High-density three-dimensional localization microscopy across large volumes

Abstract

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high–localization precision, ultrahigh–labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

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Figure 1: The effect of labeling density in localization microscopy.
Figure 2: Whole-cell 3D localization microscopy of intracellular membranes.
Figure 3: Multicolor LLS-PAINT microscopy.
Figure 4: 3D architecture of membranes and chromosomes during mitosis.
Figure 5: 3D architecture of membranes in the zebrafish neuromast sensory organ.
Figure 6: Compensation for nonlinear swelling in LLS-PAINT data sets.

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Acknowledgements

We gratefully acknowledge M. Davidson (National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA) for the cell line stably expressing calnexin and H2B and for the plasmids used for comparative staining, D. Li for discussion of resolution metrics and localization density, B. Hoeckendorf for assistance in culturing and mounting zebrafish embryos, and H. White and the shared resource teams at Janelia for assistance with cell culture. This work was supported by the Howard Hughes Medical Institute (W.R.L., L.S., J.B.G., T.A.B., L.D.L. and E.B.) and the US National Institutes of Health through the National Institute of Environmental Health Science (grant K01ES025432-01 to B.B.A.).

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Authors

Contributions

E.B. supervised the project; W.R.L., L.D.L. and E.B. conceived the idea; D.E.M., W.R.L. and E.B. developed the instrument control program; W.R.L. and L.S. designed the single-molecule fitting and plotting software; L.D.L., J.B.G. and T.A.B. designed and characterized the AzepRh and Hoechst-JF646 PAINT probes; B.B.A. developed the SyN algorithm and ANTs software used for nonlinear swelling correction; W.R.L. built the instrument and performed the imaging experiments; W.R.L. and E.B. wrote the paper with input from all other coauthors.

Corresponding author

Correspondence to Eric Betzig.

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Competing interests

Portions of the technology described herein are covered by one or more pending US patent applications filed by E.B. and W.R.L. (provisional applications 62,088,593 and 62/088,681; utility application 14/961,873) or by L.D.L. and J.B.G. (provisional applications 61/973,795 and 61/991,109; Trademark Intellectual Property application 86386733; Patent Cooperation Treaty application 15/23953) and assigned to Howard Hughes Medical Institute.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–28, Supplementary Table 1 and Supplementary Notes 1–11 (PDF 17526 kb)

Simulated localization microscopy images compared to ground truth images of the actin cytoskeleton.

A ground truth, 31 nm resolution image of actin in the lamella of a cell was generated from electron microscopy data as described in Supplementary Note 2. For comparison, we simulated the process of localization microscopy by plotting individual localizations along the same actin cytoskeleton with a localization precision of 10 nm. The bottom corner shows the amount of oversampling (i.e. the fold increase in the number of molecules) present in the image compared 1X Nyquist sampling at 20 nm resolution. For example, 1.00 fold oversampling would correspond to an average spacing between localized molecules of 10 nm. When combined with the 10 nm localization precision, this would provide an estimate of 31 nm resolution according to Eq. 2 in Supplementary Note 1. This would imply then that both the ground truth and 1.00 fold oversample localization microscopy images have equivalent resolution. In contrast, 5.00 fold oversampling would provide an estimate of 31 nm resolution according to Eq. 6 of Supplementary Note 1. We note that localization microscopy images with increased localization density and thus greater levels of oversampling compared to Nyquist more closely approximate the ground truth image. Actin in this image does not fill the entire field of view. This has explicitly accounted for when reporting Nyquist resolution as outlined in Supplementary Note 2. (MOV 11308 kb)

Raw image data as acquired by lattice light sheet-PAINT microscopy.

Raw data from the LLCPK1 cell shown in Supplementary Figure 15. Freely diffusing wheat germ agglutinin-Alexafluor 555 molecules can be seen as transient blurs outside of the cell while stable bound molecules appear as bright spots. The varying ellipticity of the stable spots as the sample is scanned through the focal plane is caused by the astigmatic lens used to encode the z-position of the molecules. (MOV 1241 kb)

Ultra-high-density 3D imaging of intracellular membranes.

Maximum intensity projections, volume renderings and orthoslices of the COS-7 cell shown in Figure 2 under both lattice light sheet-PAINT microscopy and diffraction-limited, deconvolved, dithered lattice light sheet microscopy. Bounding box = 75 × 50.5 × 5 μm. (MOV 45520 kb)

Complete characterization of the data set presented in Figure 2.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with an oversample factor = 5) as functions of position within the sample. (MOV 17600 kb)

3D multicolor super-resolution imaging of intracellular and plasma membranes.

Volume renderings and orthoslices of the COS-7 cell shown in Figure 3 a-c imaged by lattice light sheet-PAINT microscopy. Intracellular membranes are stained by the AzepRh dye and the plasma membrane is stained by wheat germ agglutinin–Alexafluor 555. Multiple internalized vesicles peripherally stained with wheat germ agglutinin are visible. Volume rendering encompasses of a 50 × 60 × 6.5 μm field of view. (MOV 42339 kb)

Complete characterization of the data set presented in Figure 3a–c.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with an oversample factor = 5) as functions of position within the sample. (MOV 23594 kb)

Combination lattice light sheet–PAINT with lattice light sheet–PALM.

Volume renderings of the COS-7 cell shown in Figure 3d-f as acquired with lattice light sheet-PAINT (Hoechst-JF646 and AzepRh) and lattice light sheet-PALM (Dendra-2 Lamin A) microscopy. The middle of the movie zooms in on nucleoids of mitochondrial DNA as stained by Hoechst-JF646. Bounding box = 60 × 60 × 7.5 μm. (MOV 33314 kb)

Complete characterization of the data set presented in Figure 3d–f.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with and oversample factor = 5) as functions of position within the sample. Note that the data for Dendra-2 Lamin A were contaminated with residual amounts of AzepRh dye from a previous experiment and thus show a mix of labeling for intracellular membranes and nuclear lamins. (MOV 24035 kb)

High-density 3D imaging of intracellular membranes and DNA in prophase and metaphase cells.

Orthoslices of the prophase and metaphase LLCPK1 cells from Figure 4 showing intracellular membranes labeled with AzepRh and nuclear and mitochondrial DNA labeled with Hoechst-JF646. It is particularly clear from this dataset that the mitochondrial DNA nucleoids reside within the mitochondrial membranes. Small protrusions at the periphery of metaphase chromosomes resemble loops of decondensed chromatin visible in previously published EM images (c.f. Main Text References 28 and 29). (MOV 25515 kb)

3D volume rendering of intracellular membranes and DNA in prophase and metaphase cells.

Volume rendering of the prophase and metaphase LLCPK1 cells from Figure 4 showing intracellular membranes labeled with AzepRh and nuclear and mitochondrial DNA labeled with Hoechst-JF646. Bounding box = 46 × 47 × 18 μm. (MOV 25063 kb)

Complete characterization of the data set presented in Figure 4.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with an oversample factor = 5) as functions of position within the sample. (MOV 46783 kb)

High-density 3D imaging of intracellular and plasma membranes in a telophase cell.

Orthoslices of the telophase LLCPK1 cell from Supplementary Figure 15 showing intracellular membranes labeled with BODIPY TR methyl ester and plasma membrane labeled with wheat germ agglutinin–Alexafluor 555. Similar to the interphase COS-7 cell from Supplementary Video 5, internalized plasma membrane lined vesicles are also visible in this dividing LLCPK1 cell. (MOV 10561 kb)

3D volume rendering of intracellular and plasma membranes in a telophase cell.

Volume rendering of the telophase LLCPK1 cell from Supplementary Figure 15 showing intracellular membranes labeled with BODIPY TR methyl ester and plasma membrane labeled with wheat germ agglutinin–Alexafluor 555. Also shown is a diffraction-limited, deconvolved dithered lattice light sheet image of mCherry-Histone H2B. Thin retraction fibers that anchor the cell to the substrate are clearly visible. Volume rendering encompasses a 50 × 26.8 × 19.7 μm field of view. (MOV 21825 kb)

Complete characterization of the data set presented in Supplementary Figure 15.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with an oversample factor = 5) as functions of position within the sample. (MOV 60005 kb)

Lattice light sheet–PAINT imaging using commercially available BODIPY-TR methyl ester.

Orthoslices of a COS-7 cell showing the staining pattern of BODIPY TR methyl ester dye. The hollow tubular structure of numerous mitochondria is clearly visible, as are dense networks of peripheral ER tubules. (MOV 4765 kb)

Mitochondrial DNA residing within mitochondrial membranes.

10 nm (xy, top) or 5 nm (xz, bottom) thick section through a mitochondria adjacent to the nuclear envelope. A ring shaped nucleoid of mitochondrial DNA (magenta) is embedded adjacent to mitochondrial membranes (green). Alternating images show membranes or DNA and reveal the close apposition and mutual exclusion of these two components. (MOV 247 kb)

High-density 3D localization microscopy of a neuromast organ on the zebrafish lateral line.

Volume rendering and orthoslices showing AzepRh staining of a zebrafish neuromast from Figure 5. Due to the large field of view and high labeling density, this dataset consisted of over 1 billion individual localized molecules. Volume rendering encompasses an 87.8 × 98.7 × 27.5 μm field of view. (MOV 25368 kb)

Complete characterization of the data set presented in Figure 5.

Orthoslices showing the mean number of photons/molecule, density of linked localization events, localization precision, and estimated lower bound on the 3D resolution (computed via equation 6 of Supplementary Note 1 with an oversample factor = 5) as functions of position within the sample. (MOV 44050 kb)

Correction for nonlinear swelling in the data presented in Figure 4.

Volume renderings and orthoslices showing the sample after translation only, and translation + SyN correction. The orthoslice location is indicated by the yellow rectangle in the volume rendering. At right, are the applied x (into/out of the screen), s (horizontal), and cz (vertical) displacement values of the SyN transformation at the same orthoslice. Note that the first time point in the movie was used as the stationary image for registration, thus the displacements are those required to warp the subsequent (more swollen) volumes onto the initial (less swollen) volume. (MOV 1295 kb)

Correction for nonlinear swelling in the data presented in Supplementary Figure 15.

Volume renderings and orthoslices showing the sample after translation only, and translation + SyN correction. The orthoslice location is indicated by the yellow rectangle in the volume rendering. At right, are the applied s (into/out of the screen), x (horizontal), and cz (vertical) displacement values of the SyN transformation at the same orthoslice. Note that the last time point in the movie was used as the stationary image for registration, thus the displacements are those required to warp the previous (less swollen) volumes onto the final (more swollen) volume. (MOV 988 kb)

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Legant, W., Shao, L., Grimm, J. et al. High-density three-dimensional localization microscopy across large volumes. Nat Methods 13, 359–365 (2016). https://doi.org/10.1038/nmeth.3797

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