Abstract
Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution 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 apparatus 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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Code availability
Custom MATLAB code about the color assignment for the salvaged fluorescence approach used during the current study is available at: https://github.com/bewersdorflab/salvaged-fluorescence.
References
Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. & Staehelin, L. A. Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144, 1135–1149 (1999).
Schroeder, L. K. et al. Dynamic nanoscale morphology of the ER surveyed by STED microscopy. J. Cell Biol. 218, 83–96 (2019).
Baddeley, D. & Bewersdorf, J. Biological insight from super-resolution microscopy: what we can learn from localization-based images. Annu. Rev. Biochem. 87, 965–989 (2018).
Sigal, Y. M., Zhou, R. & Zhuang, X. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361, 880–887 (2018).
von Middendorff, C., Egner, A., Geisler, C., Hell, S. W. & Schonle, A. Isotropic 3D nanoscopy based on single emitter switching. Opt. Express 16, 20774–20788 (2008).
Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).
Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).
Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016).
Van Engelenburg, S. B. et al. Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science 343, 653–656 (2014).
Buttler, C. A. et al. Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation. Nat. Commun. 9, 1861 (2018).
Carlini, L., Holden, S. J., Douglass, K. M. & Manley, S. Correction of a depth-dependent lateral distortion in 3D super-resolution imaging. PLoS One 10, e0142949 (2015).
Li, Y. M., Wu, Y. L., Hoess, P., Mund, M. & Ries, J. Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization. Biomed. Opt. Express 10, 2708–2718 (2019).
Burke, D., Patton, B., Huang, F., Bewersdorf, J. & Booth, M. J. Adaptive optics correction of specimen-induced aberrations in single-molecule switching microscopy. Optica 2, 177–185 (2015).
Schonle, A. & Hell, S. W. Fluorescence nanoscopy goes multicolor. Nat. Biotechnol. 25, 1234–1235 (2007).
Bossi, M. et al. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463–2468 (2008).
Baddeley, D. et al. 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One 6, e20645 (2011).
Lampe, A., Haucke, V., Sigrist, S. J., Heilemann, M. & Schmoranzer, J. Multi-colour direct STORM with red emitting carbocyanines. Biol. Cell 104, 229–237 (2012).
Zhang, Z., Kenny, S. J., Hauser, M., Li, W. & Xu, K. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nat. Methods 12, 935–938 (2015).
Mlodzianoski, M. J., Curthoys, N. M., Gunewardene, M. S., Carter, S. & Hess, S. T. Super-resolution imaging of molecular emission spectra and single molecule spectral fluctuations. PLoS One 11, e0147506 (2016).
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 8, 1027–1036 (2011).
Nieuwenhuizen, R. P. et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557–562 (2013).
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).
Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).
Schucker, K., Holm, T., Franke, C., Sauer, M. & Benavente, R. Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution. Proc. Natl Acad. Sci. USA 112, 2029–2033 (2015).
Schmitt, J. et al. Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope. Proc. Natl Acad. Sci. USA 104, 7426–7431 (2007).
Barr, F. A., Puype, M., Vandekerckhove, J. & Warren, G. GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 91, 253–262 (1997).
Fernandez-Busnadiego, R., Saheki, Y. & De Camilli, P. Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites. Proc. Natl Acad. Sci. USA 112, E2004–E2013 (2015).
Chung, J. et al. PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER–plasma membrane contacts. Science 349, 428–432 (2015).
Giordano, F. et al. PI(4,5)P2-dependent and Ca2+-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 153, 1494–1509 (2013).
Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).
Wang, Y. et al. Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm. Opt. Express 22, 15982–15991 (2014).
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).
Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).
Takakura, H. et al. Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat. Biotechnol. 35, 773–780 (2017).
Douglass, K. M., Sieben, C., Archetti, A., Lambert, A. & Manley, S. Super-resolution imaging of multiple cells by optimised flat-field epi-illumination. Nat. Photonics 10, 705–708 (2016).
Deschamps, J., Rowald, A. & Ries, J. Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy. Opt. Express 24, 28080–28090 (2016).
Pallikkuth, S. et al. Sequential super-resolution imaging using DNA strand displacement. PLoS One 13, e0203291 (2018).
Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).
Nemoto, Y. & De Camilli, P. Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein. EMBO J. 18, 2991–3006 (1999).
Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat. Methods 10, 653–658 (2013).
Hanser, B. M., Gustafsson, M. G., Agard, D. A. & Sedat, J. W. Phase-retrieved pupil functions in wide-field fluorescence microscopy. J. Microsc. 216, 32–48 (2004).
Hoess, P., Mund, M., Reitberger, M. & Ries, J. Dual-color and 3D super-resolution microscopy of multi-protein assemblies. Methods Mol. Biol. 1764, 237–251 (2018).
Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed. Opt. Express 2, 1377–1393 (2011).
Acknowledgements
We thank R. Benavente (University of Wuerzburg) for sharing the anti-SYCP1-N antibody, A. E. S. Barentine and A. M. Ernst (Yale University) for comments on the manuscript and C. G. Ebeling (Bruker Corp.) for help with the Vutara SRX software. This work was primarily supported by grants from the Wellcome Trust (grant no. 203285/B/16/Z), the G. Harold and Leila Y. Mathers Foundation and the NIH (grant nos. R01 GM118486, P30 DK045735 and NS36251). Y.L. acknowledges support from the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND. J.B.G. and L.D.L. were funded by the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Contributions
Y.Z. and J.B. conceived the idea. Y.Z. implemented the hardware. Y.Z., Y.L., J.R. and D.B. wrote the software. L.K.S., J.C., P.D.C., J.E.R. and J.B. designed biological experiments. L.K.S. developed sample preparation protocols. L.K.S. prepared and optimized microtubule, ER, mitochondria, Golgi and ER–PM contact sites samples. Y.S. and L.B. provided the synaptonemal complex samples. M.D.L. prepared and optimized the synaptonemal complex samples. P.K. prepared and optimized microtubule samples. Y.Z. imaged the samples and analyzed the images. J.B.G. and L.D.L. synthesized PA-JF646 and PA-JF669. Y.Z., L.K.S. and J.B. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.
Corresponding author
Ethics declarations
Competing interests
J.B. has financial interests in Bruker Corp. and Hamamatsu Photonics. J.B. is co-inventor of a US patent application (US20170251191A1) related to the 4Pi-SMS system and image analysis used in this work. Y.Z. and J.B. have filed a US patent application about the salvaged fluorescence multicolor imaging method described in this work.
Additional information
Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Multicolor 4Pi-SMS microscope design and setup.
(a) Schematic drawing of the front of the 4Pi-SMS system. The excitation laser (green solid line) emerges from the back side of the system (IN), is reflected by the upper dichroic beamsplitter, passes through the objectives and sample, is reflected by the lower dichroic beamsplitter and returns to the back side of the system (OUT). The conventional fluorescence (red solid line) is collected by camera 1. (b) Schematic of positions and information about the dichroic beamsplitters and emission filters used in the system. (c) Photograph of the backside of the 4Pi-SMS system. The positions where the excitation laser goes to (IN) and comes back from (OUT) the front of the system are marked. The salvaged fluorescence is collected by camera 2.
Supplementary Figure 2 Simulation of the SF approach performance for different transition wavelengths of the dichroic beamsplitter.
(a) Simulation results for AF647 and CF660C, the dyes used in two-color imaging. The blue line shows the fraction of rejected molecules when 1% cross-talk is achieved. The green line shows the localization precision of AF647. (b) Simulation results for DL650 and CF680, two of the dyes used in three-color imaging. The blue line shows the fraction of rejected molecules when 2% cross-talk is achieved. The green line shows the localization precision of DL650.
Supplementary Figure 3 Transmission profiles for the conventional and salvaged fluorescence.
(a) Transmission profiles of the dichroic beamsplitter and emission filters for the conventional fluorescence beam path. The magenta line shows the combined transmission profile of the dichroic beamsplitter and emission filters. (b) Transmission profiles of the dichroic beamsplitter and emission filters for the salvaged fluorescence beam path. The green line shows the combined profiles. (c-g) The emission spectra and the fraction of conventional and salvaged fluorescence for each dye used in this work. The blue dashed line indicates the average wavelength of each dye in the detected spectral window of the conventional fluorescence channel. The numerical value is listed in each panel. Spectra were obtained from the respective manufacturers’ websites.
Supplementary Figure 4 Two-color 4Pi-SMS imaging performance on microtubule samples.
(a-f) 4Pi-SMS images of microtubules labeled with AF647 or CF660C in COS-7 cells and the 3D localization precision. (a,d) z-projections of the data sets. Fourier shell correlation (FSC) estimations of the boxed-regions were provided. (b,e) 20-nm thick x-y slices of the same data. (c,f) Clusters featuring at least 10 localizations from the same dye molecule (emitters that appeared in consecutive frames within a radius of two times the localization precision) were aligned by their center of mass to generate the shown 3D plots. Histograms of the distributions in x, y, z were fit with Gaussian functions, and the standard deviations (𝜎x, 𝜎y and 𝜎z) are reported. (g) The binned 2D intensity histogram of AF647 and CF660C from more than 2 million localizations of each dye based on the same data as shown in (Fig. 1d). The plot shows salvaged fluorescence intensity (y-axis) versus conventional fluorescence intensity (x-axis). The solid lines show the threshold where the intensity value is 2% of each peak intensity. (h) Cross-talk between AF647 and CF660C on a logarithmic scale representing the same data as shown in (Fig. 1e). Localizations outside the two threshold boundaries in (g) are rejected. (i) Two-color images of microtubules (anti-α-tubulin antibody labeled with AF647 and CF660C together) in a COS-7 cell. The lower left and upper right corners show the CF660C and AF647 labeling, respectively. (j) Merged image of the two labels in (i). (k) A 20-nm thick x-y slice of the boxed region in (j). Inset: a 100-nm thick cross-section along the dashed line in (k). Representative images of two (a-b,d-e) or seven (i-k) independent experiments are shown.
Supplementary Figure 5 Three-color 4Pi-SMS imaging performance on microtubule samples.
(a-i) 4Pi-SMS images of microtubules labeled with DY634, DL650 or CF680 in COS-7 cells and the 3D localization precision. (a,d,g) z-projections of the data sets. Fourier shell correlation (FSC) estimations of the boxed-regions were provided. (b,e,h) 20-nm thick x-y slices of the same data. (c,f,i) Clusters featuring at least 10 localizations from the same dye molecule (emitters that appeared in consecutive frames within a radius of two times the localization precision) were aligned by their center of mass to generate the shown 3D plots. Histograms of the distributions in x, y, z were fit with Gaussian functions, and the standard deviations (𝜎x, 𝜎y and 𝜎z) are reported. (j) The binned 2D intensity histogram from more than 2 million localizations of each dye based on the same data as shown in (Fig. 1h). The plot shows salvaged fluorescence intensity (y-axis) versus conventional fluorescence intensity (x-axis). The solid lines show the threshold where the intensity value is 2% of each peak intensity. (k) Cross-talk between each dye pair on a logarithmic scale representing the same data as shown in (Fig. 1i). Localizations outside the three threshold boundaries in (j) are rejected. (l) Three-color image of microtubules co-labeled with all three dyes in a COS-7 cell. (m) Magnified image of the boxed region in (l) with the three color channels overlaid. Inset: a 20-nm thick cross-section taken at the dashed line in (m). Representative images of three independent experiments are shown (a-b,d-e,g-h,l-m).
Supplementary Figure 6 Two-color 4Pi-SMS images of ER in COS-7 cells.
(a-c) Two-color images of ER membrane (overexpressed GFP-Sec61β labeled with anti-GFP antibody) and ER lumen (overexpressed mCherry-KDEL labeled with anti-RFP nanobody) (Supplementary Video 2). Merged image of the two labels in (a) and (b) is shown in (c). The boxed region in (c) is shown in (Fig. 2b). (d-f) Two-color images of ER membrane (overexpressed GFP-Sec61β labeled both with antibodies and nanobody). (g) Magnified image of the boxed region in (f). The lower left corner shows the nanobody labeling while the upper right corner shows the antibody labeling. (h) A 50-nm thick x-y slice of (f). (i) Intensity profile along the dashed line in (h) averaged over a thickness of 100 nm. Representative images of one (a-c) or two (d-h) independent experiments are shown.
Supplementary Figure 7 Two-color 4Pi-SMS images of mitochondria in HeLa cells.
(a-c) Two-color images of the outer membrane of mitochondria (anti-TOM20 antibody) and mitochondrial DNA (anti-dsDNA antibody) (Supplementary Video 3). Merged image of the two labels in (a) and (b) is shown in (c). The boxed region in (c) is shown in (Fig. 2f). (d-f) Two-color image of the outer membrane of mitochondria (overexpressed GFP-OMP25 labeled with anti-GFP antibody alongside anti-TOM20 antibody). The dataset was reconstructed from 4 optical sections with 500-nm step sizes. (g) A 200-nm thick x-y slice of the boxed region in (f). (h) y-z view of the orange dashed box (200 nm wide) in (g). (i) x-z view of the blue dashed box (200 nm wide) in (g). (j) 3D chromatic shifts at different offset from the focus center (100-nm thick volumes) of each optical section averaged over the 4 sections. (k) 3D chromatic shifts at different z-positions from non-overlapping 200-nm thick volumes after the 4 optical sections were aligned and combined. Chromatic shifts stay within a 3-nm range in both cases. Representative images of four independent experiments are shown (a-c,d-i).
Supplementary Figure 8 Multicolor 4Pi-SMS images of synaptonemal complexes in mouse spermatocytes.
(a,b) Two-color images of synaptonemal complexes labeled with anti-SYCP3 antibody and anti-SYCP1 C-terminal antibody (Supplementary Video 4, part I). 3D overlay of the synaptonemal complex marked by a white asterisk in (a) and (b) is shown in (Fig. 2j). (c,d) Two-color images of synaptonemal complexes labeled with anti-SYCP3 antibody and anti-SYCP1 N-terminal antibody (Supplementary Video 4, part II). 3D overlay image of the synaptonemal complex marked by a white asterisk in (a) and (b) is shown in (Fig. 2m). (e) Three-color image of synaptonemal complexes labeled with anti-SYCP3 antibody, anti-SYCP1 C-terminal antibody and anti-Lamin B antibody (Supplementary Video 4, part III). (f) A 2-µm thick x-y slice through the middle of the volume in (e). The white dashed circles indicate the positions where synaptonemal complexes connect to the nuclear lamina. Synaptonemal complexes not marked by dashed circles make contact with the nuclear lamina above or below the slice. The size of the white 3D boundary boxes (x × y × z) is provided in the lower right corner of the images (a,c,e). Representative images of five (a-b), two (c-d) or four (e-f) independent experiments are shown.
Supplementary Figure 9 Three-color 4Pi-SMS images of the Golgi apparatus using cis, medial and trans markers in a HeLa cell.
(a-d) 3D images of cis Golgi (anti-GRASP65 antibody), medial Golgi (overexpressed ManII-GFP and labeled with anti-GFP nanobody), and trans Golgi (anti-p230 antibody) markers (Supplementary Video 5, part I). Merged image of the three labels in (a-c) is shown in (d) and is the same as (Fig. 3a). The size of the white 3D boundary boxes (x × y × z) is provided in the lower left corner of the images (a,d). Representative images of three independent experiments are shown (a-d).
Supplementary Figure 10 Three-color 4Pi-SMS images of the Golgi apparatus using one medial and two cis markers in a HeLa cell.
(a-d) 3D overview images of cis Golgi (anti-GRASP65 antibody), cis Golgi (anti-GM130 antibody), and medial Golgi (overexpressed ManII-GFP labeled with anti-GFP nanobody) (Supplementary Video 5, part II). The size of the white 3D boundary boxes (x × y × z) is provided in the lower right corner of the images (a). (e) 1-µm thick y-z cross-section centered at the red dashed region in (d). (f) 1-µm thick x-z cross-section centered at the yellow dashed region in (d). (g) Intensity profile along the dashed line in (f). Representative images of three independent experiments are shown (a-f).
Supplementary Figure 11 Two-color 4Pi-SMS images of the ER and contact site proteins in COS-7 cells.
(a) Two-color image of ER (overexpressed GFP-Sec61β labeled with anti-GFP antibody) and PM (labeled with WGA) in a cell overexpressing mCherry-E-Syt2 (Supplementary Video 6, part III). y-z view is taken from the boxed regions shown in blue and yellow, respectively (3 × 1 μm). (b) Image of ORP5 (overexpressed GFP-ORP5 labeled with anti-GFP antibody) from a two-color image with the PM. x-z view of the boxed region in (b) is shown in (Fig. 4c). (c) Image of E-Syt2 (overexpressed GFP-E-Syt2 labeled with anti-GFP antibody) from a two-color image with the PM. y-z view of the boxed region in (c) is shown in (Fig. 4e). (d) Two-color image of E-Syt2 (overexpressed GFP-E-Syt2 labeled with anti-GFP antibody) and the PM (labeled with WGA). The white arrows indicate some small patch-like structures of E-Syt2. (e) x-z view of the boxed region (3 × 1 μm) in (d). (f) Axial intensity profile averaged across the dashed box in (e). The blue arrow indicates the small distance between WGA and E-Syt2. Representative images of two (a,c,d-e) or three (b) independent experiments are shown.
Supplementary Figure 12 Multicolor 4Pi-SMS images of the ER and contact site proteins in COS-7 cells.
(a) Two-color image of the ER membrane (overexpressed GFP-Sec61β labeled with anti-GFP antibody; shown in the upper right half) and ORP5 (overexpressed mCherry-ORP5 labeled with anti-mCherry antibody; shown in the lower left half). (b) y-z view of the boxed region (3 × 1 μm) in (a). (c) Axial intensity profile averaged across the dashed box in (b). (d) Two-color image of the ER membrane (overexpressed GFP-Sec61β labeled with anti-GFP antibody; shown in the upper right half) and E-Syt2 (overexpressed mCherry-E-Syt2 labeled with anti-mCherry antibody; shown in the lower left half). (e) y-z view of the boxed region (3 × 1 μm) in (d). (f) Axial intensity profile averaged across the dashed box in (e). (g) Three-color image of Sec61β, ORP5 and WGA (Supplementary Video 7, part I). y-z view of the boxed region in (g) is shown in (Fig. 4h). (h) Three-color image of Sec61β, E-Syt2 and WGA (Supplementary Video 7, part II). y-z view of the boxed region in (h) is shown in (Fig. 4j). Representative images of five (a-b), three (d-e) or two (g-h) independent experiments are shown.
Supplementary Figure 13 Alternative implementations of salvaged fluorescence detection and single-objective 2D and 3D images.
(a,b) Salvaged fluorescence can be collected in two configurations in a single-objective microscope by using an emission filter tilted at a small angle. (c) The top emission beam path of the 4Pi-SMS system was blocked to mimic the detection of a single-objective system. (d) Two-color 2D image of ER and microtubules in a COS-7 cell acquired using the configuration in (c). (e) Magnified image of the boxed region in (d). (f) Two-color 3D astigmatic image of microtubules and mitochondria in a COS-7 cell acquired using the configuration in (c). The top right corner shows the data in a rainbow color table denoting z positions, and the bottom left corner shows the two-color overlay. (g) A 100-nm thick x-y slice of the boxed region in (f). (h) x-z view of the blue dashed box (200 nm wide) in (g). (i) y-z view of the orange dashed box (200 nm wide) in (g). Representative images of three (d-e) or two (f-i) independent experiments are shown.
Supplementary information
Supplementary Information
Supplementary Figs. 1–13 and Supplementary Notes 1 and 2.
Supplementary Video 2: Two-color 4Pi-SMS images of ER in a COS-7 cell.
Video rendering of the data shown in Fig. 2a–d and Supplementary Fig. 6a–c. Two-color images of ER membrane (overexpressed GFP-Sec61β labeled with anti-GFP antibody; magenta) and ER lumen (overexpressed mCherry-KDEL labeled with anti-RFP nanobody; green). Representative images of one experiment are shown.
Supplementary Video 3: Two-color 4Pi-SMS images of mitochondria in a HeLa cell
Video rendering of the data shown in Fig. 2f–i and Supplementary Fig. 7a–c. Two-color images of the outer mitochondrial membrane (anti-TOM20 antibody; magenta) and mitochondrial nucleoids (anti-dsDNA antibody; green). The dataset is reconstructed from four optical sections with 500-nm step sizes. Representative images of four independent experiments are shown.
Supplementary Video 4: Multicolor 4Pi-SMS images of the synaptonemal complex in mouse spermatocytes.
Part I: two-color images of synaptonemal complexes labeled with anti-SYCP3 antibody and anti-SYCP1 C-terminal antibody (same data as shown in Supplementary Fig. 8a,b). Part II: two-color images of synaptonemal complexes labeled with anti-SYCP3 antibody and anti-SYCP1 N-terminal antibody (same data as shown in Supplementary Fig. 8c,d). Part III: three-color image of synaptonemal complexes labeled with anti-SYCP3 antibody, anti-SYCP1 C-terminal antibody and anti-Lamin B antibody (same data as shown in Supplementary Fig. 8e,f). Each dataset is reconstructed from 20–21 optical sections with 500-nm step sizes. Representative images of five (Part I), two (Part II) or four (Part III) independent experiments are shown.
Supplementary Video 5: Three-color 4Pi-SMS images of the Golgi apparatus in HeLa cells.
Part I: three-color imaging of cis Golgi (anti-GRASP65 antibody), medial Golgi (overexpressed ManII-GFP and labeled with anti-GFP nanobody) and trans Golgi (anti-p230 antibody) markers (same data as shown in Fig. 3 and Supplementary Fig. 9). Part II: three-color imaging of cis Golgi (anti-GRASP65 antibody), cis Golgi (anti-GM130 antibody) and medial Golgi (overexpressed ManII-GFP labeled with anti-GFP nanobody) (same data as shown in Supplementary Fig. 10). Each dataset is reconstructed from four optical sections with 500-nm step sizes. Representative images of three independent experiments are shown.
Supplementary Video 6: Two-color 4Pi-SMS images of ER–PM contact sites in COS-7 cells.
Video rendering of the data shown in Fig. 4a,b and Supplementary Fig. 11a). Two-color images of ER (overexpressed GFP-Sec61β labeled with anti-GFP antibody) and PM (labeled with WGA) in control cells (Part I), and in cells overexpressing mCherry-ORP5 (Part II) or mCherry-E-Syt2 (Part III). Representative images of four (Part I), three (Part II) or two (Part III) independent experiments are shown.
Source data
Rights and permissions
About this article
Cite this article
Zhang, Y., Schroeder, L.K., Lessard, M.D. et al. Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging. Nat Methods 17, 225–231 (2020). https://doi.org/10.1038/s41592-019-0676-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-019-0676-4
