Toward Absolute Molecular Numbers in DNA-PAINT

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.

Parameters for analysis steps DNA origami design, assembly and purification DNA origami structures were designed using the design module of Picasso 1 (see Figure 4, top for docking strand positions). Folding of structures was performed using the following components: single-stranded DNA scaffold (0.01 µM), core staples (0.5 µM), biotin staples (0.5 µM), modified staples (each 0.5 µM), 1× folding buffer in a total of 50 µl for each sample. Annealing was done by cooling the mixture from 80 to 25 °C in 3 h in a thermocycler. Structures were purified using gel electrophoresis (3 h at 60 V). For detailed instructions see 1,2 .
DNA origami sample preparation DNA origami samples were prepared as described before 1 . A glass slide was glued onto a coverslip with the help of double-sided tape (Scotch, cat. no. 665D) to form a flow chamber with inner volume of ~20 μl. First, 20 µl of biotin-labeled bovine albumin (1 mg/ml, dissolved in buffer A+) was flushed into the chamber and incubated for 3 min. The chamber was then washed with 40 µl of buffer A+. 20 µl of streptavidin (0.5 mg/ml, dissolved in buffer A+) was then flushed through the chamber and incubated for 3 min. After washing with 40 µl of buffer A+ and subsequently with 40 µl of buffer B+, 20 µl of biotin-labeled DNA structures (dilution from DNA origami stock dependent on origami yield after gel purification. Adjusted for each origami species individually to obtain sparse DNA origami surface density. Starting dilution ~1:4) were flushed into the chamber and incubated for 10 min. The chamber was washed with 40 µl of buffer B+. Finally, 40 µl of the imager solution was flushed into the chamber, which was subsequently sealed with two-component epoxy glue before imaging. Adjustment of imager concentrations: The imager concentrations used for all experiments were c = 5, 10 and 20 nM. As described in Supplementary Figure 6, we first prepared a larger volume of 20 nM imager solution, from which in two subsequent 1:1 dilution steps the 10 nM and 5 nM solutions were prepared. Sequence design of imager and docking strands can be found in Supplementary Table 2.

Super-resolution microscopy setup
Fluorescence imaging was carried out on an inverted custom-built microscope 3 (see setup sketch in Supplementary Figure 2a) in an objective-type TIRF configuration with an oil-immersion objective (Olympus UAPON, 100×, NA 1.49). One laser was used for excitation: 561 nm (1 W, DPSS-system, MPB). Laser power was adjusted by polarization rotation with a half-wave plate (Thorlabs, WPH05M-561) before passing a polarizing beam-splitter cube (Thorlabs, PBS101). To spatially clean the beam-profile the laser light was coupled into a single-mode polarization-maintaining fiber (Thorlabs, P3-488PM-FC-2) using an aspheric lens (Thorlabs, C610TME-A). The coupling polarization into the fiber was adjusted using a zero-order half wave plate (Thorlabs, WPH05M-561). The laser light was re-collimated after the fiber using an achromatic doublet lens (Thorlabs, AC254-050-A-ML) resulting in a collimated FWHM beam diameter of ~6 mm. The Gaussian laser beam profile was transformed into a collimated flat-top profile using a refractive beam shaping device (AdlOptica, piShaper 6_6_VIS). The laser beam diameter was magnified by a factor of 2.5 using a custom-built telescope (Thorlabs, AC254-030-A-ML and Thorlabs, AC508-075-A-ML). The laser light was coupled into the microscope objective using an achromatic doublet lens (Thorlabs, AC508-180-A-ML) and a dichroic beam splitter (AHF, F68-785). Fluorescence light was spectrally filtered with a laser notch filter (AHF, F40-072) and a bandpass filter (AHF Analysentechnik, 605/64) and imaged on a sCMOS camera (Andor, Zyla 4.2) without further magnification (Thorlabs, TTL180-A) resulting in an effective pixel size of 130 nm (after 2 × 2 binning). Microscopy samples were mounted into a closed water-based temperature chamber (Okolab, H101-CRYO-BL) on a x-y-z stage (ASI, S31121010FT and ASI, FTP2050) that was used for focusing with the microscope objective being at fixed position. The temperature of the objective was actively controlled using the same water cycle as the temperature chamber. Focus stabilization was achieved via the CRISP autofocus system (ASI @ 850 nm) in a feedback loop with a piezo actuator (Piezoconcept, Z-INSERT100) moving the sample. The CRISP was coupled into the excitation path of the microscope using a long pass dichroic mirror (Thorlabs, DMLP650L). Our custom TIRF setup was used for all Figures.

Imaging conditions
All fluorescence microscopy data was recorded with our sCMOS camera (2048 × 2048 pixels, pixel size: 6.5 µm). The camera was operated with the open source acquisition software µManager 4 at 2x2 binning and cropped to the center 700 × 700 pixel FOV. The exposure time was set to 200 ms, the read out rate to 200 MHz and the dynamic range to 16 bit. For lbFCS measurements the laser power was set to 1.4 mW (see Supplementary Figure 2), corresponding to an average intensity of ~10 W/cm 2 over the circular illuminated area of 130 m in diameter. The acquisition lengths for lbFCS measurements were set to: 9,000 frames (c = 20 & 10 nM) and 18,000 frames (c = 5 nM). Longer acquisition lengths at lower imager concentrations ensure that sufficient imager binding events are registered from each DS cluster as a prerequisite for robust autocorrelation analysis 5 . For high resolution imaging the laser power was set to 70 mW (intensity of ~500 W/cm 2 ) and the acquisition length to 5,000 frames.

Super-resolution reconstruction & data analysis
Refer to Supplementary Figure 1 for a detailed step-by-step guide through all processing steps after data acquisition. The lbFCS software package and installation instructions are available at https://github.com/schwillepaint/lbFCS. A full integration in the Picasso 1 software package is currently under construction.   filter n.a.