum chemical prediction of intramolecular spirocyclization
    Tachibana, Ryo; Kamiya, Mako; Suzuki, Satoshi; Morokuma, Keiji; Nanjo, Aika; Urano, Yasuteru
    Communications Chemistry (2020), 3 (1), 82CODEN: CCOHCT; ISSN:2399-3669. (Nature Research)
    Abstr.: Fluorogenic probes are essential tools for real-time visualization of dynamic intracellular processes in living cells, but so far, their design has been largely dependent on trial-and-error methods. Here we propose a quantum chem. calcn.-based method for rational prediction of the fluorescence properties of hydroxymethyl rhodamine (HMR)-based fluorogenic probes. Our computational anal. of the intramol. spirocyclization reaction, which switches the fluorescence properties of HMR derivs., reveals that consideration of the explicit water mols. is essential for accurate estn. of the free energy difference between the open (fluorescent) and closed (non-fluorescent) forms. We show that this approach can predict the open-closed equil. (pKcycl values) of unknown HMR derivs. in aq. media. We validate this pKcycl prediction methodol. by designing red and yellow fluorogenic peptidase probes that are highly activated by γ-glutamyltranspeptidase, without the need for prior synthesis of multiple candidates.
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    Uno, S.; Kamiya, M.; Morozumi, A.; Urano, Y. A Green-Light-Emitting, Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Dual-Colour Super-Resolution Imaging. Chem. Commun. 2018, 54 (1), 102– 105,  DOI: 10.1039/C7CC07783A
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    18
    A green-light-emitting, spontaneously blinking fluorophore based on intramolecular spirocyclization for dual-colour super-resolution imaging
    Uno, Shin-nosuke; Kamiya, Mako; Morozumi, Akihiko; Urano, Yasuteru
    Chemical Communications (Cambridge, United Kingdom) (2018), 54 (1), 102-105CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The authors have developed the first green-light-emitting, spontaneously blinking fluorophore (SBF), HEtetTFER. In combination with the near-IR-light-emitting SBF (HMSiR), HEtetTFER allows dual-color single-mol. localization microscopy (SMLM) in buffer soln. without any additive and without photoactivation.
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    Halabi, E. A.; Pinotsi, D.; Rivera-Fuentes, P. Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching. Nat. Commun. 2019, 10 (1), 1232,  DOI: 10.1038/s41467-019-09217-7
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    19
    Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching
    Halabi Elias A; Rivera-Fuentes Pablo; Pinotsi Dorothea
    Nature communications (2019), 10 (1), 1232 ISSN:.
    Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.
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    van de Linde, S.; Heilemann, M.; Sauer, M. Live-Cell Super-Resolution Imaging with Synthetic Fluorophores. Annu. Rev. Phys. Chem. 2012, 63 (1), 519– 540,  DOI: 10.1146/annurev-physchem-032811-112012
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    Live-cell super-resolution imaging with synthetic fluorophores
    van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus
    Annual Review of Physical Chemistry (2012), 63 (), 519-540CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)
    A review. Super-resoln. imaging methods now can provide spatial resoln. that is well below the diffraction limit approaching virtually mol. resoln. They can be applied to biol. samples and provide new and exciting views on the structural organization of cells and the dynamics of biomol. assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chem. modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiol. conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resoln. imaging methods.
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    Chi, W.; Qiao, Q.; Wang, C.; Zheng, J.; Zhou, W.; Xu, N.; Wu, X.; Jiang, X.; Tan, D.; Xu, Z.; Liu, X. Descriptor ΔGC-O Enables the Quantitative Design of Spontaneously Blinking Rhodamines for Live-Cell Super-Resolution Imaging. Angew. Chem. 2020, 132 (45), 20390– 20398,  DOI: 10.1002/ange.202010169
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    Dempsey, G. T.; Vaughan, J. C.; Chen, K. H.; Bates, M.; Zhuang, X. Evaluation of Fluorophores for Optimal Performance in Localization-Based Super-Resolution Imaging. Nat. Methods 2011, 8 (12), 1027– 1036,  DOI: 10.1038/nmeth.1768
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    Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging
    Dempsey, Graham T.; Vaughan, Joshua C.; Chen, Kok Hao; Bates, Mark; Zhuang, Xiaowei
    Nature Methods (2011), 8 (12), 1027-1036CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    One approach to super-resoln. fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resoln. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and no. of switching cycles, largely dictate the quality of super-resoln. images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resoln. image quality has been described in only a few cases. Here we quant. characterized the switching properties of 26 org. dyes and directly related these properties to the quality of super-resoln. images. This anal. provides guidelines for characterization of super-resoln. probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resoln. imaging.
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    Jones, S. A.; Shim, S.-H.; He, J.; Zhuang, X. Fast, Three-Dimensional Super-Resolution Imaging of Live Cells. Nat. Methods 2011, 8 (6), 499– 505,  DOI: 10.1038/nmeth.1605
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    Fast, three-dimensional super-resolution imaging of live cells
    Jones, Sara A.; Shim, Sang-Hee; He, Jiang; Zhuang, Xiaowei
    Nature Methods (2011), 8 (6), 499-505CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    We report super-resoln. fluorescence imaging of live cells with high spatiotemporal resoln. using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resoln. images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolns. of ∼25 nm and temporal resolns. as fast as 0.5 s. We also demonstrated live-cell 3D super-resoln. imaging. We obtained 3D spatial resoln. of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolns. as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resoln. imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
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    Wäldchen, S.; Lehmann, J.; Klein, T.; van de Linde, S.; Sauer, M. Light-Induced Cell Damage in Live-Cell Super-Resolution Microscopy. Sci. Rep. 2015, 5, 15348,  DOI: 10.1038/srep15348
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    Light-induced cell damage in live-cell super-resolution microscopy
    Waldchen Sina; Lehmann Julian; Klein Teresa; van de Linde Sebastian; Sauer Markus
    Scientific reports (2015), 5 (), 15348 ISSN:.
    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
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    Zheng, Y.; Ye, Z.; Liu, Z.; Yang, W.; Zhang, X.; Yang, Y.; Xiao, Y. Nitroso-Caged Rhodamine: A Superior Green Light-Activatable Fluorophore for Single-Molecule Localization Super-Resolution Imaging. Anal. Chem. 2021, 93, 7833– 7842,  DOI: 10.1021/acs.analchem.1c00175
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    Nitroso-Caged Rhodamine: A Superior Green Light-Activatable Fluorophore for Single-Molecule Localization Super-Resolution Imaging
    Zheng, Ying; Ye, Zhiwei; Liu, Zengjin; Yang, Wei; Zhang, Xinfu; Yang, Youjun; Xiao, Yi
    Analytical Chemistry (Washington, DC, United States) (2021), 93 (22), 7833-7842CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The evolution of super-resoln. imaging techniques, esp. single-mol. localization microscopy, demands the engineering of switchable fluorophores with labeling functionality. Yet, the switching of these fluorophores depends on the exterior conditions of UV light and enhancing buffers, which is bioincompatible for living-cell applications. Herein, to surpass these limitations, a nitroso-caging strategy is employed to cage rhodamines into leuco forms, which for the first time, is discovered to uncage highly bright zwitterions by green light. Further, clickable construction grants the specificity and versatility for labeling various components in living cells. The simultaneous photoactivation and excitation of these novel probes allow for single-laser super-resoln. imaging without any harmful additives. Super-resoln. imaging of microtubules in fixed cells or mitochondria and the distribution of glycans and H2B proteins in living cells are achieved at a mol. scale with robust integrity. We envision that our nitroso-caging probes would set a platform for the development of future visible-activatable probes.
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    Takakura, H.; Zhang, Y.; Erdmann, R. S.; Thompson, A. D.; Lin, Y.; McNellis, B.; Rivera-Molina, F.; Uno, S.; Kamiya, M.; Urano, Y.; Rothman, J. E.; Bewersdorf, J.; Schepartz, A.; Toomre, D. Long Time-Lapse Nanoscopy with Spontaneously Blinking Membrane Probes. Nat. Biotechnol. 2017, 35 (8), 773– 780,  DOI: 10.1038/nbt.3876
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    Long time-lapse nanoscopy with spontaneously blinking membrane probes
    Takakura, Hideo; Zhang, Yongdeng; Erdmann, Roman S.; Thompson, Alexander D.; Lin, Yu; McNellis, Brian; Rivera-Molina, Felix; Uno, Shin-nosuke; Kamiya, Mako; Urano, Yasuteru; Rothman, James E.; Bewersdorf, Joerg; Schepartz, Alanna; Toomre, Derek
    Nature Biotechnology (2017), 35 (8), 773-780CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Imaging cellular structures and organelles in living cells by long time-lapse super-resoln. microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-d., environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resoln. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resoln. images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.
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    Chu, L.-A.; Lu, C.-H.; Yang, S.-M.; Liu, Y.-T.; Feng, K.-L.; Tsai, Y.-C.; Chang, W.-K.; Wang, W.-C.; Chang, S.-W.; Chen, P.; Lee, T.-K.; Hwu, Y.-K.; Chiang, A.-S.; Chen, B.-C. Rapid Single-Wavelength Lightsheet Localization Microscopy for Clarified Tissue. Nat. Commun. 2019, 10 (1), 4762,  DOI: 10.1038/s41467-019-12715-3
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    Rapid single-wavelength lightsheet localization microscopy for clarified tissue
    Chu Li-An; Chang Wei-Kun; Chiang Ann-Shyn; Chen Bi-Chang; Chu Li-An; Lu Chieh-Han; Yang Shun-Min; Lee Ting-Kuo; Hwu Yeu-Kuang; Chiang Ann-Shyn; Lu Chieh-Han; Liu Yen-Ting; Tsai Yun-Chi; Wang Wen-Cheng; Chang Shu-Wei; Chen Peilin; Chen Bi-Chang; Lu Chieh-Han; Feng Kuan-Lin; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn
    Nature communications (2019), 10 (1), 4762 ISSN:.
    Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 10(7) μm(3)) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 μm(3) per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.
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    Werther, P.; Yserentant, K.; Braun, F.; Kaltwasser, N.; Popp, C.; Baalmann, M.; Herten, D.; Wombacher, R. Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe. Angew. Chem., Int. Ed. 2020, 59 (2), 804– 810,  DOI: 10.1002/anie.201906806
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    Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe
    Werther, Philipp; Yserentant, Klaus; Braun, Felix; Kaltwasser, Nicolai; Popp, Christoph; Baalmann, Mathis; Herten, Dirk-Peter; Wombacher, Richard
    Angewandte Chemie, International Edition (2020), 59 (2), 804-810CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Recent developments in fluorescence microscopy call for novel small-mol.-based labels with multiple functionalities to satisfy different exptl. requirements. A current limitation in the advancement of live-cell single-mol. localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell expts. At the same time, quality of super-resoln. imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, the authors present the design and synthesis of a small-mol. label comprising both fluorogenic and self-blinking features. Bioorthogonal click chem. ensures fast and highly selective attachment onto a variety of biomol. targets. Along with spectroscopic characterization, the probe improves quality and conditions for regular and single-mol. localization microscopy on live-cell samples.
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    Morozumi, A.; Kamiya, M.; Uno, S.; Umezawa, K.; Kojima, R.; Yoshihara, T.; Tobita, S.; Urano, Y. Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-Resolution Imaging. J. Am. Chem. Soc. 2020, 142 (21), 9625– 9633,  DOI: 10.1021/jacs.0c00451
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    Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging
    Morozumi, Akihiko; Kamiya, Mako; Uno, Shin-nosuke; Umezawa, Keitaro; Kojima, Ryosuke; Yoshihara, Toshitada; Tobita, Seiji; Urano, Yasuteru
    Journal of the American Chemical Society (2020), 142 (21), 9625-9633CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Single-mol. localization microscopy (SMLM) allows the reconstruction of super-resoln. images but generally requires prior intense laser irradn. and in some cases additives to induce blinking of conventional fluorophores. The authors previously introduced a spontaneously blinking rhodamine fluorophore based on an intramol. spirocyclization reaction for live-cell SMLM under physiol. conditions. Here, the authors report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equil. (between the fluorescent dissocd. form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-IR (NIR) and green ranges, the authors succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.
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    Lampe, A.; Haucke, V.; Sigrist, S. J.; Heilemann, M.; Schmoranzer, J. Multi-colour Direct STORM with Red Emitting Carbocyanines. Biol. Cell 2012, 104 (4), 229– 237,  DOI: 10.1111/boc.201100011
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    Multi-colour direct STORM with red emitting carbocyanines
    Lampe, Andre; Haucke, Volker; Sigrist, Stephan J.; Heilemann, Mike; Schmoranzer, Jan
    Biology of the Cell (2012), 104 (4), 229-237CODEN: BCELDF; ISSN:0248-4900. (Wiley-Blackwell)
    Background information. Single mol.-based super-resoln. methods have become important tools to study nanoscale structures in cell biol. However, the complexity of multi-color applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy (dSTORM) offers a simple way to perform single mol. super-resoln. imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-color dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-color imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent (OFF) states. Results. We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM (SD-dSTORM). In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biol. nanostructures. In addn., SD-dSTORM allows the use of com. available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localization. Conclusions. SD-dSTORM presents a significant advance towards user-friendly single mol. localisation-based super-resoln. microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-color dSTORM.
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    Winterflood, C. M.; Platonova, E.; Albrecht, D.; Ewers, H. Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging. Biophys. J. 2015, 109 (1), 3– 6,  DOI: 10.1016/j.bpj.2015.05.026
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    Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging
    Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge
    Biophysical Journal (2015), 109 (1), 3-6CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Multicolor three-dimensional (3D) superresoln. techniques allow important insight into the relative organization of cellular structures. While a no. of innovative solns. have emerged, multicolor 3D techniques still face significant tech. challenges. In this Letter we provide a straightforward approach to single-mol. localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a no. of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compd. resoln. routinely achieved only in a single color.
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    Koide, Y.; Urano, Y.; Hanaoka, K.; Piao, W.; Kusakabe, M.; Saito, N.; Terai, T.; Okabe, T.; Nagano, T. Development of NIR Fluorescent Dyes Based on Si-Rhodamine for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134 (11), 5029– 5031,  DOI: 10.1021/ja210375e
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    Development of NIR Fluorescent Dyes Based on Si-rhodamine for in Vivo Imaging
    Koide, Yuichiro; Urano, Yasuteru; Hanaoka, Kenjiro; Piao, Wen; Kusakabe, Moriaki; Saito, Nae; Terai, Takuya; Okabe, Takayoshi; Nagano, Tetsuo
    Journal of the American Chemical Society (2012), 134 (11), 5029-5031CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The authors have developed a series of novel near-IR (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si-rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 (I) and SiR700 (II) showed sufficiently high quantum efficiency in aq. media. Both antibody-bound and free dye exhibited high tolerance to photobleaching in aq. soln. S.c. xenograft tumors were successfully visualized in a mouse tumor model using SiR700-labeled anti-tenascin-C (TN-C) antibody, SiR700-RCB1. SiR-NIRs are expected to be useful as labeling agents for in vivo imaging studies including multicolor imaging, and also as scaffolds for NIR fluorescence probes.
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    Lukinavičius, G.; Reymond, L.; Umezawa, K.; Sallin, O.; D’Este, E.; Göttfert, F.; Ta, H.; Hell, S. W.; Urano, Y.; Johnsson, K. Fluorogenic Probes for Multicolor Imaging in Living Cells. J. Am. Chem. Soc. 2016, 138 (30), 9365– 9368,  DOI: 10.1021/jacs.6b04782
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    Fluorogenic Probes for Multicolor Imaging in Living Cells
    Lukinavicius, Grazvydas; Reymond, Luc; Umezawa, Keitaro; Sallin, Olivier; D'Este, Elisa; Gottfert, Fabian; Ta, Haisen; Hell, Stefan W.; Urano, Yasuteru; Johnsson, Kai
    Journal of the American Chemical Society (2016), 138 (30), 9365-9368CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, resp. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresoln. microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresoln. microscopy in the far-red, thus significantly expanding our capacity for imaging living cells.
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    Butkevich, A. N.; Ta, H.; Ratz, M.; Stoldt, S.; Jakobs, S.; Belov, V. N.; Hell, S. W. Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins. ACS Chem. Biol. 2018, 13 (2), 475– 480,  DOI: 10.1021/acschembio.7b00616
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    Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins
    Butkevich, Alexey N.; Ta, Haisen; Ratz, Michael; Stoldt, Stefan; Jakobs, Stefan; Belov, Vladimir N.; Hell, Stefan W.
    ACS Chemical Biology (2018), 13 (2), 475-480CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resoln. imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.
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    Grimm, J. B.; Brown, T. A.; Tkachuk, A. N.; Lavis, L. D. General Synthetic Method for Si-Fluoresceins and Si-Rhodamines. ACS Cent. Sci. 2017, 3 (9), 975– 985,  DOI: 10.1021/acscentsci.7b00247
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    General Synthetic Method for Si-Fluoresceins and Si-Rhodamines
    Grimm, Jonathan B.; Brown, Timothy A.; Tkachuk, Ariana N.; Lavis, Luke D.
    ACS Central Science (2017), 3 (9), 975-985CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compds. Extensive exploration of these xanthene dyes has yielded general structure-activity relationships where the development of new probes is limited only by imagination and org. chem. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging expts. Nevertheless, efforts to tune the chem. and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient prepn. of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3-5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in soln., and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biol. imaging.
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    Belov, V. N.; Bossi, M. L.; Fölling, J.; Boyarskiy, V. P.; Hell, S. W. Rhodamine Spiroamides for Multicolor Single-Molecule Switching Fluorescent Nanoscopy. Chem. - Eur. J. 2009, 15 (41), 10762– 10776,  DOI: 10.1002/chem.200901333
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    Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy
    Belov, Vladimir N.; Bossi, Mariano L.; Foelling, Jonas; Boyarskiy, Vadim P.; Hell, Stefan W.
    Chemistry - A European Journal (2009), 15 (41), 10762-10776CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The design, synthesis, and evaluation of new rhodamine spiroamides are described. These mols. have applications in optical nanoscopy based on random switching of the fluorescent single mols. The new markers may be used in (co)localization studies of various objects and their (mutual) positions and shape can be detd. with a precision of a few tens of nanometers. Multicolor staining, good photoactivation, a large no. of emitted photons, and selective chem. binding with amino or thiol groups were achieved due to the presence of various functional groups on the rhodamine spiroamides. A rigidized sulfonated xanthene fragment fused with six-membered rings, N,N'-bis(2,2,2-trifluoroethyl) groups, and a combination of addnl. double bonds and sulfonic acid groups with a simple aliph. spiroamide residue provide multicolor properties and improve the performance of the rhodamine spiroamides in photoactivation and bioconjugation reactions. Having both essential parts of the photoswitchable assembly [the switching and the fluorescent (reporter) groups] combined in one chem. entity make this approach attractive for further development. A series of rhodamine spiroamides is presented along with characterizations of their most relevant properties for application as fluorescent probes in single-mol. switching and localization microscopy. Optical images with resolns. on the nanometer scale illustrate the potential of the labels in the colocalization of biol. objects and the two-photon activation technique with optical sectioning.
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    Vogel, M.; Rettig, W.; Sens, R.; Drexhage, K. H. Structural Relaxation of Rhodamine Dyes with Different N-Substitution Patterns: A Study of Fluorescence Decay Times and Quantum Yields. Chem. Phys. Lett. 1988, 147, 452– 460,  DOI: 10.1016/0009-2614(88)85007-3
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    Structural relaxation of rhodamine dyes with different N-substitution patterns: a study of fluorescence decay times and quantum yields
    Vogel, Martin; Rettig, Wolfgang; Sens, Ruediger; Drexhage, Karl H.
    Chemical Physics Letters (1988), 147 (5), 452-60CODEN: CHPLBC; ISSN:0009-2614.
    The viscosity- and temp.-controlled dynamical behavior of rhodamine dyes in the excited state was investigated by stationary and time-resolved fluorescence measurements using synchrotron radiation. An efficient deactivation pathway is linked with rotation of the amino groups presumably towards a state with charge localization. It is shown how electronic and steric factors influence the relaxation rate.
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    Grimm, J. B.; English, B. P.; Chen, J.; Slaughter, J. P.; Zhang, Z.; Revyakin, A.; Patel, R.; Macklin, J. J.; Normanno, D.; Singer, R. H.; Lionnet, T.; Lavis, L. D. A General Method to Improve Fluorophores for Live-Cell and Single-Molecule Microscopy. Nat. Methods 2015, 12 (3), 244– 250,  DOI: 10.1038/nmeth.3256
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    A general method to improve fluorophores for live-cell and single-molecule microscopy
    Grimm, Jonathan B.; English, Brian P.; Chen, Jiji; Slaughter, Joel P.; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J.; Normanno, Davide; Singer, Robert H.; Lionnet, Timothee; Lavis, Luke D.
    Nature Methods (2015), 12 (3), 244-250CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Specific labeling of biomols. with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small no. of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by mol. modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addn. of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-mol. measurements to super-resoln. imaging. The novel substitution is generalizable, yielding a palette of chem. dyes with improved quantum efficiencies that spans the UV and visible range.
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    Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures. Chem. Rev. 2003, 103 (10), 3899– 4032,  DOI: 10.1021/cr940745l
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    Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures
    Grabowski, Zbigniew R.; Rotkiewicz, Krystyna; Rettig, Wolfgang
    Chemical Reviews (Washington, DC, United States) (2003), 103 (10), 3899-4031CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    He aim of this review is to summarize, on the background of other types of ICT states, the exptl. and theor. findings concerning the excited- state structures of the much discussed compds. in which the electron donor (D) and the electron acceptor (A) moieties are linked by a formally single bond: the D-A mols. A major part of this review (sections II-IX) concerns the most discussed compd. 4-(dimethylamino)benzonitrile, 1, and its close derivs. and analogs (usually with only a single arom. ring).
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    Karstens, T.; Kobs, K. Rhodamine B and Rhodamine 101 as Reference Substances for Fluorescence Quantum Yield Measurements. J. Phys. Chem. 1980, 84 (14), 1871– 1872,  DOI: 10.1021/j100451a030
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    Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements
    Karstens, T.; Kobs, K.
    Journal of Physical Chemistry (1980), 84 (14), 1871-2CODEN: JPCHAX; ISSN:0022-3654.
    The fluorescence quantum yield values ΦF of the dyes Rhodamine B and Rhodamine 101 were detd. by temp. dependent measurements of the fluorescence emission and the fluorescence lifetime. Both dyes are stable in soln., when O2 is excluded. If their concn. is kept below 3 × 10-6 M they fulfill the direct relation between absorption and emission. The fluorescence quantum yield ΦF of 101 approaches 100%, whereas ΦF of B is at most 50% at room temp.
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    Lv, X.; Gao, C.; Han, T.; Shi, H.; Guo, W. Improving the Quantum Yields of Fluorophores by Inhibiting Twisted Intramolecular Charge Transfer Using Electron-Withdrawing Group-Functionalized Piperidine Auxochromes. Chem. Commun. 2020, 56 (5), 715– 718,  DOI: 10.1039/C9CC09138F
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    Improving the quantum yields of fluorophores by inhibiting twisted intramolecular charge transfer using electron-withdrawing group-functionalized piperidine auxochromes
    Lv, Xin; Gao, Chunmei; Han, Taihe; Shi, Hu; Guo, Wei
    Chemical Communications (Cambridge, United Kingdom) (2020), 56 (5), 715-718CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Herein, we present that the neg. inductive effect exerted by electron-withdrawing groups, such as sulfone groups, can obviously improve the ionization potential of amino auxochromes, thereby effectively inhibiting twisted intramol. charge transfer (TICT) and markedly improving the quantum yields of several families of fluorophores in aq. soln.
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    Diekmann, R.; Kahnwald, M.; Schoenit, A.; Deschamps, J.; Matti, U.; Ries, J. Optimizing Imaging Speed and Excitation Intensity for Single-Molecule Localization Microscopy. Nat. Methods 2020, 17 (9), 909– 912,  DOI: 10.1038/s41592-020-0918-5
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    Optimizing imaging speed and excitation intensity for single-molecule localization microscopy
    Diekmann, Robin; Kahnwald, Maurice; Schoenit, Andreas; Deschamps, Joran; Matti, Ulf; Ries, Jonas
    Nature Methods (2020), 17 (9), 909-912CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    A review. High laser powers are common practice in single-mol. localization microscopy to speed up data acquisition. Here we systematically quantified how excitation intensity influences localization precision and labeling d., the two main factors detg. data quality. We found a strong trade-off between imaging speed and quality and present optimized imaging protocols for high-throughput, multicolor and three-dimensional single-mol. localization microscopy with greatly improved resoln. and effective labeling efficiency.
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    Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-color labeling strategy for intracellular targets in living cells. Here we demonstrate a universal labeling method based on the org., membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-color STED imaging in living cells below 50 nm resoln. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resoln.
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    Dynamic nanoscale morphology of the ER surveyed by STED microscopy.
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    Grimm, J. B.; Muthusamy, A. K.; Liang, Y.; Brown, T. A.; Lemon, W. C.; Patel, R.; Lu, R.; Macklin, J. J.; Keller, P. J.; Ji, N.; Lavis, L. D. A General Method to Fine-Tune Fluorophores for Live-Cell and In Vivo Imaging. Nat. Methods 2017, 14 (10), 987– 994,  DOI: 10.1038/nmeth.4403
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    A general method to fine-tune fluorophores for live-cell and in vivo imaging
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    Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. It was recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. This strategy was refined and extended revealing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chem. properties of rhodamine dyes with unprecedented precision. This strategy allowed to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. These results demonstrate the versatility of these new dyes in cells, tissues and animals.
  46. 46
    Grimm, J. B.; Tkachuk, A. N.; Xie, L.; Choi, H.; Mohar, B.; Falco, N.; Schaefer, K.; Patel, R.; Zheng, Q.; Liu, Z.; Lippincott-Schwartz, J.; Brown, T. A.; Lavis, L. D. A General Method to Optimize and Functionalize Red-Shifted Rhodamine Dyes. Nat. Methods 2020, 17 (8), 815– 821,  DOI: 10.1038/s41592-020-0909-6
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    Nature Methods (2020), 17 (8), 815-821CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Expanding the palette of fluorescent dyes is vital to push the frontier of biol. imaging. Although rhodamine dyes remain the premier type of small-mol. fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-IR light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. Herein a framework for rationalizing rhodamine behavior in biol. environments and a general chem. modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chem. groups is reported. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biol. imaging expts. in cells and in vivo.

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  • Abstract

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    Figure 1

    Figure 1. (a) Structure and pH-dependent equilibrium of the spontaneously blinking fluorophore HMSiR. (15) (b–d) Structures of previously reported fluorophores considered as potential HMSiR partners for multicolor live-cell SMLM (16,18,21,29) (e) Structure of the spontaneously blinking fluorophore reported herein, Yale676sb.

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    Figure 2

    Figure 2. (a) Structures and photophysical properties (ƛabs, ƛem, and Φ) of previously reported SiR fluorophores with red-shifted absorption and emission spectra and HMSiR analogs HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ. Normalized (b) absorption and (c) emission spectra of HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ in 0.2 M sodium phosphate (pH = 4.5 for HMSiR, pH = 2.0 for HMSiRindol, HMSiRjulol and HMSiRTHQ). (d) pH-dependent change in absorbance of 2 μM HMSIR (650 nm), HMSiRindol (697 nm), HMSiRjulol (684 nm), and HMSiRTHQ (678 nm) as a function of pH in 0.2 M sodium phosphate buffer at room temperature. The absorbance of each fluorophore was monitored at the wavelength of maximal absorbance in panel b.

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    Figure 3

    Figure 3. Structures and photophysical properties (ƛabs, ƛem, and Φ) of (a) HMSiR and HMSiR2FlEt, and (b) HMSiRTHQ, Yale676sb, and Cal664sb. Normalized (c) absorption and (d) emission spectra of HMSiR, HMSiRindol, HMSiRjulol, and HMSiRTHQ in 0.2 M sodium phosphate (pH = 4.5 for HMSiR; pH = 2.0 for HMSiRindol, HMSiRjulol, and HMSiRTHQ). (e) pH-dependent spirocyclization equilibria. Normalized absorption of open form of 2 μM HMSiR, HMSiR2-FlEt, HMSiRTHQ, Yale676sb, and Cal664sb as a function of pH in 0.2 M sodium phosphate buffer at room temperature.

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    Figure 4

    Figure 4. (a) Super-resolution image of the ER in U2-OS cells using Yale676sb. Average reconstructed signal as a function of position along the seven line profiles indicated by yellow lines is shown. Insets: (b) dynamic ER network remodeling; histograms illustrating range in no. of photons (c) and localization precision (d) associated with single molecules in panel a. (e) Two-color super-resolution image of the ER and mitochondria in U2-OS cells using Yale676sb (magenta) in conjunction with HMSiR (green). Insets: (f, g) super-resolved mitochondrial and ER networks in close proximity. Scale bars: 5 μm for panels a and e; 1 μm for panels b and f. All reconstructions using 5 s of acquired frames.

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      5
      Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging
      Zhang, Yongdeng; Schroeder, Lena K.; Lessard, Mark D.; Kidd, Phylicia; Chung, Jeeyun; Song, Yuanbin; Benedetti, Lorena; Li, Yiming; Ries, Jonas; Grimm, Jonathan B.; Lavis, Luke D.; De Camilli, Pietro; Rothman, James E.; Baddeley, David; Bewersdorf, Joerg
      Nature Methods (2020), 17 (2), 225-231CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Combining the mol. specificity of fluorescent probes with three-dimensional imaging at nanoscale resoln. is crit. for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-mol. switching super-resoln. microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi app. and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.
    6. 6
      Stehr, F.; Stein, J.; Schueder, F.; Schwille, P.; Jungmann, R. Flat-Top TIRF Illumination Boosts DNA-PAINT Imaging and Quantification. Nat. Commun. 2019, 10 (1), 1268,  DOI: 10.1038/s41467-019-09064-6
      6
      Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification
      Stehr Florian; Stein Johannes; Schueder Florian; Schwille Petra; Jungmann Ralf; Schueder Florian; Jungmann Ralf
      Nature communications (2019), 10 (1), 1268 ISSN:.
      Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation.
    7. 7
      Wang, L.; Frei, M. S.; Salim, A.; Johnsson, K. Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy. J. Am. Chem. Soc. 2019, 141 (7), 2770– 2781,  DOI: 10.1021/jacs.8b11134
      7
      Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy
      Wang, Lu; Frei, Michelle S.; Salim, Aleksandar; Johnsson, Kai
      Journal of the American Chemical Society (2019), 141 (7), 2770-2781CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A review. Super-resoln. fluorescence microscopy is a powerful tool to visualize biomols. and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resoln. Different phys. approaches to super-resoln. microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomols. In this perspective, the role of small-mol. fluorescent probes for live-cell super-resoln. microscopy and the challenges that need to be overcome for their generation are discussed. Recent trends in the development of labeling strategies are reviewed together with the required chem. and spectroscopic properties of the probes. Finally, selected examples of the use of small-mol. fluorescent probes in live-cell super-resoln. microscopy are given.
    8. 8
      Huang, B.; Bates, M.; Zhuang, X. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem. 2009, 78 (1), 993– 1016,  DOI: 10.1146/annurev.biochem.77.061906.092014
      8
      Super-resolution fluorescence microscopy
      Huang, Bo; Bates, Mark; Zhuang, Xiaowei
      Annual Review of Biochemistry (2009), 78 (), 993-1016CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review. Achieving a spatial resoln. that is not limited by the diffraction of light, recent developments of super-resoln. fluorescence microscopy techniques allow the observation of many biol. structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resoln. fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biol. structures and processes.
    9. 9
      Toomre, D.; Bewersdorf, J. A New Wave of Cellular Imaging. Annu. Rev. Cell Dev. Biol. 2010, 26 (1), 285– 314,  DOI: 10.1146/annurev-cellbio-100109-104048
      9
      A new wave of cellular imaging
      Toomre, Derek; Bewersdorf, Joerg
      Annual Review of Cell and Developmental Biology (2010), 26 (), 285-314CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)
      A review. Fluorescence imaging methods that push or break the diffraction limit of resoln. (approx. 200 nm) have grown explosively. These super-resoln. nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resoln., speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity-down to single mols. with tens of nanometers resoln. Nanoscopes have revealed transient intermediate states of organelles and mols. in living cells and have led to new discoveries but also biol. controversies. We highlight common unifying principles behind nanoscopy such as the conversion of a subset of probes between states (ground or excited) and the use of scanning (ordered or stochastic). We emphasize major advances, biol. applications, and promising new developments.
    10. 10
      Schermelleh, L.; Ferrand, A.; Huser, T.; Eggeling, C.; Sauer, M.; Biehlmaier, O.; Drummen, G. P. C. Super-Resolution Microscopy Demystified. Nat. Cell Biol. 2019, 21 (1), 72– 84,  DOI: 10.1038/s41556-018-0251-8
      10
      Super-resolution microscopy demystified
      Schermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C.
      Nature Cell Biology (2019), 21 (1), 72-84CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
      Super-resoln. microscopy (SRM) bypasses the diffraction limit, a phys. barrier that restricts the optical resoln. to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
    11. 11
      Schermelleh, L.; Heintzmann, R.; Leonhardt, H. A Guide to Super-Resolution Fluorescence Microscopy. J. Cell Biol. 2010, 190 (2), 165– 175,  DOI: 10.1083/jcb.201002018
      11
      A guide to super-resolution fluorescence microscopy
      Schermelleh, Lothar; Heintzmann, Rainer; Leonhardt, Heinrich
      Journal of Cell Biology (2010), 190 (2), 165-175CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      A review. For centuries, cell biol. has been based on light microscopy and at the same time been limited by its optical resoln. However, several new technologies have been developed recently that bypass this limit. These new super-resoln. technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single mols. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.
    12. 12
      Jradi, F. M.; Lavis, L. D. Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy. ACS Chem. Biol. 2019, 14, 1077– 1090,  DOI: 10.1021/acschembio.9b00197
      12
      Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy
      Jradi, Fadi M.; Lavis, Luke D.
      ACS Chemical Biology (2019), 14 (6), 1077-1090CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A review. The development of single-mol. localization microscopy (SMLM) has sparked a revolution in biol. imaging, allowing 'super-resoln.' fluorescence microscopy below the diffraction limit of light. The last decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-mol. fluorescent probes for this technique. In this review, written by chemists for chemists, the authors detail the history of single-mol. localization microscopy and collate the collection of probes with demonstrated utility in SMLM. The authors hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biol. samples with exquisite detail.
    13. 13
      Li, H.; Vaughan, J. C. Switchable Fluorophores for Single-Molecule Localization Microscopy. Chem. Rev. 2018, 118 (18), 9412– 9454,  DOI: 10.1021/acs.chemrev.7b00767
      13
      Switchable Fluorophores for Single-Molecule Localization Microscopy
      Li, Honglin; Vaughan, Joshua C.
      Chemical Reviews (Washington, DC, United States) (2018), 118 (18), 9412-9454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The past decade has witnessed an explosion in the use of super-resoln. fluorescence microscopy methods in biol. and other fields. Single-mol. localization microscopy (SMLM) is one of the most widespread of these methods and owes its success in large part to the ability to control the on-off state of fluorophores through various chem., photochem., or binding-unbinding mechanisms. The authors provide here a comprehensive overview of switchable fluorophores in SMLM including a detailed review of all major classes of SMLM fluorophores, and the authors also address strategies for labeling specimens, considerations for multichannel and live-cell imaging, potential pitfalls, and areas for future development.
    14. 14
      Fernández-Suárez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9 (12), 929– 943,  DOI: 10.1038/nrm2531
      14
      Fluorescent probes for super-resolution imaging in living cells
      Fernandez-Suarez, Marta; Ting, Alice Y.
      Nature Reviews Molecular Cell Biology (2008), 9 (12), 929-943CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resoln. imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resoln. Current research is focused on further improving spatial resoln. in an effort to reach the goal of video-rate imaging of live cells with mol. (1-5 nm) resoln. Here, the authors describe the contributions of fluorescent probes to far-field super-resoln. imaging, focusing on fluorescent proteins and org. small-mol. fluorophores. The authors describe the features of existing super-resoln. fluorophores and highlight areas of importance for future research and development.
    15. 15
      Uno, S.; Kamiya, M.; Yoshihara, T.; Sugawara, K.; Okabe, K.; Tarhan, M. C.; Fujita, H.; Funatsu, T.; Okada, Y.; Tobita, S.; Urano, Y. A Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Live-Cell Super-Resolution Imaging. Nat. Chem. 2014, 6 (8), 681– 689,  DOI: 10.1038/nchem.2002
      15
      A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging
      Uno, Shin-nosuke; Kamiya, Mako; Yoshihara, Toshitada; Sugawara, Ko; Okabe, Kohki; Tarhan, Mehmet C.; Fujita, Hiroyuki; Funatsu, Takashi; Okada, Yasushi; Tobita, Seiji; Urano, Yasuteru
      Nature Chemistry (2014), 6 (8), 681-689CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Single-mol. localization microscopy is used to construct super-resoln. images, but generally requires prior intense laser irradn. and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodol. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramol. spirocyclization reaction. Optimization of the intramol. nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equil. between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-mol. localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resoln. imaging of microtubules in live cells for up to 1 h.
    16. 16
      Zheng, Q.; Ayala, A. X.; Chung, I.; Weigel, A. V.; Ranjan, A.; Falco, N.; Grimm, J. B.; Tkachuk, A. N.; Wu, C.; Lippincott-Schwartz, J.; Singer, R. H.; Lavis, L. D. Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging. ACS Cent. Sci. 2019, 5 (9), 1602– 1613,  DOI: 10.1021/acscentsci.9b00676
      16
      Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging
      Zheng, Qinsi; Ayala, Anthony X.; Chung, Inhee; Weigel, Aubrey V.; Ranjan, Anand; Falco, Natalie; Grimm, Jonathan B.; Tkachuk, Ariana N.; Wu, Carl; Lippincott-Schwartz, Jennifer; Singer, Robert H.; Lavis, Luke D.
      ACS Central Science (2019), 5 (9), 1602-1613CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Rhodamine dyes exist in equil. between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equil. toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compds.-ligands that shift to the fluorescent zwitterion upon binding a biomol. target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, the authors develop a quant. framework for the development of new fluorogenic dyes, detg. that the lactone-zwitterion equil. const. (KL-Z) is sufficient to predict fluorogenicity. This rubric emerged from the anal. of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging expts. The authors then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resoln. immunofluorescence. JF526 constitutes a new label for advanced microscopy expts. and the quant. framework will enable the rational design of other fluorogenic probes for bioimaging.
    17. 17
      Tachibana, R.; Kamiya, M.; Suzuki, S.; Morokuma, K.; Nanjo, A.; Urano, Y. Molecular Design Strategy of Fluorogenic Probes Based on Quantum Chemical Prediction of Intramolecular Spirocyclization. Commun. Chem. 2020, 3 (1), 82,  DOI: 10.1038/s42004-020-0326-x
      17
      Molecular design strategy of fluorogenic probes based on quantum chemical prediction of intramolecular spirocyclization
      Tachibana, Ryo; Kamiya, Mako; Suzuki, Satoshi; Morokuma, Keiji; Nanjo, Aika; Urano, Yasuteru
      Communications Chemistry (2020), 3 (1), 82CODEN: CCOHCT; ISSN:2399-3669. (Nature Research)
      Abstr.: Fluorogenic probes are essential tools for real-time visualization of dynamic intracellular processes in living cells, but so far, their design has been largely dependent on trial-and-error methods. Here we propose a quantum chem. calcn.-based method for rational prediction of the fluorescence properties of hydroxymethyl rhodamine (HMR)-based fluorogenic probes. Our computational anal. of the intramol. spirocyclization reaction, which switches the fluorescence properties of HMR derivs., reveals that consideration of the explicit water mols. is essential for accurate estn. of the free energy difference between the open (fluorescent) and closed (non-fluorescent) forms. We show that this approach can predict the open-closed equil. (pKcycl values) of unknown HMR derivs. in aq. media. We validate this pKcycl prediction methodol. by designing red and yellow fluorogenic peptidase probes that are highly activated by γ-glutamyltranspeptidase, without the need for prior synthesis of multiple candidates.
    18. 18
      Uno, S.; Kamiya, M.; Morozumi, A.; Urano, Y. A Green-Light-Emitting, Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Dual-Colour Super-Resolution Imaging. Chem. Commun. 2018, 54 (1), 102– 105,  DOI: 10.1039/C7CC07783A
      18
      A green-light-emitting, spontaneously blinking fluorophore based on intramolecular spirocyclization for dual-colour super-resolution imaging
      Uno, Shin-nosuke; Kamiya, Mako; Morozumi, Akihiko; Urano, Yasuteru
      Chemical Communications (Cambridge, United Kingdom) (2018), 54 (1), 102-105CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The authors have developed the first green-light-emitting, spontaneously blinking fluorophore (SBF), HEtetTFER. In combination with the near-IR-light-emitting SBF (HMSiR), HEtetTFER allows dual-color single-mol. localization microscopy (SMLM) in buffer soln. without any additive and without photoactivation.
    19. 19
      Halabi, E. A.; Pinotsi, D.; Rivera-Fuentes, P. Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching. Nat. Commun. 2019, 10 (1), 1232,  DOI: 10.1038/s41467-019-09217-7
      19
      Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching
      Halabi Elias A; Rivera-Fuentes Pablo; Pinotsi Dorothea
      Nature communications (2019), 10 (1), 1232 ISSN:.
      Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.
    20. 20
      van de Linde, S.; Heilemann, M.; Sauer, M. Live-Cell Super-Resolution Imaging with Synthetic Fluorophores. Annu. Rev. Phys. Chem. 2012, 63 (1), 519– 540,  DOI: 10.1146/annurev-physchem-032811-112012
      20
      Live-cell super-resolution imaging with synthetic fluorophores
      van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus
      Annual Review of Physical Chemistry (2012), 63 (), 519-540CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)
      A review. Super-resoln. imaging methods now can provide spatial resoln. that is well below the diffraction limit approaching virtually mol. resoln. They can be applied to biol. samples and provide new and exciting views on the structural organization of cells and the dynamics of biomol. assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chem. modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiol. conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resoln. imaging methods.
    21. 21
      Chi, W.; Qiao, Q.; Wang, C.; Zheng, J.; Zhou, W.; Xu, N.; Wu, X.; Jiang, X.; Tan, D.; Xu, Z.; Liu, X. Descriptor ΔGC-O Enables the Quantitative Design of Spontaneously Blinking Rhodamines for Live-Cell Super-Resolution Imaging. Angew. Chem. 2020, 132 (45), 20390– 20398,  DOI: 10.1002/ange.202010169
      There is no corresponding record for this reference.
    22. 22
      Dempsey, G. T.; Vaughan, J. C.; Chen, K. H.; Bates, M.; Zhuang, X. Evaluation of Fluorophores for Optimal Performance in Localization-Based Super-Resolution Imaging. Nat. Methods 2011, 8 (12), 1027– 1036,  DOI: 10.1038/nmeth.1768
      22
      Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging
      Dempsey, Graham T.; Vaughan, Joshua C.; Chen, Kok Hao; Bates, Mark; Zhuang, Xiaowei
      Nature Methods (2011), 8 (12), 1027-1036CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      One approach to super-resoln. fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resoln. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and no. of switching cycles, largely dictate the quality of super-resoln. images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resoln. image quality has been described in only a few cases. Here we quant. characterized the switching properties of 26 org. dyes and directly related these properties to the quality of super-resoln. images. This anal. provides guidelines for characterization of super-resoln. probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resoln. imaging.
    23. 23
      Jones, S. A.; Shim, S.-H.; He, J.; Zhuang, X. Fast, Three-Dimensional Super-Resolution Imaging of Live Cells. Nat. Methods 2011, 8 (6), 499– 505,  DOI: 10.1038/nmeth.1605
      23
      Fast, three-dimensional super-resolution imaging of live cells
      Jones, Sara A.; Shim, Sang-Hee; He, Jiang; Zhuang, Xiaowei
      Nature Methods (2011), 8 (6), 499-505CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      We report super-resoln. fluorescence imaging of live cells with high spatiotemporal resoln. using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resoln. images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolns. of ∼25 nm and temporal resolns. as fast as 0.5 s. We also demonstrated live-cell 3D super-resoln. imaging. We obtained 3D spatial resoln. of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolns. as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resoln. imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
    24. 24
      Wäldchen, S.; Lehmann, J.; Klein, T.; van de Linde, S.; Sauer, M. Light-Induced Cell Damage in Live-Cell Super-Resolution Microscopy. Sci. Rep. 2015, 5, 15348,  DOI: 10.1038/srep15348
      24
      Light-induced cell damage in live-cell super-resolution microscopy
      Waldchen Sina; Lehmann Julian; Klein Teresa; van de Linde Sebastian; Sauer Markus
      Scientific reports (2015), 5 (), 15348 ISSN:.
      Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
    25. 25
      Zheng, Y.; Ye, Z.; Liu, Z.; Yang, W.; Zhang, X.; Yang, Y.; Xiao, Y. Nitroso-Caged Rhodamine: A Superior Green Light-Activatable Fluorophore for Single-Molecule Localization Super-Resolution Imaging. Anal. Chem. 2021, 93, 7833– 7842,  DOI: 10.1021/acs.analchem.1c00175
      25
      Nitroso-Caged Rhodamine: A Superior Green Light-Activatable Fluorophore for Single-Molecule Localization Super-Resolution Imaging
      Zheng, Ying; Ye, Zhiwei; Liu, Zengjin; Yang, Wei; Zhang, Xinfu; Yang, Youjun; Xiao, Yi
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (22), 7833-7842CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The evolution of super-resoln. imaging techniques, esp. single-mol. localization microscopy, demands the engineering of switchable fluorophores with labeling functionality. Yet, the switching of these fluorophores depends on the exterior conditions of UV light and enhancing buffers, which is bioincompatible for living-cell applications. Herein, to surpass these limitations, a nitroso-caging strategy is employed to cage rhodamines into leuco forms, which for the first time, is discovered to uncage highly bright zwitterions by green light. Further, clickable construction grants the specificity and versatility for labeling various components in living cells. The simultaneous photoactivation and excitation of these novel probes allow for single-laser super-resoln. imaging without any harmful additives. Super-resoln. imaging of microtubules in fixed cells or mitochondria and the distribution of glycans and H2B proteins in living cells are achieved at a mol. scale with robust integrity. We envision that our nitroso-caging probes would set a platform for the development of future visible-activatable probes.
    26. 26
      Takakura, H.; Zhang, Y.; Erdmann, R. S.; Thompson, A. D.; Lin, Y.; McNellis, B.; Rivera-Molina, F.; Uno, S.; Kamiya, M.; Urano, Y.; Rothman, J. E.; Bewersdorf, J.; Schepartz, A.; Toomre, D. Long Time-Lapse Nanoscopy with Spontaneously Blinking Membrane Probes. Nat. Biotechnol. 2017, 35 (8), 773– 780,  DOI: 10.1038/nbt.3876
      26
      Long time-lapse nanoscopy with spontaneously blinking membrane probes
      Takakura, Hideo; Zhang, Yongdeng; Erdmann, Roman S.; Thompson, Alexander D.; Lin, Yu; McNellis, Brian; Rivera-Molina, Felix; Uno, Shin-nosuke; Kamiya, Mako; Urano, Yasuteru; Rothman, James E.; Bewersdorf, Joerg; Schepartz, Alanna; Toomre, Derek
      Nature Biotechnology (2017), 35 (8), 773-780CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Imaging cellular structures and organelles in living cells by long time-lapse super-resoln. microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-d., environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resoln. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resoln. images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.
    27. 27
      Chu, L.-A.; Lu, C.-H.; Yang, S.-M.; Liu, Y.-T.; Feng, K.-L.; Tsai, Y.-C.; Chang, W.-K.; Wang, W.-C.; Chang, S.-W.; Chen, P.; Lee, T.-K.; Hwu, Y.-K.; Chiang, A.-S.; Chen, B.-C. Rapid Single-Wavelength Lightsheet Localization Microscopy for Clarified Tissue. Nat. Commun. 2019, 10 (1), 4762,  DOI: 10.1038/s41467-019-12715-3
      27
      Rapid single-wavelength lightsheet localization microscopy for clarified tissue
      Chu Li-An; Chang Wei-Kun; Chiang Ann-Shyn; Chen Bi-Chang; Chu Li-An; Lu Chieh-Han; Yang Shun-Min; Lee Ting-Kuo; Hwu Yeu-Kuang; Chiang Ann-Shyn; Lu Chieh-Han; Liu Yen-Ting; Tsai Yun-Chi; Wang Wen-Cheng; Chang Shu-Wei; Chen Peilin; Chen Bi-Chang; Lu Chieh-Han; Feng Kuan-Lin; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn; Chiang Ann-Shyn
      Nature communications (2019), 10 (1), 4762 ISSN:.
      Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 10(7) μm(3)) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 μm(3) per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.
    28. 28
      Werther, P.; Yserentant, K.; Braun, F.; Kaltwasser, N.; Popp, C.; Baalmann, M.; Herten, D.; Wombacher, R. Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe. Angew. Chem., Int. Ed. 2020, 59 (2), 804– 810,  DOI: 10.1002/anie.201906806
      28
      Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe
      Werther, Philipp; Yserentant, Klaus; Braun, Felix; Kaltwasser, Nicolai; Popp, Christoph; Baalmann, Mathis; Herten, Dirk-Peter; Wombacher, Richard
      Angewandte Chemie, International Edition (2020), 59 (2), 804-810CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Recent developments in fluorescence microscopy call for novel small-mol.-based labels with multiple functionalities to satisfy different exptl. requirements. A current limitation in the advancement of live-cell single-mol. localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell expts. At the same time, quality of super-resoln. imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, the authors present the design and synthesis of a small-mol. label comprising both fluorogenic and self-blinking features. Bioorthogonal click chem. ensures fast and highly selective attachment onto a variety of biomol. targets. Along with spectroscopic characterization, the probe improves quality and conditions for regular and single-mol. localization microscopy on live-cell samples.
    29. 29
      Morozumi, A.; Kamiya, M.; Uno, S.; Umezawa, K.; Kojima, R.; Yoshihara, T.; Tobita, S.; Urano, Y. Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-Resolution Imaging. J. Am. Chem. Soc. 2020, 142 (21), 9625– 9633,  DOI: 10.1021/jacs.0c00451
      29
      Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging
      Morozumi, Akihiko; Kamiya, Mako; Uno, Shin-nosuke; Umezawa, Keitaro; Kojima, Ryosuke; Yoshihara, Toshitada; Tobita, Seiji; Urano, Yasuteru
      Journal of the American Chemical Society (2020), 142 (21), 9625-9633CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Single-mol. localization microscopy (SMLM) allows the reconstruction of super-resoln. images but generally requires prior intense laser irradn. and in some cases additives to induce blinking of conventional fluorophores. The authors previously introduced a spontaneously blinking rhodamine fluorophore based on an intramol. spirocyclization reaction for live-cell SMLM under physiol. conditions. Here, the authors report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equil. (between the fluorescent dissocd. form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-IR (NIR) and green ranges, the authors succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.
    30. 30
      Lampe, A.; Haucke, V.; Sigrist, S. J.; Heilemann, M.; Schmoranzer, J. Multi-colour Direct STORM with Red Emitting Carbocyanines. Biol. Cell 2012, 104 (4), 229– 237,  DOI: 10.1111/boc.201100011
      30
      Multi-colour direct STORM with red emitting carbocyanines
      Lampe, Andre; Haucke, Volker; Sigrist, Stephan J.; Heilemann, Mike; Schmoranzer, Jan
      Biology of the Cell (2012), 104 (4), 229-237CODEN: BCELDF; ISSN:0248-4900. (Wiley-Blackwell)
      Background information. Single mol.-based super-resoln. methods have become important tools to study nanoscale structures in cell biol. However, the complexity of multi-color applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy (dSTORM) offers a simple way to perform single mol. super-resoln. imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-color dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-color imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent (OFF) states. Results. We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM (SD-dSTORM). In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biol. nanostructures. In addn., SD-dSTORM allows the use of com. available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localization. Conclusions. SD-dSTORM presents a significant advance towards user-friendly single mol. localisation-based super-resoln. microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-color dSTORM.
    31. 31
      Winterflood, C. M.; Platonova, E.; Albrecht, D.; Ewers, H. Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging. Biophys. J. 2015, 109 (1), 3– 6,  DOI: 10.1016/j.bpj.2015.05.026
      31
      Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging
      Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge
      Biophysical Journal (2015), 109 (1), 3-6CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Multicolor three-dimensional (3D) superresoln. techniques allow important insight into the relative organization of cellular structures. While a no. of innovative solns. have emerged, multicolor 3D techniques still face significant tech. challenges. In this Letter we provide a straightforward approach to single-mol. localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a no. of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compd. resoln. routinely achieved only in a single color.
    32. 32
      Koide, Y.; Urano, Y.; Hanaoka, K.; Piao, W.; Kusakabe, M.; Saito, N.; Terai, T.; Okabe, T.; Nagano, T. Development of NIR Fluorescent Dyes Based on Si-Rhodamine for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134 (11), 5029– 5031,  DOI: 10.1021/ja210375e
      32
      Development of NIR Fluorescent Dyes Based on Si-rhodamine for in Vivo Imaging
      Koide, Yuichiro; Urano, Yasuteru; Hanaoka, Kenjiro; Piao, Wen; Kusakabe, Moriaki; Saito, Nae; Terai, Takuya; Okabe, Takayoshi; Nagano, Tetsuo
      Journal of the American Chemical Society (2012), 134 (11), 5029-5031CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors have developed a series of novel near-IR (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si-rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 (I) and SiR700 (II) showed sufficiently high quantum efficiency in aq. media. Both antibody-bound and free dye exhibited high tolerance to photobleaching in aq. soln. S.c. xenograft tumors were successfully visualized in a mouse tumor model using SiR700-labeled anti-tenascin-C (TN-C) antibody, SiR700-RCB1. SiR-NIRs are expected to be useful as labeling agents for in vivo imaging studies including multicolor imaging, and also as scaffolds for NIR fluorescence probes.
    33. 33
      Lukinavičius, G.; Reymond, L.; Umezawa, K.; Sallin, O.; D’Este, E.; Göttfert, F.; Ta, H.; Hell, S. W.; Urano, Y.; Johnsson, K. Fluorogenic Probes for Multicolor Imaging in Living Cells. J. Am. Chem. Soc. 2016, 138 (30), 9365– 9368,  DOI: 10.1021/jacs.6b04782
      33
      Fluorogenic Probes for Multicolor Imaging in Living Cells
      Lukinavicius, Grazvydas; Reymond, Luc; Umezawa, Keitaro; Sallin, Olivier; D'Este, Elisa; Gottfert, Fabian; Ta, Haisen; Hell, Stefan W.; Urano, Yasuteru; Johnsson, Kai
      Journal of the American Chemical Society (2016), 138 (30), 9365-9368CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, resp. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresoln. microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresoln. microscopy in the far-red, thus significantly expanding our capacity for imaging living cells.
    34. 34
      Butkevich, A. N.; Ta, H.; Ratz, M.; Stoldt, S.; Jakobs, S.; Belov, V. N.; Hell, S. W. Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins. ACS Chem. Biol. 2018, 13 (2), 475– 480,  DOI: 10.1021/acschembio.7b00616
      34
      Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins
      Butkevich, Alexey N.; Ta, Haisen; Ratz, Michael; Stoldt, Stefan; Jakobs, Stefan; Belov, Vladimir N.; Hell, Stefan W.
      ACS Chemical Biology (2018), 13 (2), 475-480CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resoln. imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.
    35. 35
      Grimm, J. B.; Brown, T. A.; Tkachuk, A. N.; Lavis, L. D. General Synthetic Method for Si-Fluoresceins and Si-Rhodamines. ACS Cent. Sci. 2017, 3 (9), 975– 985,  DOI: 10.1021/acscentsci.7b00247
      35
      General Synthetic Method for Si-Fluoresceins and Si-Rhodamines
      Grimm, Jonathan B.; Brown, Timothy A.; Tkachuk, Ariana N.; Lavis, Luke D.
      ACS Central Science (2017), 3 (9), 975-985CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compds. Extensive exploration of these xanthene dyes has yielded general structure-activity relationships where the development of new probes is limited only by imagination and org. chem. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging expts. Nevertheless, efforts to tune the chem. and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient prepn. of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3-5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in soln., and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biol. imaging.
    36. 36
      Belov, V. N.; Bossi, M. L.; Fölling, J.; Boyarskiy, V. P.; Hell, S. W. Rhodamine Spiroamides for Multicolor Single-Molecule Switching Fluorescent Nanoscopy. Chem. - Eur. J. 2009, 15 (41), 10762– 10776,  DOI: 10.1002/chem.200901333
      36
      Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy
      Belov, Vladimir N.; Bossi, Mariano L.; Foelling, Jonas; Boyarskiy, Vadim P.; Hell, Stefan W.
      Chemistry - A European Journal (2009), 15 (41), 10762-10776CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The design, synthesis, and evaluation of new rhodamine spiroamides are described. These mols. have applications in optical nanoscopy based on random switching of the fluorescent single mols. The new markers may be used in (co)localization studies of various objects and their (mutual) positions and shape can be detd. with a precision of a few tens of nanometers. Multicolor staining, good photoactivation, a large no. of emitted photons, and selective chem. binding with amino or thiol groups were achieved due to the presence of various functional groups on the rhodamine spiroamides. A rigidized sulfonated xanthene fragment fused with six-membered rings, N,N'-bis(2,2,2-trifluoroethyl) groups, and a combination of addnl. double bonds and sulfonic acid groups with a simple aliph. spiroamide residue provide multicolor properties and improve the performance of the rhodamine spiroamides in photoactivation and bioconjugation reactions. Having both essential parts of the photoswitchable assembly [the switching and the fluorescent (reporter) groups] combined in one chem. entity make this approach attractive for further development. A series of rhodamine spiroamides is presented along with characterizations of their most relevant properties for application as fluorescent probes in single-mol. switching and localization microscopy. Optical images with resolns. on the nanometer scale illustrate the potential of the labels in the colocalization of biol. objects and the two-photon activation technique with optical sectioning.
    37. 37
      Vogel, M.; Rettig, W.; Sens, R.; Drexhage, K. H. Structural Relaxation of Rhodamine Dyes with Different N-Substitution Patterns: A Study of Fluorescence Decay Times and Quantum Yields. Chem. Phys. Lett. 1988, 147, 452– 460,  DOI: 10.1016/0009-2614(88)85007-3
      37
      Structural relaxation of rhodamine dyes with different N-substitution patterns: a study of fluorescence decay times and quantum yields
      Vogel, Martin; Rettig, Wolfgang; Sens, Ruediger; Drexhage, Karl H.
      Chemical Physics Letters (1988), 147 (5), 452-60CODEN: CHPLBC; ISSN:0009-2614.
      The viscosity- and temp.-controlled dynamical behavior of rhodamine dyes in the excited state was investigated by stationary and time-resolved fluorescence measurements using synchrotron radiation. An efficient deactivation pathway is linked with rotation of the amino groups presumably towards a state with charge localization. It is shown how electronic and steric factors influence the relaxation rate.
    38. 38
      Grimm, J. B.; English, B. P.; Chen, J.; Slaughter, J. P.; Zhang, Z.; Revyakin, A.; Patel, R.; Macklin, J. J.; Normanno, D.; Singer, R. H.; Lionnet, T.; Lavis, L. D. A General Method to Improve Fluorophores for Live-Cell and Single-Molecule Microscopy. Nat. Methods 2015, 12 (3), 244– 250,  DOI: 10.1038/nmeth.3256
      38
      A general method to improve fluorophores for live-cell and single-molecule microscopy
      Grimm, Jonathan B.; English, Brian P.; Chen, Jiji; Slaughter, Joel P.; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J.; Normanno, Davide; Singer, Robert H.; Lionnet, Timothee; Lavis, Luke D.
      Nature Methods (2015), 12 (3), 244-250CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Specific labeling of biomols. with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small no. of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by mol. modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addn. of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-mol. measurements to super-resoln. imaging. The novel substitution is generalizable, yielding a palette of chem. dyes with improved quantum efficiencies that spans the UV and visible range.
    39. 39
      Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures. Chem. Rev. 2003, 103 (10), 3899– 4032,  DOI: 10.1021/cr940745l
      39
      Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures
      Grabowski, Zbigniew R.; Rotkiewicz, Krystyna; Rettig, Wolfgang
      Chemical Reviews (Washington, DC, United States) (2003), 103 (10), 3899-4031CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      He aim of this review is to summarize, on the background of other types of ICT states, the exptl. and theor. findings concerning the excited- state structures of the much discussed compds. in which the electron donor (D) and the electron acceptor (A) moieties are linked by a formally single bond: the D-A mols. A major part of this review (sections II-IX) concerns the most discussed compd. 4-(dimethylamino)benzonitrile, 1, and its close derivs. and analogs (usually with only a single arom. ring).
    40. 40
      Karstens, T.; Kobs, K. Rhodamine B and Rhodamine 101 as Reference Substances for Fluorescence Quantum Yield Measurements. J. Phys. Chem. 1980, 84 (14), 1871– 1872,  DOI: 10.1021/j100451a030
      40
      Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements
      Karstens, T.; Kobs, K.
      Journal of Physical Chemistry (1980), 84 (14), 1871-2CODEN: JPCHAX; ISSN:0022-3654.
      The fluorescence quantum yield values ΦF of the dyes Rhodamine B and Rhodamine 101 were detd. by temp. dependent measurements of the fluorescence emission and the fluorescence lifetime. Both dyes are stable in soln., when O2 is excluded. If their concn. is kept below 3 × 10-6 M they fulfill the direct relation between absorption and emission. The fluorescence quantum yield ΦF of 101 approaches 100%, whereas ΦF of B is at most 50% at room temp.
    41. 41
      Lv, X.; Gao, C.; Han, T.; Shi, H.; Guo, W. Improving the Quantum Yields of Fluorophores by Inhibiting Twisted Intramolecular Charge Transfer Using Electron-Withdrawing Group-Functionalized Piperidine Auxochromes. Chem. Commun. 2020, 56 (5), 715– 718,  DOI: 10.1039/C9CC09138F
      41
      Improving the quantum yields of fluorophores by inhibiting twisted intramolecular charge transfer using electron-withdrawing group-functionalized piperidine auxochromes
      Lv, Xin; Gao, Chunmei; Han, Taihe; Shi, Hu; Guo, Wei
      Chemical Communications (Cambridge, United Kingdom) (2020), 56 (5), 715-718CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Herein, we present that the neg. inductive effect exerted by electron-withdrawing groups, such as sulfone groups, can obviously improve the ionization potential of amino auxochromes, thereby effectively inhibiting twisted intramol. charge transfer (TICT) and markedly improving the quantum yields of several families of fluorophores in aq. soln.
    42. 42
      Diekmann, R.; Kahnwald, M.; Schoenit, A.; Deschamps, J.; Matti, U.; Ries, J. Optimizing Imaging Speed and Excitation Intensity for Single-Molecule Localization Microscopy. Nat. Methods 2020, 17 (9), 909– 912,  DOI: 10.1038/s41592-020-0918-5
      42
      Optimizing imaging speed and excitation intensity for single-molecule localization microscopy
      Diekmann, Robin; Kahnwald, Maurice; Schoenit, Andreas; Deschamps, Joran; Matti, Ulf; Ries, Jonas
      Nature Methods (2020), 17 (9), 909-912CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      A review. High laser powers are common practice in single-mol. localization microscopy to speed up data acquisition. Here we systematically quantified how excitation intensity influences localization precision and labeling d., the two main factors detg. data quality. We found a strong trade-off between imaging speed and quality and present optimized imaging protocols for high-throughput, multicolor and three-dimensional single-mol. localization microscopy with greatly improved resoln. and effective labeling efficiency.
    43. 43
      Bottanelli, F.; Kromann, E. B.; Allgeyer, E. S.; Erdmann, R. S.; Wood Baguley, S.; Sirinakis, G.; Schepartz, A.; Baddeley, D.; Toomre, D. K.; Rothman, J. E.; Bewersdorf, J. Two-Colour Live-Cell Nanoscale Imaging of Intracellular Targets. Nat. Commun. 2016, 7 (1), 10778,  DOI: 10.1038/ncomms10778
      43
      Two-colour live-cell nanoscale imaging of intracellular targets
      Bottanelli, Francesca; Kromann, Emil B.; Allgeyer, Edward S.; Erdmann, Roman S.; Wood Baguley, Stephanie; Sirinakis, George; Schepartz, Alanna; Baddeley, David; Toomre, Derek K.; Rothman, James E.; Bewersdorf, Joerg
      Nature Communications (2016), 7 (), 10778CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-color labeling strategy for intracellular targets in living cells. Here we demonstrate a universal labeling method based on the org., membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-color STED imaging in living cells below 50 nm resoln. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resoln.
    44. 44
      Schroeder, L. K.; Barentine, A. E. S.; Merta, H.; Schweighofer, S.; Zhang, Y.; Baddeley, D.; Bewersdorf, J.; Bahmanyar, S. Dynamic Nanoscale Morphology of the ER Surveyed by STED Microscopy. J. Cell Biol. 2019, 218 (1), 83– 96,  DOI: 10.1083/jcb.201809107
      44
      Dynamic nanoscale morphology of the ER surveyed by STED microscopy.
      Schroeder, Lena K.; Barentine, Andrew E. S.; Merta, Holly; Schweighofer, Sarah; Zhang, Yongdeng; Baddeley, David; Bewersdorf, Joerg; Bahmanyar, Shirin
      Journal of Cell Biology (2019), 218 (1), 83-96CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresoln. microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resoln. in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resoln. We det. the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quant. anal. of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.
    45. 45
      Grimm, J. B.; Muthusamy, A. K.; Liang, Y.; Brown, T. A.; Lemon, W. C.; Patel, R.; Lu, R.; Macklin, J. J.; Keller, P. J.; Ji, N.; Lavis, L. D. A General Method to Fine-Tune Fluorophores for Live-Cell and In Vivo Imaging. Nat. Methods 2017, 14 (10), 987– 994,  DOI: 10.1038/nmeth.4403
      46
      A general method to fine-tune fluorophores for live-cell and in vivo imaging
      Grimm, Jonathan B.; Muthusamy, Anand K.; Liang, Yajie; Brown, Timothy A.; Lemon, William C.; Patel, Ronak; Lu, Rongwen; Macklin, John J.; Keller, Philipp J.; Ji, Na; Lavis, Luke D.
      Nature Methods (2017), 14 (10), 987-994CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. It was recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. This strategy was refined and extended revealing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chem. properties of rhodamine dyes with unprecedented precision. This strategy allowed to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. These results demonstrate the versatility of these new dyes in cells, tissues and animals.
    46. 46
      Grimm, J. B.; Tkachuk, A. N.; Xie, L.; Choi, H.; Mohar, B.; Falco, N.; Schaefer, K.; Patel, R.; Zheng, Q.; Liu, Z.; Lippincott-Schwartz, J.; Brown, T. A.; Lavis, L. D. A General Method to Optimize and Functionalize Red-Shifted Rhodamine Dyes. Nat. Methods 2020, 17 (8), 815– 821,  DOI: 10.1038/s41592-020-0909-6
      47
      A general method to optimize and functionalize red-shifted rhodamine dyes
      Grimm, Jonathan B.; Tkachuk, Ariana N.; Xie, Liangqi; Choi, Heejun; Mohar, Boaz; Falco, Natalie; Schaefer, Kathy; Patel, Ronak; Zheng, Qinsi; Liu, Zhe; Lippincott-Schwartz, Jennifer; Brown, Timothy A.; Lavis, Luke D.
      Nature Methods (2020), 17 (8), 815-821CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Expanding the palette of fluorescent dyes is vital to push the frontier of biol. imaging. Although rhodamine dyes remain the premier type of small-mol. fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-IR light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. Herein a framework for rationalizing rhodamine behavior in biol. environments and a general chem. modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chem. groups is reported. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biol. imaging expts. in cells and in vivo.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00670.

    • Description of all synthetic and imaging procedures and characterization of all fluorophores (PDF)


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