Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy

Abstract

Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.

Keywords: CP: Imaging; DNA-PAINT; SMLM; STORM; crosstalk; dSTORM; fluorescence; multicolor; multitarget; spectral demixing; super resolution microscopy.

Graphical abstract

Figure 1

Simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) and crosstalk evaluation (A) In S2C-DNA-PAINT, blinking events appear only on either one camera or the other (left panels): fluorophores emitting in the red (here Cy3B, orange curve on top right spectra graph) and far-red (here Atto643, red curve) are separated by the 662-nm cutoff dichroic mirror (gray line). Ratiometric analysis (bottom right graph) shows fluorophores appearing on only one camera, leading to the chosen ratio ranges (orange and red colored areas): 0.00–0.01 for Cy3B and 0.99–1.0 for Atto643, used in (B), (C), and (D). (B) Crosstalk measurement from nanorulers (40-nm and 80-nm spacing) bearing P1 and P3 docking strands using I1-Atto655 and I3-Cy3B imager strands. Nanorulers were classified by total length (top distributions, peaks at 80 and 160 nm) before determining the crosstalk (see Figure S2D). (C) Crosstalk measurement from a single-target cellular sample: zoomed image of a COS-7 cell labeled for microtubules with a secondary antibody carrying an F1 docking strand. Acquisition A (top, left column) uses a mixture of non-target (IF2) imager strands to measure the background signal in both channels (see Figure S3). Acquisition B (top, right column) is done over the same field of view with a mixture of a target (IF1-Cy3B) and a non-target (IF2-Atto643) imager strands. An ROI enclosing the microtubule network (bottom row, white is an ROI overlay on the background images) is obtained from the target (IF1-Cy3B) image and used to measure the number of localizations for acquisitions A and B in both channels (middle graph). The crosstalk from Cy3B into Atto643 is based on the difference between the number of localizations in acquisition B, channel 2 (right red bar) and the number of localizations in acquisition A, ch2 (left red bar). Conversely, the crosstalk of Atto643 into Cy3B can be measured using a target IF2-Cy3B acquisition, evaluating the difference induced in ch1 between acquisition A and B (orange bars in bottom graph). (D) Crosstalk measurement from a two-target cellular sample: COS-7 cells labeled for tubulin (F1 docking strand) and clathrin (F2 docking strand), simultaneously imaged with IF1-Atto643 and IF2-Atto565. Insets show zoomed isolated channels. Crosstalk was calculated from exclusive ROIs containing only microtubules or only clathrin-coated pits excluding overlapping areas. (E) Crosstalk values obtained from the three different approaches, grouped by crosstalk direction (far-red into red on the left, orange; red into far-red on the right, red): nanorulers (B), single-target cellular sample (C), and two-target cellular sample (D). Points correspond to individual images or datapoints, error bars are SEM.

Figure 2

Spectral demixing STORM (SD-dSTORM) and crosstalk evaluation (A) In SD-dSTORM, blinking events from two far-red fluorophores (AF647 and CF680) appear on both cameras (left panels) due to their spectral proximity (top right graph). Ratiometric analysis of photon ratios for blinking events from each fluorophore leads to a 0.01–0.38 range for AF647 (yellow area), and 0.42–0.99 for CF680 (blue area), used in (B), (C), and (D). (B) Crosstalk measurement from nanorulers (40-nm and 80-nm spacing) bearing P1 and P3 docking strands and imaged with I1-Atto680 and I3-655 imager strands. Nanorulers were classified by total length (top distribution, peaks 80 and 160 nm) before determining the crosstalk. (C) Crosstalk measurement from a single-target cellular sample: COS-7 cells labeled for microtubules with a secondary antibody conjugated to either AF647 or CF680 with ratiometric analysis of the localizations (top). After demixing (yellow and blue areas), images were reconstructed (bottom panels). ROIs were drawn around the microtubules with an intensity threshold and used on both channels to calculate the crosstalk. (D) Crosstalk measurement from a two-target cellular sample: COS cells labeled for tubulin with AF647 and for clathrin with CF680. Insets show zoomed isolated channels. Crosstalk was calculated from exclusive ROIs containing only microtubules or only clathrin-coated pits excluding overlapping areas. (E) Crosstalk values obtained from the three different approaches, grouped by crosstalk direction (far-red into red on the left, orange; red into far-red on the right, red): nanorulers (see B), single-target cellular sample (C), and two-target cellular sample (D). Points correspond to individual images or datapoints, error bars are SEM.

Figure 3

Effect of crosstalk on the detection of biologically relevant patterns by S2C-DNA-PAINT (A) Reconstructed images of axons stained for β2-spectrin (orange) and adducin (blue), imaged by S2C-DNA-PAINT using IF1-Cy3B and IF2-Atto643, respectively. On the same field of view, the imager concentration of IF1-Cy3B (β2-spectrin) was kept constant, while the concentration of IF2-Atto643 (adducin) was increased successively from 1% to 10% and 100% of the reference concentration. (B) Autocorrelation curves from 1-μm-long intensity profiles along axons for the constant β2-spectrin-IF1-Cy3B (orange) and varying adducin-IF2-Atto643 (blue) channels for each imager concentration conditions. (C) Amplitude of the autocorrelation first peak for each imager concentration value. (D) Adducin imaging done at constant 100% IF2-Atto643 imager concentration (blue), while varying the β2-spectrin IF1-Cy3B imager concentration (from 1% to 100%, orange). (E) Autocorrelation curves from 1-μm-long intensity profiles along axons for the varying β2-spectrin-IF1-Cy3B (orange) and constant adducin-IF2-Atto643 (blue) channels for each imager concentration condition. (F) Amplitude of the autocorrelation first peak for each imager concentration value. For (C) and (F), dots are individual axonal segments, error bars are SEM; stars show a significant difference with the 100%–1% condition (first bar) by post hoc Tukey-Kramer test, p < 0.05.

Figure 4

Effect of crosstalk on the detection of biologically relevant patterns by SD-dSTORM (A) Reconstructed images of axons of stained for adducin (orange) and β2-spectrin (blue) imaged by SD-dSTORM using secondary antibodies conjugated with AF647 and CF680, respectively. During immunolabeling, the concentration of AF647-conjugated antibody was kept constant, while the concentration of CF680-conjugated antibody was increased successively from 3.3% to 10% and 100% of the total secondary antibody concentration. (B) Autocorrelation curves from 1-μm-long intensity profiles along axons for the constant adducin-AF647 (orange) and varying β2-spectrin-CF680 (bottom) channels for each secondary antibody concentration condition. (C) Amplitude of the autocorrelation first peak for each secondary antibody concentration value. (D) β2-spectrin imaging done at constant 100% CF680-conjugated secondary antibody concentration (blue) while varying the adducin AF647-conjugated secondary antibody concentration (from 3.3% to 100%, orange). (E) Autocorrelation curves from 1-μm-long intensity profiles along axons for the varying adducin-AF647 (orange) and constant β2-spectrin-CF680 (blue) channels for each secondary antibody concentration conditions. (F) Amplitude of the autocorrelation first peak for each secondary antibody concentration value. For (C) and (F), dots are individual axonal segments, bars are SEM; stars show a significant difference compared with the first condition (left bar) by post hoc Tukey-Kramer test, p < 0.05.

Figure 5

Extension of S2C-DNA-PAINT and SD-dSTORM to astigmatism-based 3D SMLM (A) 3D S2C-DNA-PAINT: image of a COS-7 cell labeled for clathrin and tubulin, imaged with IF2-Cy3B (orange) and IF1-Atto643 (red), respectively. Insets show zoomed isolated channels, color-coded for Z. (B) Distribution of the photon ratios for the acquisition in (A), with colored areas for the ratios chosen for demixing: 0–0.01 for Cy3B (orange), 0.99–1 for Atto643 (red). (C) Full-width half-maximum (FWHM) analysis of the average intensity profile for transverse section along three isolated microtubules in (A). Inset, average transverse section obtained after alignment of individual sections. (D) SD-dSTORM image of a COS-7 cell labeled for tubulin and clathrin, revealed with secondary antibodies conjugated to AF647 (yellow) and CF680 (cyan), respectively. Insets show zoomed isolated channels, color-coded for Z. (E) Distribution of the photon ratios for the acquisition in (A), with colored areas for the ratios chosen for demixing: 0.01–0.38 for AF647 (yellow area), 0.42–0.99 for CF680 (blue area). (F) FWHM analysis of the average intensity profile for transverse section along three isolated microtubules in (A). Inset, average transverse section obtained after alignment of individual sections.

Figure 6

Extension of SD-dSTORM to three targets and crosstalk evaluation (A) Emission spectra of the fluorophores used for three-color SD-dSTORM: AF647 (yellow), CF660C (green), and CF680 (blue), and transmission of the 700-nm long-pass dichroic inserted in the detection pathway (gray). (B) Photon ratios for each fluorophore determined by ratiometric analysis from single-fluorophore-stained microtubules in COS-7 cells. Colored areas highlight the ratio ranges chosen for AF647 (0.01–0.29, yellow), CF660C (0.31–0.40, green), and CF680 (0.50–0.99, blue). (C) SD-dSTORM images of COS cells stained for microtubules using secondary antibodies conjugated to AF647 (yellow), CF660C (green), or CF680 (blue). ROIs segmented from the microtubule channel were used to measure the number of localizations in each channel and calculate the crosstalk between channels. (D) Three-channel SD-dSTORM image of a COS cell labeled for the endoplasmic reticulum (ER; AF647), clathrin (CF660C), and tubulin (CF680). Insets show zoomed isolated channels. (E) Photon ratios for each fluorophore from the three-channel acquisition in (D). Colored areas highlight the ratio ranges chosen to demix AF647 (0.01–0.30, yellow), CF660C (0.35–0.45, green), and CF680 (0.50–0.99, blue). (F) Crosstalk between the channels calculated from the single-fluorophore staining (C) and the three-target staining (D) cellular samples. For the three-target sample, exclusive ROIs were used to delineate regions containing one target, but not the two others. Points correspond to individual images or datapoints; error bars are SEM.