Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

Fluorescence excitation and emission spectra: Fluorescence excitation and emission spectra of synthetic probes were measured in living U-2 OS cells by confocal microscopy. Acquisition settings were as follows: Green excitation: excitation between 470-534 nm in 2 nm steps; collection at 555 -600 nm. Green emission: excitation at 470 nm and collection between 480-567 nm in 3 nm steps with a bandwidth of 10 nm. Orange excitation: excitation between 475-575 nm in 2 nm steps; collection at 595-700 nm. Orange emission: excitation at 520 nm and collection between 530-617 nm in 3 nm steps with a bandwidth of 10 nm. Red excitation: excitation between 550-650 nm in 2 nm steps; collection at 670-780 nm. Red emission: excitation at 600 nm and collection between 610-697 nm in 3 nm steps with a bandwidth of 10 nm. Except LysoTracker-Red excitation: excitation between 500-600 nm in 2 nm steps; collection at 620-700 nm. LysoTracker-Red emission: excitation at 540 nm and collection between 550-647 nm in 3 nm steps with a bandwidth of 10 nm. NIR excitation: excitation between 630-670 nm in 2 nm steps; collection at 720-780 nm. NIR emission: excitation at 670 nm and collection between 690-768 nm in 3 nm steps with a bandwidth of 10 nm.
Cell viability test U-2 OS cells stably expressing CalR-HaloTag7-KDEL-T2A-Lyn11-SNAPf (induced with 100 μg ml -1 doxycycline) were transiently transfected with a plasmid expressing Golgi-HaloTag11 one day prior labeling. The cells were labeled with MaP618-CA (0.5 μM), MaP555-BG (2.0 μM) for 1 h whereby after 30 min LysoTracker-Green (75 nM) and MitoTracker-Green (200 nM) were added. Subsequently the cells were washed with growth medium and then labeled with SPY555-Actin (500 nM), SiR700-Tubulin (500 nM), and SPY700-DNA (1 μM) for ~20 h. The medium was collected from unlabeled and labeled cells. Additionally, the adherent cells were trypsinised and also added into the same tube to collect all dead and live cells. After spinning the cells at 300 g for 3 min, the cell pellet was resuspended in 2% FBS/PBS containing 1 μM SYTOX Blue dead cell stain (ThermoFisher). Technical triplicates were measured at the BD LSRFortessa™ X-20 Flow Cytometer (Becton, Dickinson and Company) using the software BD FACSDiva™. The following settings were used to detect the various channels: Software and image processing: All images were processed in LASX and ImageJ/Fiji 10,11 unless otherwise stated. Excitation and emission spectra were visualized using OriginLab 12 .

Supporting Figures
Supporting Figure S1: Phasor plots of the 18 synthetic probes tested. A Green spectral region. B Red spectral region. C Orange spectral region. D Far-red spectral region. E NIR spectral region.  Table S3). To visualize the crosstalk and its relation to the pure species, the separated images were given in two different brightness scaling. Scale bars, 10 μm. Figure S4: Live-cell fluorescence lifetime multiplexing using synthetic probes in different spectral regions. A Green spectral region labeling U-2 OS cells with MitoTracker-Green and LysoTracker-Green. B Red spectral region labeling U-2 OS cells with SPY620-DNA and SPY620-Actin. C-D NIR spectral region labeling U-2 OS cells with SPY700-DNA and SiR700-Actin (C) or SPY700-DNA and SiR700-Tubulin (D). The composite, the FastFLIM image with the respective color scale, the total fluorescence intensity, the two individual separated species as well as the corresponding wavelet-filtered phasor plot used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm. The composite, the FastFLIM image with the respective color scale, the total fluorescence intensity, the two individual separated species as well as the corresponding wavelet-filtered phasor plot used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm. Figure S8: Multiplexing synthetic probes and HaloTag in the red channel. A U-2 OS cells stably expressing CalR-HaloTag7-SNAP-tag-KDEL were labeled with MaP618-CA and SPY620-DNA. The composite, the FastFLIM image with the respective color scale, the total fluorescence intensity, the two individual separated species as well as the corresponding wavelet-filtered phasor plot used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm.

Supporting
Supporting Figure S9: Four species images combining fluorescence lifetime multiplexing in the green and orange spectral channel. A-B U-2 OS cells were labeled with MitoTracker-Green, LysoTracker-Green, SPY555-DNA, and either SPY555-Tubulin (A) or SPY555-Actin (B) and imaged in the two spectral channels giving access to four species images. The composite, the total fluorescence intensity, the four individual separated species as well as the corresponding wavelet-filtered phasor plots used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm.
Supporting Figure S10: Four species images combining fluorescence lifetime multiplexing in the green and red spectral channel. U-2 OS cells were labeled with MitoTracker-Green, LysoTracker-Green, SPY620-DNA, and SPY620-Actin and imaged in the two spectral channels giving access to a four species image. The composite, the total fluorescence intensity, the four individual separated species as well as the corresponding wavelet-filtered phasor plots used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm.
Supporting Figure S11: Six species images combining fluorescence lifetime multiplexing in the green, orange, and NIR spectral channel. A-C U-2 OS cells stably expressing CalR-HaloTag7-SNAP-tag-KDEL were labeled with MitoTracker-Green, LysoTracker-Green, MaP555-CA, SPY555-Actin, SPY700-DNA and SiR700-Tubulin and imaged in the three spectral channels giving access to six species images. The composite, the total fluorescence intensity, the six individual separated species as well as the corresponding wavelet-filtered phasor plots used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm. Figure S12: Dynamic six species images combining fluorescence lifetime multiplexing in the green, orange and NIR spectral channel. U-2 OS cells stably expressing CalR-HaloTag7-SNAP-tag-KDEL were labeled with MitoTracker-Green, LysoTracker-Green, MaP555-CA, SPY555-Actin, SPY700-DNA, and SiR700-Tubulin and repeatedly imaged in the three spectral channels giving access to six species images. Composites at the four different time points, a zoom as well as the corresponding summed wavelet-filtered phasor plots used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm.

Supporting
Supporting Figure S13: Eight species image combining fluorescence lifetime multiplexing in the green, orange, red, and NIR spectral channel. U-2 OS cells stably expressing CalR-HaloTag7-KDEL and Lyn11-SNAP-tag were transiently transfected with β-4-Gal-T1-HaloTag11 and labeled with MitoTracker-Green, LysoTracker-Green, MaP555-BG, SPY555-Actin, MaP618-CA, SPY700-DNA, and SiR700-Tubulin and imaged in the four spectral channels giving access to eight species images. The composite, the total fluorescence intensity, the eight individual separated species as well as the corresponding wavelet-filtered phasor plots used for separation are given. Species separation was achieved using the phasor approach by positioning the cluster circles on the phasor plot at the position of the pure species. Scale bars, 10 μm. Figure S14: Flow cytometry plots and statistical analysis of cell viability. U-2 OS cells stably expressing CalR-HaloTag7-KDEL and Lyn11-SNAP-tag were transiently transfected with β-4-Gal-T1-HaloTag11 and labeled with MitoTracker-Green, LysoTracker-Green, MaP555-BG, SPY555-Actin, MaP618-CA, SPY700-DNA, and SiR700-Tubulin. After 20 h, all cells both dead (supernatant) and living (trypsination) were collected and labeled with SYTOX Blue. Flow cytometry analysis of four channels clearly showed labeled and unlabeled (Green, Orange, and the combined Red and NIR) as well as living and dead cells (SYTOX Blue). The difference in cell viability of labeled and unlabeled cells was not statistically significant (two sided t-test, p = 0.27, degrees of freedom = 4). The mean ± 95%CI and the three individual data points from technical replicates are given.

Supporting Tables
Supporting Table S1: Structures of all 18 synthetic probes tested. In addition, the vendor/reference and their excitation and emission maximum (as specified by the vendor) are given.  Table S5: Plasmids used and generated as well as the stable cell line derived thereof.