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Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Nature Biotechnology volume 34, pages 987–992 (2016)Cite this article

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Abstract

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.

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Figure 1: Retention of FP and antibody fluorescence signals in proExM and proExM of FP fusions.
Figure 2: Validation of proExM in different mammalian tissue types.
Figure 3: proExM of mammalian brain circuitry.
Figure 4: Three workflows for expansion microscopy with protein retention.

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Acknowledgements

We acknowledge N. Pak for assistance with perfusions. For funding, E.S.B. acknowledges the MIT Media Lab, the MIT Brain and Cognitive Sciences Department, the New York Stem Cell Foundation-Robertson Investigator Award, National Institutes of Health (NIH) Transformative Award 1R01GM104948, NIH Director's Pioneer Award 1DP1NS087724, NIH 1R01EY023173 and NIH 1U01MH106011, the MIT McGovern Institute, and the Open Philanthropy Project. F.C. acknowledges the National Science Foundation fellowship and Poitras Fellowship. A.M. was supported by T32 GM007484, Integrative Neuronal Systems, P.W.T. acknowledges the Hertz Fellowship. B.P.E. was supported by the Howard Hughes Medical Institute. E.S.B. and S.R.T. acknowledge the MIT McGovern Institute MINT program.

Author information

Author notes

  1. Paul W Tillberg and Fei Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Paul W Tillberg

  2. Massachusetts Institute of Technology Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Paul W Tillberg, Fei Chen, Kiryl D Piatkevich, Yongxin Zhao, Chih-Chieh (Jay) Yu, Ho-Jun Suk & Edward S Boyden

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Fei Chen, Chih-Chieh (Jay) Yu, Linyi Gao, Guanyu Gong, Uthpala Seneviratne, Steven R Tannenbaum & Edward S Boyden

  4. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Brian P English

  5. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Anthony Martorell, Ellen M DeGennaro, Robert Desimone & Edward S Boyden

  6. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Ho-Jun Suk

  7. Osaka University Medical School, Suita, Osaka, Japan.,

    Fumiaki Yoshida

  8. McGovern Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Fumiaki Yoshida, Ellen M DeGennaro, Robert Desimone & Edward S Boyden

  9. University of Michigan Medical School, Ann Arbor, Michigan, USA

    Douglas H Roossien & Dawen Cai

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  1. Paul W Tillberg
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Contributions

P.W.T., F.C., K.D.P., Y.Z., C.-C.Y. and E.S.B. all contributed key ideas, analyzed data and wrote the paper. P.W.T. designed and performed antigen retrieval experiments. F.C. designed and performed AcX-secondary antibody retention experiments and Brainbow experiments. K.D.P. and F.C. designed and performed fluorophore preservation experiments. Y.Z. designed and performed LysC experiments. C.-C.Y. and P.W.T. designed and performed depth modulation of AcX experiments and preliminary characterizations of autoclave treatments. L.G. created code for data analysis. B.P.E. carried out PALM experiments. A.M. and H.-J.S. carried out mouse surgeries. F.Y. carried out primate surgeries under the supervision of R.D. with assistance from E.M.D. D.H.R. and D.C. assisted with Brainbow experiment design. G.G., U.S. and S.R.T. designed and performed the SNOTRAP experiment. F.C. and Y.Z. designed and performed experiments for validation of proExM on non-brain mouse tissue. E.S.B. supervised the project.

Corresponding author

Correspondence to Edward S Boyden.

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Competing interests

P.W.T., F.C., E.S.B. and C.-C.Y. have filed for patent protection (US provisional patent application no. 62/202,423) on a subset of the technologies here described. E.S.B. has helped co-found a company (Expansion Technologies, Inc.) to help disseminate kits to the community.

Integrated supplementary information

Supplementary Figure 1 Post-expansion antibody delivery, after epitope-preserving homogenization.

(a, b) Wide-field fluorescence images of Thy1-YFP-expressing mouse brain hemisphere slice before expansion (a), and after autoclave treatment and antibody staining (b). (c-h) Confocal micrographs of cortex from Thy1-YFP-expressing mouse brain treated with different disruption methods and antibodies, with anti-GFP (green, staining YFP) as a reference. (c) Autoclave method followed by staining against bassoon (blue) and homer (red). (d) Autoclaving followed by myelin basic protein staining. (e) Autoclaving followed by vimentin (red) and glial fibrillar acidic protein (blue) staining. (f) Staining for Lamin A/C after autoclave (i) or LysC (ii) treatment, or with secondary antibodies applied after LysC homogenization (with primaries previously anchored to the gel using AcX). (g-h) Comparison of staining before gelation (g) versus after disruption (h) using the autoclave method for Tom20 (i) and YFP (ii, shown in the red channel in the bottom panel because the endogenous YFP is green), and after disruption using LysC for homer (red) and PSD-95 (blue) (iii). Scale bars: (a) 1 mm, (b) 1 mm (3.96 mm), (c-h) 5 μm (~21 μm).

Supplementary Figure 2 Pre- and post-expansion images of a Thy1-YFP mouse brain slice treated with AcX and LysC mild digestion method.

(a) Pre-expansion wide-field image. (b) Post-expansion wide-field image. The arrow indicates the location of images (c-e). The bright edge surrounding the slice was the result of scattering at the gel-air interface. (c) Pre-expansion confocal image of a selected region of interest in hippocampus. (d) Post-expansion confocal image of the same selected region as (c). (e) Post-expansion DIC image of the same selected region as shown in (d). Scale bars: (a) 1 mm, (b) 4 mm (post-expansion units), (c) 5 μm, (d-e) 20 μm (post-expansion units).

Supplementary Figure 3 Incomplete homogenization with autoclave and LysC methods.

Fluorescence images of Thy1-YFP expressing mouse cerebral cortex, with YFP stained with anti-GFP using confocal imaging after autoclave treatment and antibody staining, showing a discontinuous neurite not residing at the surface of the imaged volume (a), and using widefield imaging after LysC treatment and antibody staining, showing defects in the expansion regions containing white matter tracts (b). Scale bars; (a) 5 μm (~20 μm), (b) 0.5 mm (~2 mm).

Supplementary Figure 4 Comparison of immunostaining methods with autoclave, LysC, and pre-gelation antibody treatment.

Confocal images of Thy1-YFP expressing mouse cerebral cortex, immunostained pre-gelation followed by AcX treatment, gelation, and proteinase K digestion (proExM), column (i). Thy1-YFP brain samples immunostained after AcX treatment and gelation followed by autoclave treatment, column (ii), or by LysC digestion column (iii). Autoclave and LysC specimens all have YFP stained with anti-GFP (green) in addition to TOM20 (row (a)), homer (red) and bassoon (blue) (row (b)), homer (red) and post-synaptic density 95 (PSD95, blue) (row (c)), glutamic acid decarboxylase (GAD) 65/67 (row (d)), myelin basic protein (MBP, row (e)), and vimentin (red) and glial fibrillary acidic protein (GFAP, blue) (row (f)). Scale bars; 5 μm (~20 μm).

Supplementary Figure 5 Control experiments of retention of EGFP and EYFP fluorescence in HEK293FT cells after proExM.

(a) Representative images of EGFP-H2B fusion in live HEK293FT cells and following proExM treatment without (top) or with (bottom) the AcX treatment. Scale bar: 20 μm. (b) Percentage of EGFP fluorescence retained following proExM treatment without (left) or with (right) AcX treatment relative to live cells (mean ± standard deviation, n = 4). (c) Representative images of EGFP-H2B fusion in live HEK293FT cells (top left) and following proExM treatment in shrunk (top left) and fully expanded gel (bottom). Scale bar 5 μm. (d) Percentage of EGFP fluorescence retained following proExM treatment in shrunk (left) and fully (right) expanded gel relative to live cells (mean ± standard deviation, n = 4 samples). (e) Normalized curves of photobleaching of EGFP under wide-field illumination (475/34 nm, ~60mW/mm2 light power) measured in live (dashed line, n = 8 cells) and proExM treated fully expanded HEK293FT cells (solid line, n = 7 cells). (f) Normalized curves of photobleaching of EYFP under wide-field illumination (512/10 nm, ~8.4mW/mm2 light power) measured in live (dashed line, n = 14 cells) and proExM treated fully expanded HEK293FT cells (solid line, n = 5 cells). (g) Retention of EGFP and EYFP fluorescence in proExM treated HEK293FT cells upon long term storage in 1x PBS at 4°C (n = 3 samples).

Supplementary Figure 6 ProExM imaging of S-nitrosylation.

(a) ProExM of tubulin fibers stained with Anti-Tubulin in primary neuron culture. (b) ProExM of fluorescently labeled streptavidin bound to biotinylated cysteine S-nitrosylated proteins chemically tagged via the SNOTRAP method. (c) Color composite of (a) and (b) (tubulin, red; SNOTRAP, green).

Supplementary Figure 7 Performance of selected photoswitchable and photoactivatable FPs in proExM.

(a) Representative images of selected photoswitchable/photoactivatable FP-histone fusions in live HEK293FT cells (live, upper image for each FP) and in the same cells after proExM treatment (proExM, lower image for each FP). (b) Fluorescence of selected FP-histone fusions in HEK293FT cells before (live, open bars) and after proExM treatment (proExM, crosshatched bars, mean ± standard deviation, n = 4 transfection replicates each). Fluorescence of selected FPs normalized to their molecular brightness relative to EGFP. (c) Averaged intensity image of 100 consecutive frames of unconverted H3.3-Dendra2 within a nucleus of a HEK293 cell after proExM, excited by a 488 nm laser. (d) PALM image derived from 10,000 consecutive frames of cell in c, which was photoconverted using low-power continuous 405 nm laser excitation. The 196,441 detected particles are displayed using Gaussian mask estimation according to their localization full-width at half-maximum. The mean and median localization errors for the H3.3-Dendra2 fusion were 23.3 nm. (e) Distribution of the total number of photons from mEos2-α-tubulin (mean 196.6, median 169.6). (f) The mean and median localization errors for the mEos2-α-tubulin fusion were 26.1 and 25.9 nm, respectively. (g) PALM image derived from 15,000 consecutive frames of proExM treated HeLa cell expressing mEos2-α-tubulin, which was photoconverted using low-power continuous 405 nm laser excitation. The 3.15 million detected particles are displayed using Gaussian mask estimation according to their localization full-width at half-maximum. Scale bars: (a) 10 μm, (c-d, g) 2.2 μm (physical size post-expansion, 10 μm).

Supplementary Figure 8 Pre- and post- expansion images of a Thy1-YFP mouse brain slice, and mouse brain with Brainbow 3.0 fluorescent proteins, and treated with proExM.

(a) Pre-expansion wide-field image of Thy1-YFP brain slice. (b) Post-expansion wide-field image of the slice from a. (c) Post-expansion maximum intensity projection image (~ 10 μm in Z) of membrane bound GFP in Brainbow 3.0 mouse brain tissue. (d) One Z slice of the image from c. (e) Post-expansion imaging of two color imaging of membrane bound GFP and membrane bound mCherry in in Brainbow 3.0 mouse tissue. Scale bars: (a), (b) 500 μm (20.5 μm). (c-e) 5 μm (~20 μm).

Supplementary Figure 9 Optimizing AcX penetration depth in fixed brain tissue

(a) Chamber assay for measuring penetration depth of a NHS-ester mixture (99% AcX + 1% NHS-biotin, which has similar molecular weight and charge as AcX) from the side of a tissue slice. After overnight treatment with the NHS-ester mixture, slices were retrieved, washed and treated with fluorophore-conjugated streptavidin to visualize penetration of NHS-ester mixture. (b) Representative image of a 100-μm-thick mouse brain slice stained under the chamber assay conditions. Scale bar 1mm. (c) Fluorescent intensity along the line-cut represented as the white dashed line in b. The distance over which the intensity drops from maximum to half of its value (D1/2) is a characteristic length for the depth of NHS-ester penetration. (d, e) Staining with MES-based saline (MBS; 100 mM MES + 150 mM NaCl) yields significantly improved depth of NHS-ester penetration than phosphate-based saline (PBS) over all pH levels tested. Scale bar 1 mm. (f, g) Staining at 4oC yields moderately greater depth of penetration than at RT. Scale bar 1 mm. (h) Representative images of native YFP fluorescence in a 500-μm-thick Thy1-YFP mouse brain slice, before (left) and after (right) proExM. Scale bar 1 mm (pre-expansion units). (i) Confocal imaging demonstrates YFP fluorescence retention at the center of the 500-μm-thick slice after an overnight AcX treatment with MBS, pH 6.0. Scale bar 100 μm (post-expansion units).

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Tillberg, P., Chen, F., Piatkevich, K. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34, 987–992 (2016). https://doi.org/10.1038/nbt.3625

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