© 2016 Nature America, Inc. All rights reserved. this this work. should Correspondence be addressed to E.S.B. ( Institute of Technology, Cambridge, USA. Massachusetts, Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, and Cognitive Sciences, Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, of Technology, Cambridge, USA. Massachusetts, of Technology Media Lab, Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, 1 posite. All of the chemicals required for ExM can be purchased except com -specimen the of expansion physical uniform mediate to homogenize protease its mechanical properties, followed by nonspecific dialysis in a water to with treated is sample the the Finally, incorporates it labels. that so sample, the in synthesized is gel polyelec trolyte swellable a Then, fluorophore. gel-anchorable a with est inter of biomolecules labeling involves protocol ExM original The to and water, index-matched is >99% water.material as the expanded transparent, are samples Expanded microscope. super-resolution a of sizes voxel voxel the with the but at microscope specimens diffraction-limited a of rates biological image to used be can ExM that We demonstrated had microscope. a by diffraction-limited conventional viewable range size the into structures bringing thus subdiffraction-limited imaging, before specimen biological resolution a of lateral expansion nm ~70 with physical via is circumvented limit diffraction optical Using the ExM, specimens preserved thick of imaging enables which ExM, technology, a developed recently We on conventional microscopes. resolution (~70 nm) imaging of cells and mammalian tissues demonstrated the utility of proExM for multicolor super- and streptavidin, fluorescent . We validated and the use of conventional labeled fluorescently antibodies and in which proteins are anchored to the swellable gel, allowing describe retention ExM (proExM), a variant of ExM custom-made reagents that are not widely available. Here we in the retention of native proteins in the gel and relied on them to a swellable gel. The first ExM method did not result by physically separating fluorescent probes after anchoring instead of specialized super-resolution microscopes. ExM works specimens with nanoscale precision on diffraction-limited Expansion (ExM) enables imaging of preserved Dawen Cai H Douglas Roossien Brian P English Paul W Tillberg and antibodies tissues labeled using standard fluorescent proteins expansion microscopy of Protein-retention cells and nature biotechnology nature Received 17 November 2015; accepted 30 May 2016; published online 4 July 2016; Department Department of Electrical Engineering and Computer Science, Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, 9 & Edward S Boyden 4 1 , , Linyi Gao , 2 , 10 9 , , Fei Chen , , Guanyu Gong advance online publication online advance 3 , , Anthony Martorell 2 , 3 2 , 4 , 10 3 Janelia Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. , 5 , Kiryl D , Piatkevich Kiryl , 8 3 , , Uthpala Seneviratne 9 [email protected] University University of Michigan Medical School, Ann Arbor, Michigan, USA. 5

, , Ho-Jun Suk 7 Osaka Osaka University Medical School, Suita, Osaka, Japan.

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, , Yongxin Zhao for the gel-anchorable label, which requires custom synthesis and and synthesis for raises the barrier to researchers adopt the Another draw method. custom requires which label, gel-anchorable the for residues (in contrast to nonspecific proteinase K) (in proteinase residues contrast to nonspecific lysine at proteins cuts which LysC, to tissues gel-embedded exposed and ( treatment expansion after delivered be before indeed could antibodies Wethat found YFP anti-GFP; with endogenous expansion after labeling imaged (we samples brain mouse Thy1-YFP of expansion × ~4 found and autoclave, an in h 1 for buffer detergent-rich alkaline an in tissues gel-embedded from Borrowing group. SDS-PAGE functional denaturing acrylamide on an amines with modifies proteins which Technologies), Life SE; AcX; (acryloyl-X, abbreviated acid 6-((acryloyl)amino)hexanoic of ester imidyl succin the used we gel, polymeric the into proteins Toincorporate epitopes. preserve to proteolysis reduced with approach modified a we used As experiment, state. a first expanded in the antibodies with stained and gel ExM the to anchored chemically be could native proteins whether asked We protocol. ExM original the in expansion adoption. its enable should that of standard methods used to histological prepare samples for imaging extension simple a is proExM protocol. ExM original the from tion diges proteolytic nonspecific the to subjected when even preserved are gel the to anchored directly are that streptavidin and antibodies proteins (FPs) as well as conventional fluorescently labeled secondary fluorescent encoded genetically from signals fluorescence that strate Wedemon molecule. cross-linking available commercially a using gel, swellable the to anchored are labels, than rather proteins, which labeling. antibody without imaged be cannot fluorophores encoded genetically that is protocol ExM the of back upeetr Fg 1c Fig. Supplementary d 3 oi:10.1038/nbt.362 Strong protease digestion (i.e., with proteinase K) enables isotropic in proExM, ExM, of variant a of development the report we Here , , Steven R Tannenbaum 2 , 6 , , Fumiaki Yoshida 6 Harvard-MIT Harvard-MIT Division of Health Sciences and Technology, 3 Department Department of Biological Engineering, Institute Massachusetts 2 , Chih-Chieh (Jay) , Chih-Chieh Yu 5 2 and antigen retrieval protocols retrieval antigen and − e ). As a second treatment strategy, we we strategy, treatment second a As ). 7 3 , 8 , Robert , Desimone Robert , Ellen , M Ellen DeGennaro 10 These These authors contributed equally to 8 McGovern McGovern Institute, Massachusetts Supplementary Fig. 1a Fig. Supplementary 5 2 Department Department of Brain Massachusetts Institute Massachusetts 2 , 3 ,

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e 20 © 2016 Nature America, Inc. All rights reserved. various FPs in the proExM workflow. We selected 20 widely used used widely 20 selected We workflow. proExM the in FPs various expansion retention (proExM). microscopy protein as imaging and expansion digestion, strong gelation, by followed specimen, fixed a of treatment AcX of 5 Fig. mean preservation; in GFP (65% the gel fluorescence expanded with high efficiency ExM workflow,standard preserved K proteinase digestion, including ite using AcX. We discovered that treatment with AcX followed by the ExM method, if they were retained in the their polymer-specimen original compos the in retain required digestion might proteolytic the FPs after fluorescence GFP-like and GFP that hypothesized We proteases to stability extraordinary exhibits GFP that known Itis ience of protein direct anchoring with strong K proteinase treatment. expansion. reliable for necessary was protocol ExM original ( ( ability PSD-95; and GFP (Tom20, staining pre-gelation to compared signal brighter in result ( respectively), s.d.; in Tom20 for staining pre-expansion SR-SIM vs. proExM for length measurement of a function as error measurement length R.m.s. in used specimens three the of example a representative is here imaged specimen The ( image confocal post-expansion ( arrows). (white overlaid field vector distortion with microscopy confocal conventional with (purple) proExM and (green) SR-SIM with Tom20of in ( samples). s.d.; area, shaded mean; line, (blue pancreas the in staining vimentin for pre-expansion SR-SIM vs. proExM for length measurement of a function as error in region ( image confocal post-expansion ( arrows). (white overlaid field vector distortion with microscopy confocal conventional with (purple) proExM and (green) SR-SIM with imaged pancreas mouse in vimentin of image ( lung). and spleen, s.d.; area, in types tissue different the for images pre-expansion widefield vs. proExM for length measurement of function in ( image post-expansion ( Methods). Online arrows; (white overlaid field vector distortion with expansion (purple) after and (green) before pancreas mouse in vimentin of image composite fluorescence ( lung and ( pancreas mouse immunostained Thy1-YFP of samples post-expansion and expansion pre- of images widefield magnification, ( types. tissue mammalian 2 Figure FPs with spectra ranging from the blue to the near-infrared biotechnology near-infrared nature the to blue the from ranging spectra with FPs f n j Supplementary Fig. 4 Fig. Supplementary , , , k g e We systematically examined persistence of fluorescence for for fluorescence of persistence examined systematically We We next sought to devise a strategy that would combine the conven o ) Pre-expansion SR-SIM image ( image SR-SIM ) Pre-expansion ) Pre-expansion widefield image ( image widefield ) Pre-expansion . . ( ) Pre-expansion SR-SIM image ( image SR-SIM ) Pre-expansion n h = 3 mouse brain cortex samples). Scale bars: ( bars: Scale samples). cortex = brain 3 mouse ). Because of the utility of this protocol, we termed the process process the termed we protocol, this of utility the of Because ). ) R.m.s. length measurement error as a as error measurement length ) R.m.s.

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© 2016 Nature America, Inc. All rights reserved. after room-temperature proteinase K proteinase Weafter room-temperature digestion. antibody-stained brain (for example, more connective tissue), which hinders expansion than properties mechanical different have lung and spleen Pancreas, (1,980 treatment with proteinase K as described for cultured cells ( cells after for cultured K described as scale proteinase with treatment millimeter the at distortion without expanded neurons the under YFP ing spleen, and lung pancreas, (brain, types tissue mouse different four on proExM performed we tissues, paper). this of scope the resolution validating (although claims for PALMpost-ExM is beyond PALMwith compatible fluorophores of photoswitching preservation (PALM) ( microscopy zation locali photoactivated with fusions and mEos2-tubulin H2B-Dendra and ( proExM during FPs photoswitchable four of actin-mRuby2, and mEmerald and (keratin-mRuby2 clathrin-mEmerald, expected encoded fluorophores in our screen, yielded excellent image quality genetically as the the of two mRuby2, in and mEmerald nulls containing proteins the of ( definition pits the of excellent middle obtaining cells HeLa in nm 70 than better of lution ( a microtubule full-width at half-maximum (FWHM) of 67 nm resolution imaging of cells have commonly used to as structures stereotyped demonstrate super- ( keratin and clathrin fusions tubulin, of examined first We cells. HeLa cultured fluorophores in encoded unstained) (i.e., genetically bearing proteins fusion several ( distance ment and 20 0 between after proExM over feature scales measurements length protocol ExM ( proExM after imaging ( (SR-SIM) microscopy illumination structured super-resolution with cells cultured in bules preserved, we validated proExM by imaging immunostained microtu isotropy would be that nanoscale ExM protocol, suggesting validated tion, expansion and imaging, as a potentially strategy.generalized other protease-resistant tags to the polymer, followed by gelation, diges 6 Fig. ( proExM with signals S-nitrosation of imaging the viously developed chemical method, SNOTRAP imaged probes to designed capture S-nitrosation cysteine using a pre 413 image of one showing optical section of the boxed region in from neurons the boxed in region of Brainbow3.0 labeled expansion membrane stack following microscopy in ( Brainbow3.0. delivered virally expressing mouse hippocampus immunostained showing imaging ( spines. dendritic showing of images a of subregion ( cortex. macaque rhesus ( post-expansion 3 Figure s r e t t e l  n

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g m), m), ( proExM of proExM brain circuitry.mammalian ( m, and found that r.m.s. errors were ~1−2% of measure the m, r.m.s. were that and ~1−2% found errors . Scale bars: ( c ) 300 f ) ) 5 1 . We quantified the root-mean-square (r.m.s.) oferror . We the root-mean-square quantified b Fig. 1 Fig. µ ) widefield ) image of in widefield injected GFP virally fluorescence µ m × 254 m, m, ( Thy1 Fig. 1 Fig. g a f ) 100 ) ) 5 ). e b c ) Post-expansion widefield image of widefield the ) sample Post-expansion ) ) Volume of microscopy rendering confocal . . Inset is of a the boxed magnification region, Fig. 1 Fig. d promoter ( promoter Fig. 1 Fig. µ ) Low-magnification widefield fluorescence fluorescence widefield ) Low-magnification µ 9– j m m (19.8 m × 25 ). Dual-color proExM imaging of fusion fusion of imaging proExM Dual-color ). 1 µ 1 2 m, ( Supplementary Fig. 7 Fig. Supplementary 1 . . The tubulin-mClover fusion presented ). We imaged cells expressing histone histone expressing cells imaged We ). d . We also imaged clathrin-mEmerald clathrin-mEmerald imaged also We . e e . . ( ) before proExM and with confocal confocal with and proExM before ) ), as we did to validate our original original our validate to did we as ), Fig. 1 Fig. b g ) 100 µ ) Pre-expansion confocal image confocal ) Pre-expansion Fig. 1g Fig. µ Fig. 2a Fig. m, ( m), m), ( Thy1-YFP l c µ ). We examined the stability stability the examined We ). ) 1 Fig. 1h Fig. m (physical size post-expansion, h ) ) 50 – – µ k d m, ( a ), which we and others others and we which ), ). Mouse brain express brain Mouse ). f , . ( b µ Supplementary Fig. 7 Fig. Supplementary ) in a sparse subset of of subset sparse a in ) ) Pre-expansion ( ) Pre-expansion m m (198 d , 8 h i , , thus demonstrating ) 500 ) Post-expansion ), suggesting a reso ), suggesting ), demonstrating demonstrating ), Fig. Fig. 1 Supplementary Supplementary µ µ m, ( m). k e ; ; paxillin- ) 500 Fig. 2 Fig. ± a 8 nm ) ) and µ µ a m m

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proteins and cells in intact tissues and organisms. We applied proExM FPs gene after viral delivery, are routinely for in used biology labeling n 0 between length, of 1−3% the measurement were type for errors tissue this at R.m.s. room temperature. digestion ( after microscopy ( before SR-SIM with imaged and marker, cortical tissue stained with antibodies against Tom20, a mitochondrial brain on mouse analysis Wea similar samples). two from performed for length 0 between small measurements measurement the of observed 1−5% we of order Again the on errors pancreas. r.m.s. the in staining vimentin on ( SR-SIM formed ( types tissue the of three all among similar and distance) measurement the (1 small were errors r.m.s. The tissue. spleen and lung creas, (<100 microscale the at proExM feature after of measurements error r.m.s. the measuring by expansion of isotropy the quantified We proExM. with improvement resolution the showed ( before tissue the of scopy ( scale length millimeter the at morphology tissue of preservation excellent with tissue, spleen and lung pancreas, of expansion for allowed proExM h), 4 for °C (60 enzyme K proteinase the of optimum the to tem perature digestion the in modification slight a with that, types. Weobserved tissue diverse these in expansion of isotropy the examine to tissue connective of marker a as vimentin filament intermediate the = 3 specimens). 3 = e d b a f Transgenic animals expressing FPs, as well as animals expressing expressing animals as well as FPs, expressing animals Transgenic per we nanoscale, the at expansion of isotropy the examine To Fig. 2 Fig. advance online publication online advance h Fig. 2b Fig. ) at this length scale. length at ) this Fig. 2i Fig. Fig. 2 Fig. – d ). High-resolution diffraction-limited micro diffraction-limited High-resolution ). c , j o ) and proExM confocal imaging ( imaging confocal proExM and ) µ ) proExM processing using proteinase K proteinase using processing proExM ) m and 25 m 25 and g Fig. 2e Fig. , f µ ) vs. after ( after vs. ) m m ( nature biotechnology nature Fig. 2 Fig. Fig. 2m Fig. µ m m and 40 l h ; ; Fig. 2e Fig. n = 4 fields of view of view = 4 fields , n ) and confocal confocal and ) µ µ m) for pan for m) , g m m ( ) proExM proExM ) Fig. 2i Fig. Fig. Fig. 2 − 3% of of 3% , k p - - - - ) ,

© 2016 Nature America, Inc. All rights reserved. access ( access antibody and staining of brightness increasing potentially delivery, ( FPs immunostained cells and tissues as well as samples expressing various of imaging nanoscale perform to used be can strategy proExM This before gelation. cross-linker molecule small available a commercially sample, a throughout by gel biological swellable synthesized applying after ( resolved expansion clearly be can expansion before resolve to close too dendrites and how axons showed expansion after and before images ( sample Brainbow the of imaging super-resolved multicolor volume, large for allowed ( tissues morphol brain and in preserved staining were ogy antibody proExM, Following via antibodies. amplification subsequent for allow to distinct antigenically are FPs These FPs. membrane-anchored of combinations random with (refs. Brainbow3.0 delivered ing virally ( visible necks spine thin even with expansion, after resolved easily was ogy lens on a conventional confocal microscope, dendritic spine morphol scale distortion after expansion ( ( macaque ( mice of brains the including tissue, brain mammalian intact in expressed FPs of visualization for (bottom). state expanded the in staining antibody finally and LysC), with digestion or denaturation, and hydrolysis alkaline example, (for procedure homogenization a gentle with processed then are gelation, by followed AcX, with treated Samples (middle). processing ExM subsequent and treatment AcX before permeabilized) optionally (and fixed chemically are FPs expressing Samples top). water; in expansion and K treatment proteinase (gelation, processing ExM subsequent (RT) and temperature room at treatment AcX before protocols, immunostaining 4 Figure nature biotechnology nature epitope and functionality be labeled after expansion if the nonspecific We showed that native proteins anchored in this way can retain retain can way this in anchored proteins native that showed We a to proteins anchor directly to possible is it that report Wehere express We circuitry brain of mouse for imaging proExM applied Fig. Fig.

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RT RT conventional super-resolution methods. As with any super-resolution or of other ized hardware, enabling aspects post-processing extensive of rates acquisition voxel conventional microscopy. at super- Like of ExM, proExM imaging does not sizes require with special voxel microscopy enable protocols resolution ExM as win, net a is there may require longer working distance objectives and tiled acquisition), thus (and expansion before than size in prepared larger are protocols ExM by samples Although imaging. of speed great exhibit tocols pro ExM modality, imaging subdiffraction-limited effective an As lens. objective the of distance working by only limited microscopes, fluorescence conventional on samples, into deep imaging resolution are optically clear and index-matched to water immunostaining. without all constructs, FP of transfection and vectors expression viral animals, transgenic of use the for allows fluorescence endogenous of Preservation workflows. circuit for useful mapping. be ProExM is simple and easily may integrated into existing sample which samples, Brainbow expanding by of WeproExM capability the multicolor, large-volume furthermore demonstrated and treatment. gel, proteolysis nonspecific the after signals in fluorescence exhibit retained also are methods standard ( after expansion antibodies access better but tissue dense in poorly antibodies post-gelation tissue native of modified with tations inherent in to antibodies the delivering crowded environment replaced is homogenization treatments. ExM Such approaches may overcome the limi of proteolysis As with our original ExM protocol, samples processed with proExM using delivered antibodies fluorescent and FPs that found We Supplementary Fig. 1 Fig. Supplementary Protein Antibody 1 h,RT 1 h,RT 15– H stain H 2 2 O O 1 9 . For example, closely packed epitopes may bind bind may epitopes packed closely example, For . AcX ). 1 . . This allows for super- Protein s r e t t e l  - - -

© 2016 Nature America, Inc. All rights reserved. mouse tissue. the E.S.B.project. supervised Y.Z. anddesigned performed experiments for validation of proExM on non-brain G.G., U.S. and S.R.T. anddesigned performed the SNOTRAP experiment. F.C. and assistance from E.M.D. D.H.R. and D.C. assisted with Brainbow experiment design. surgeries. F.Y. outcarried primate surgeries under ofthe supervision R.D. with analysis. B.P.E. outcarried PALM experiments. A.M. and H.-J.S. outcarried mouse preliminary characterizations of autoclave treatments. L.G. created code for data and P.W.T. anddesigned performed depth modulation of AcX experiments and preservation experiments. Y.Z. anddesigned performed LysC experiments. C.-C.Y. and Brainbow experiments. K.D.P. and F.C. anddesigned performed fluorophore F.C. anddesigned antibody performed AcX-secondary retention experiments and wrote the paper. P.W.T. anddesigned performed antigen experiments. retrieval P.W.T., F.C., K.D.P., Y.Z., C.-C.Y. and E.S.B. all contributed key ideas, analyzed data the MIT McGovern Institute MINT program. by the Howard Hughes Medical Institute. E.S.B. and S.R.T. acknowledge Systems, P.W.T. acknowledges the Hertz Fellowship. B.P.E. was supported Fellowship. A.M. was supported by T32 GM007484, Integrative Neuronal Project. F.C. acknowledges the National FoundationScience fellowship and Poitras the 1U01MH106011, MIT McGovern Institute, and the PhilanthropyOpen NIH Director’s Pioneer Award 1DP1NS087724, NIH 1R01EY023173 and NIH National Institutes of Health (NIH) Transformative Award 1R01GM104948, Department, the New York Stem Foundation-Robertson Cell Investigator Award, acknowledges the MIT Media Lab, the MIT Brain and Cognitive Sciences We acknowledge N. Pak for assistance with Forperfusions. funding, E.S.B. online version of the pape Note: Any Supplementary Information and Source Data files are available in the the of in version available are references associated any and Methods M biological precision. nanoscale with complex samples of imaging routine supporting in applications (for imaging many find likely will proExM Thus PALM, super-resolution here). example, shown as traditional with even or tomography, compatible as deconvolution such are strategies analysis samples and imaging Expanded downstream well as dimensions. axial the lateral along as well equally samples magnifies physically proExM specimen. unexpanded the of brightness the on depending loss owing reduced to scattering, though and longer exposure times are typically useful, autofluorescence of dilution concomitant the by and are thus dimmer than unexpanded specimens. This effect is offset fluorophores fewer contain they are smaller, voxels because method, s r e t t e l  AUTH A

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9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/ R version of t The authors declare competing interests:financial details are available in the 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. C eprints and permissions information is available online at at online available is information permissions and eprints O

MPETI Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals Seneviratne, U. protein. gree-fluorescent Aequorea of Renaturation W.W. Ward, & S.H. Bokman, trypsin efficiency, Maturation R.M. Wachter, & M.E. Phail, the J.A., for method Sniegowski, A III. J.R. Yates, & K.E. Howell, M.J., MacCoss, C.C., Wu, from Lys-C endoproteinase of Use J.J. Beintema, & W.J. Weijer, P.A., Jekel, loss and retrieval antigen temperature High B. Jasani, & R. Attanoos, N.C., Hunt, of head the of assembly the during proteins structural of Cleavage U.K. Laemmli, microscopy. Expansion imaging. Optical E.S. Boyden, P.W. & Tillberg, F., Chen, aioo K, aeoh, . Myjm, . Oaua RY Hptei fr the for Hypothesis R.Y. Osamura, & K. Miyajima, S., Takekoshi, K., Kakimoto, immunohistochemical the for technique dual-label A G. Pearse, & K.J. Randall, for immuno-labeling 3D intracellular for approach novel A J.A. Post, & N. Jiménez, Coxiella of proteins surface to binding antibody of hindrance T. Steric Hackstadt, artifacts Immunolabeling B.N.G. Giepmans, & F.,K.A. Dijk, Sjollema, U., Schnell, J. Livet, the for tools Improved J.R. Sanes, J.W.T.,& Lichtman, Luo, K.B., Cohen, D., Cai, M. Bossi, Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X. Multicolor super-resolution imaging Rego, E.H. neatos ewe clua srcue wt nnmtrsae resolution. nanometer-scale with structures cellular between interactions stagesneurodegeneration.of analysis. sequence protein in enzymogenes Lysobacter techniques. autoclave and microwave of Pathol. Clin. J. comparison a morphology: nuclear of T4. bacteriophage Science (2008). immunohistochemistry. sections in frozen formalin-fixation fresh without on occurring retrieval antigen heat-induced for mechanism rat paraffin-embedded tissue. lymphoid formalin-fixed, in subsets T-lymphocyte of demonstration tomography. electron lipopolysaccharide. I phase by burnetti imaging. live-cell for need the and system. nervous the in proteins toolbox. Brainbow species. molecular distinct of detection photo-switchablewith fluorescent probes. 109 resolution. 50-nm at structures cellular reveals protein Methods Nat. Biochem. Biophys. Res. Commun. Res. Biophys. Biochem. of variants Gly67 and (2005). Glu222, protein. Arg96, of fluorescent properties green optical and sensitivity, proteins. (2003). 532–538 membrane of analysis proteomic comprehensive (1983). 347–354 , E135–E143 (2012). E135–E143 , index.htm advance online publication online advance he pape

N 347 t al. et G G FI et al. t al. et , 543–548 (2015). 543–548 ,

5 N et al. l

rngnc taeis o cmiaoil xrsin f fluorescent of expression combinatorial for strategies Transgenic . Nonlinear structured-illumination microscopy with a photoswitchable r , 1047–1052 (2008). 1047–1052 , 49 . A Toxicol.Pathol. Multicolor far-field fluorescence nanoscopy through isolated through nanoscopy fluorescence far-field Multicolor N Nat. Methods Nat. , 767–770 (1996). 767–770 , Nature S-nitrosation of proteins relevant to Alzheimer’s disease during early CIAL CIAL I Traffic

227 Proc.Natl. Acad. Sci.USA

N 13 ice. ipy. e. Commun. Res. Biophys. Biochem. TERESTS , 680–685 (1970). 680–685 , , 926–933 (2012). 926–933 ,

Nature

10 36

101 Nat. Methods Nat. , 540–547 (2013). 540–547 , , 795–804 (2008). 795–804 ,

, 1372–1380 (1981). 1372–1380 , 450 Nano Lett. Nano Science Infect. Immun. Infect. , 56–62 (2007). 56–62 , nature biotechnology nature

317 9

, 152–158 (2012). 152–158 , 8 . o. Histol. Mol. J.

, 2463–2468 (2008). 2463–2468 , , 1749–1753(2007)., 113 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc.

56 , 4152–4157, (2016). http://www. nl Biochem. Anal. , 802–807 (1988). 802–807 , a. Biotechnol. Nat.

332

39 nature.com/ , 657–663 657–663 , 389–399 , online

134

21 , ,

© 2016 Nature America, Inc. All rights reserved. and secondary antibodies (Rabbit Anti-Tom20, Santa Cruz Biotech sc-11415 sc-11415 Biotech Cruz Anti-Tom20, Santa (Rabbit antibodies secondary and 50- min. 5 for Technologies, 10 then were Specimens each. incubated with antibodies (donkey anti-sheep secondary Alexa Fluor min 488, Life 5 for times three PBS in washed then and 10 of concentration a at (PBT) serum donkey normal 2% Anti-Tubulin, (Sheep 1 × Tritonbuffer 0.1% ATN02) PBS with and in blocking X-100 Cytoskeleton antibodies primary with stained were HeLa fixed in Microtubules min. 15 for Triton-X 0.1% with permeabilized and PBS, with were fixed with 4% paraformaldehydeimaging. for 10 min, washed three pre-expansion times for 5 min microscopy illumination Structured dilution. fluorophore for account to area segmented by normalized were processing sample after and before urements to the determine loss of used during fluorescence proExM processing. Intensity was meas cortex the in interest of region A conditions. imaging identical with again taken were slice PBS the of images with epifluorescence extensively and min), 30 washed × (3 were slices brain the digestion, and gelation proExM Following pre-gelation. NA 4 × objective 0.13 with taken were slices in described bodies Systems; (Synaptic (50 slices brain type wild of sections Cortical and Thy1-YFP cross-talk) Fluor for 488 owing to used Thy1-YFP Alexa (only below. described as cortex mouse imaging ( Retention antibodies). ary (see vendors mercial (goat anti-rabbit, 10 antibodies secondary ProExM. during retention dye fluorescent of Quantification brightness). on effect expansion axial the mitigates sectioning to optical area epifluorescence since used was (area dilution for fluorophore account segmented by normalized were processing sample after and before nucleus for each measurements Intensity processing. sample after and before cells of set same the in intensity fluorescence of measurement automated for CellProfiler via thresholding Otsu Automatic Matlab. in algorithm, SIFT/RANSAC of the an implementation out with ried only with PBS. Registration of pre- and post-sample processing images was car shown in For control experiments chloride-sensitive is which MgCl mm 60 and NaCl M 1 in shrunk then and PBS, with below. described as overnight K proteinase with digested and gelled with h, 6 least treated at for and Technologies) (Life AcX Cellgro) mg/ml 0.1 (PBS; saline phosphate-buffered with min 5 for 10 min, with and 0.1% Triton-Xpermeabilized for 15 min, washed three times Immediately software. for AR 4% paraformaldehyde with were fixed cultures cell imaging, live-cell after NIS-Elements by controlled Zyla 5.5 (Andor), a and camera (Semrock), exciters nm 631/28 and nm, 585/29 nm, 510/25 lens, objective 0.3 NA a × SPECTRA 10 X light with engine (Lumencor) equipped with 390/22 nm, microscope 438/24 nm, inverted 475/28 nm, Ti Eclipse Nikon a using transfection after h 24 performed was cells HEK293FT live of gent Bio) (Mirus according to the manufacturer’s protocol. Widefield imaging by the manufacturer. Cells were transfected using TransIT-X2 transfection rea contamination mycoplasma for checked and profiling STR by authenticated were lines cell transfection; of ease for used were cells HeLa and HEK293FT 1% penicillin/streptomycin (Cellgro), and 1% cells sodium pyruvate (BioWhittaker). CCL-2) (ATCC (Invitrogen), FBS 10% with supplemented HeLa (Cellgro) in DMEM cultured were and (Invitrogen) HEK293FT 31871). plasmid of iRFP was swapped with LSSmKate2 gene in pNLS-LSSmKate2-N1 (Addgene NLS-iRFP fusion protein, a PCR-amplified AgeI/NotI fragment encoding gene with the LSSmOrange gene in pH2B-LSSmOrange-N1 swapped (Addgene). Toand generate fragments AgeI/NotI as PCR-amplified were genes respective the plasmids pPATagRFP-H2B-N1 and pmKate2-H2B-N1 ones, remaining ( Addgene from obtained screening. protein Fluorescent O doi: NLINE METH NLINE Following digestion, the samples were processed by extensively washing washing extensively by processed were samples the digestion, Following 10.1038/nbt.3625 µ m brain tissue slices were prepared and stained with primary primary with stained and prepared were slices tissue brain m µ µ m thick) were stained with anti-Homer primary antibody antibody primary anti-Homer with stained were thick) m g/mL) g/mL) in PBT for 1−4 h and then washed in PBS three times ODS Supplementary Table 4 Table Supplementary Supplementary Table 2 Supplementary Supplementary Table 2 Table Supplementary Supplementary Tables 1 Tables Supplementary 2 0 Fig. 1 Fig. , and thus was measured in the expanded state). state). expanded the in measured was thus and , Most of the mammalian plasmids were were plasmids mammalian the of Most c Supplementary Figure 5 Figure Supplementary ) was quantified via before-after proExM proExM before-after via quantified was ) µ g/mL) were g/mL) from purchased com . Epifluorescence images of images brain . Epifluorescence ), and different secondary anti secondary different and ), 2 for list of fluorescent second fluorescent of list for 1 of fluorescent nuclei allowed allowed nuclei fluorescent of and and 3 ). To construct the the Toconstruct ). 2 gels were washed µ (except for YFP, for (except g/mL for 1−4 h 1−4 for g/mL Fluorescent Fluorescent HeLa cells cells HeLa ------

AcX treatment. AcX and stored at 4 on staining. VT1000S) °C until a sliced vibratome (Leica at 10 all (Biotium), CF633 pig anti-guinea donkey and Technologies) (Life 546 Fluor Alexa anti-rat goat 488, Fluor Alexa anti-chicken goat were: used antibodies with min 30 PBT. for times antibodies Slices were 1 with incubated secondary d at four RT. washed Secondary then PBT,and in °C 4 at d 3 for antibodies (all primary with staining incubated were Slices antibody (RT)). temperature before room at min incubations 30 for (PBT) serum donkey normal and 2% X-100 Triton 0.1% with PBS × 1 with blocked and permeabilized were Slices USA. Michigan, Arbor, Ann School, Medical Michigan of University anti-mKate2, rat guinea-pig anti-mTFP) were by produced the Cai laboratory, below). (see perfused were then and injection weeks the 3−4 for virus at expressed Animals rest diffusion. viral to allow to 5 min for site allowed cortex, was example, needle (for the brain which after the into hippocampus), needle injection 34-gauge a through a function of measurement length. measurement of function a as measurements such for error r.m.s. the found and measurements to-point between those two points. We sampled the entire population of possible point- distance the measure to image post-expansion the using in error localization relative the found we points, two any at vectors resulting the subtracting By each point in the post-expansion image relative to an ideal uniform expansion. image to post-expansion the scaled image pre-expansion the expresses shift of to The mapping and rotation, field vector deformation translation scaling. the limited transformation geometric a estimate to used was (RANSAC) sensus library source open VLFeat the using generated keypoints (SIFT) transform feature scale-invariant using generated automatically were registration for points Control process. Matlab in package tion the registra to non-rigid but a with images, time this again B-spline-based pre-expansion registered then were images scaled These package. Viewer 3D Fiji the using scaling without a rotation 3D using corrected was this imaging, post-expansion and pre- between changed tilt specimen the case In scaling. to registered images the pre-expansion by corresponding rotation, first translation and uniform were images Post-expansion expansion. after and before quantification. error Measurement imaging. pre-expansion for matching index refractive and photobleaching of suppression for reagent antifade (Invitrogen) Gold SlowFade with imaged SIM microscope with 100 × 1.40 NA (Olympus) oil objective. Stained cells were on SR-SIM OMX a was performed (GE imaging Blaze healthcare) Deltavision below. as described Technologies)) (Life 568 Fluor Alexa and goat anti-rabbit rocker to ensure mixing during the reaction. the during mixing ensure to rocker NaCl; (100 mM MES, 150 saline mM acid (MES)-based morpholino)ethanesulfonic 2-( a in and temperature, lower at pH, lower at sample the incubating tissue by thick For RT. at h, >6 for mg/mL (>100 0.1 of concentration a at PBS in diluted AcX in incubated are slices tissue and cells anchoring, For months. 2 to up for stored be can way this prepared AcX environment. desiccated a in frozen stored and aliquoted mg/mL, 10 of concentration a at DMSO drous imidyl ester; here abbreviated AcX; Thermo-Fisher) was resuspended in anhy (50 Slices glycine. mM 100 to moved being before d, 1 for °C 4 at paraformaldehyde 4% in left dissected, were Brains formaldehyde. anesthetized with isoflurane and perfused transcardially with ice-cold 4% para perfusion. Mouse 10 × 2 (7.5 AAVmix pad. heating a on lying animal the with conditions sterile under place took Surgery apparatus. a to stereotaxic head-fixed and isoflurane with ously described previously as performed were injections Vector Penn Core) Pennsylvania, of Brainbow3.0 injection and antibody staining. Primary antibodies against Brainbow 3.0 fluorophores (chicken anti-GFP, (chicken fluorophores 3.0 Brainbow against antibodies Primary µ Supplementary Fig. 9 Fig. Supplementary m), AcX penetration depth and labeling uniformity can be improved improved be can uniformity labeling and depth penetration AcX m), µ g/ml. 1 3 . Briefly, transgenic mice (Emx1-Cre) were anesthetized continu anesthetized were (Emx1-Cre) mice transgenic . Briefly, Acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid, succin acid, (6-((acryloyl)amino)hexanoic SE Acryloyl-X, All solutions below were made up in 1 × PBS. Mice were were Mice PBS. × 1 in up made were below solutions All 12 genome copy/mL) was injected at a rate of 0.2 0.2 of rate a at injected was copy/mL) genome 2 2 to capture any non-uniformities in the expansion expansion the in non-uniformities any capture to ). Tissue slices can be incubated on a shaker or or shaker a on incubated be can slices Tissue ). The same fields of view were imaged imaged were view of fields same The Brainbow3.0 rAAV (University nature biotechnology nature 2 4 , and random sample con sample random and , 2 3 . SIFT keypoints were were keypoints SIFT . µ m and 100 100 and m µ m) were m) µ l/min l/min µ N L ------

© 2016 Nature America, Inc. All rights reserved. normal fresh-frozen tissue sections, postfixed with cold acetone, of pancreas, of pancreas, acetone, cold with postfixed sections, tissue fresh-frozen normal types. tissue of different ProExM ref. in (6) equation using determined were errors localization using analysis routines written in Igor Pro version 6.36. The mean and median counts photon to converted were intensities fluorescence integrated particle, using corrected drift were ticles par resulting The and a pixels. of PSF 1.3 GLRT 20 sensitivity with detection localization. Particle DU-897_BV,Ultra at frames/s. 20 full-chip) amplifier,EM MHz 17 500, Gain (v. 1.4.20) sample power of levels 2 kW/cm atestimated (CrystaLaser) laser DPSS 555-nm a with excited were molecules USB-6363 acquisition board (National Instruments), Photoconverted Dendra2 excitation 405-nm of the StradusStroboscopic 405-100 acquisition. laser (Vortran)image of was course achieved the using during a NI-DAQ- ms 200 every W/cm (50 nm 405 of the emitted light. Dendra2 was photoconverted by 100 filtered Semrock) and Chroma), (FF01-609/54-25, a filter emission band-pass (ZT405/488/561/640rpc, Chroma), a 2-mm-thick emission dichroic (T560lpxr, in resulting Griot), magnification Melles overall × 100 (LAO-300.0, lens tube custom a and objective, RAMM frame microscope (ASI) using an Olympus 1.4 NA PLAPON 60 × OSC PALMimaging. imaging. before gels the encase to solidify, to allowed and gel the around removed. pipetted was w/w) (2% agarose low-melt liquid liquid for immobilization, If needed excess all with plates six-well glass-bottom in placed were for used was NAlens air 0.3 × 10 40 with 710 LSM Zeiss a of exception the with (Nikon) 40 a with system Yokogawa)confocal (CSU-X1 disk spinning Andor an using imaged were presented tissues other epifluorescence on Ti-E Nikon Ti-E epifluorescence microscope with a 10 Nikon a Figs. 1a on pre-ExM 4 a with microscope imaged were comparisons, specimens after vs. before low-magnification and slices tissue for factor 60 a with system cal of cells was on performed an Andor spinning disk (CSU-X1 Yokogawa) confo after expansion. microscopy Fluorescence plateaued. sample expanding the of size the water, until fresh in times 3−5 repeated was step This gels. thicker for times longer with expand, h to for 0.25−2 water deionized of doubly volumes in excess placed next were overnight at RT (this step can also be performed at 37 °C for 4 h). Digested gels solution X-100, proteinase in Triton immersed fully gels the 0.5% with incubated EDTA, and 1 M NaCl) mM 1 8), (pH Tris mM (50 buffer digestion in °C gelation. for h 2 for 4 incubator °C 37 humidified a to then at transferred and respectively, slices, slices) brain and cells, for cultured for min 30 min, 4-hydroxy-TEMPO 1 for (and APS/TEMED plus solution monomer the with incubated were slices tissue or Cells sections. tissue into 0.5% (w/w) stock to inhibit gelation during of diffusion the monomer solution was up added to (4-hydroxy-TEMPO) from (w/w) 0.01% a ylpiperidin-1-oxyl 4-hydroxy-2,2,6,6-tetrameth inhibitor the For slices, each. (w/w) up to 0.2% solution monomer the to added were accelerator (TEMED) ethylenediamine tetramethyl and initiator (APS) persulfate ammonium of w/w) (10% stocks (w/w) Concentrated use. before 0.15% °C 4 to cooled was solution Monomer acrylamide, use. before (w/w) 2.5% acrylate, N,N sodium (w/w) 8.625% previously described as performed were expansion) and digestion (gelation, steps following the ing, Gelation, digestion and expansion. nature biotechnology nature (5−10 lung and spleen To stabilize the gels against drift during imaging following expansion, gels gels expansion, following imaging during drift against gels Tothe stabilize Bio England (New K Proteinase ′ -methylenebisacrylamide) was mixed, frozen in aliquots, and thawed thawed and aliquots, in frozen mixed, was -methylenebisacrylamide) , b , 2 6 3b with a back-illuminated EMCCD camera (Andor Technology, Ixon , 5a−g PALM data were recorded on a custom-built three-camera three-camera custom-built a on recorded were data PALM and × × 2 Localizer ) every 200 ms, which was ramped up to 1.2 ms pulses pulses ms 1.2 to up ramped was which ms, 200 every ) 0.13 NA air objective ( NAobjective air 0.13 1.40 NA oil objective ( objective oil NA 1.40 8a µ 1 . Briefly, monomer solution (1 × PBS, 2 M NaCl, NaCl, M 2 PBS, × (1 solution monomer Briefly, . × m) were obtained from US Biomax (MOFTS036, (MOFTS036, Biomax US from obtained were m) , b 1.1 NA water objective. The Zeiss LSM 710 with with 710 LSM Zeiss The objective. water NA 1.1 ). For data in 2 5 . A 2-mm-thick quad-band excitation dichroic dichroic excitation quad-band 2-mm-thick A . Supplementary Figures 1 Figures Supplementary 2 2 . Fluorescence was detected using . was detected Fluorescence 7 ad hoc ad Supplementary Figure 9i Figure Supplementary was used for eight-way adjacency particle particle adjacency eight-way for used was Standard histology preparations of mouse of mouse preparations histology Standard For AcX-anchored FPs and antibody stain l abs) was diluted 1:100 to 8 units/mL units/mL 8 to 1:100 diluted was abs) × fiducial markers. For each detected detected each For markers. fiducial 1.15 NA water immersion objective objective immersion water NA 1.15 Figure 3a Post-expansion confocal imaging Post-expansion confocal Fig. Fig. Fig. Fig. 2 , b 1 a , tissue slices were imaged × ). To quantify expansion expansion To quantify ). − µ 0.45 NA. all Otherwise, d s long excitation pulses , and and , ,

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Specimen disruption using autoclave. using disruption Specimen experiments for used in shown was Technologies) (Life 488 Fluor Alexa anti-rabbit in shown experiments for used was Technologies) (Life 546 Fluor Alexa anti-chicken goat that except (Biotium), CF633 anti-mouse goat and Technologies) (Life goat 546 Fluor Technologies), Alexa anti-rabbit (Life 488 Fluor Alexa anti-chicken goat antibod were: used Secondary PBT.ies with min 30 least at times four washed then and 20 at antibodies secondary with incubated were PBT. Specimens with min 30 times four washed then and (RT), h 4 PBT, for ( antibodies primary with incubated were Specimens permeabilization. specimens, of pre-gelation case the in and at RT0.1% (PBT) for 2 h for blocking, serum donkey with Tritonnormal 2% and X-100 PBS × 1 in incubated were treatment, LysC or autoclave after or Antibody staining of endogenous proteins. h. 4 for °C at 60 performed was digestion that exception the with above described expansion and digestion gelation, before RT at overnight PBS in AcX mg/mL 0.05 with incubated were Tissues above. described as performed was imaging Pre-expansion Technologies). Life 488, Fluor Alexa RT at (anti-chicken h 2 for antibodies secondary with incubated (Abcam) for 4 h at RT and then washed fouranti-vimentin times chicken 30 min with primary PBT. with Slices stained werewere Tissues staining. antibody with 0.1% Triton X-100 and 2% normal donkey serum (PBT) for 30 min before MOFTS051, and MOFTS031, respectively). Tissues were blocked with 1 × PBS culture were washed 3 × 5 min using PBS and fixed using cold methanol at methanol cold using fixed and PBS using min 5 × 3 washed were culture staining. SNOTRAP of ProExM C for calculated 23.1, 22.5; 27.1, 38.2, 42.5, 6H); 45.9, 62.3, (m, 63.1, 69.2, 118.3, 1.57-3.51 129.6, 129.6, 2H), 129.9, 134.8, (t, 2.15 2H), (t, 2.55 2H), 13 (m, 2.8 12H), (m, 3H), 3.67–3.51 (m, 2H), (m, 3.2 4.60–4.51 1H), (m, 7.84 4H), (m, 7.23–7.18 min. 8 further over B then and 95% min, to 20 at 90% min, 10 at 70% min, 5 for 40% was at composition initially Solvent mL/min. 2.5 of rate flow a at (A) (B) water in acetonitrile acid and formic 0.1% of gradient linear a with eluted was column cm × 10 (25 Luna mm, C18 9.4 Phenomenex a semi-preparative follows: as were systems solvent and columns HPLC Technologies). (Agilent nm 254 at detector UV array photodiode a with system HPLC 1100 an on repurified was probe SNOTRAP The 30%). (yield product desired the give to gradient) reduced pressure and purified by flash chromatography (hexane/EtOAc/MeOH under for 7 concentrated h at was then RT,stirred solution clear resulting and the mixture added resulting The successively. was mmol) 0.03 mg, (4 mL) (DMAP) ridine (5 DMF dry (70 mg, (DCC) 0.34 mmol) and dimethylaminopy dicyclohexylcarbodiimide in mmol) 0.34 biotin-PEG mg, (100 benzenethiol SNOTRAP-biotin. of Synthesis above. described those to steps identical with immunostaining to subjected were and 3 × each for min 30 buffer LysC in digestion washed were 33 with incubated for 5 min in LysC digestion buffer (25 mM Tris-HCl, 1 mM EDTA, pH 8.5) and II type Technologies) (Life lagenase in 37 °C for 2−4 h. Gels were washed then col U/ml 600 with Scientific) ThermoFisher magnesium, and calcium (with Mild digestion with LysC. buffer. disruption remove to Triton X-100) 0.1% serum, donkey normal 2% PBS, × (1 PBT in washed and imaging and staining antibody for plates well to transferred then were Gels tubes. polypropylene as such vessel autoclave-safe an in out carried be must liquid on treatment This h. 1 for autoclave held °C 121 by of temperature a with treated mode and sterilization buffer disruption fresh in placed then for washed SDS), 1% were X-100, Triton 5% base, Tris Gels mM (100 NaCl. buffer M disruption in min 1 15 in washed and chambers gelation from C NMR (125 MHz, CD MHz, (125 NMR C 31 P NMR (202 MHz, CD MHz, (202 NMR P Figure 1 Figure 3 poinc cd 10 g 02 mo, hme, n,, N,N Inc,), ChemPep, mmol, 0.22 mg, (100 acid -propionic 37 H µ e . 47 g/ml LysC (Promega) in LysC 37 °C g/ml for (Promega) at 8 h. least Finally, gels N 1 3 H NMR (500 MHz, CD MHz, (500 NMR H O 6 3 After gelation, gels were pre-treated in HBSS buffer PS CN, CN, 2 , 724.2638; found, 724.2632. found, 724.2638; , 3 CN, CN, δ ) 199.19, 172.5, 164.5, 144.8, 138.1, 137.0, 137.0, 138.1, 144.8, 164.5, 172.5, 199.19, ) For SNOTRAP staining, primary neuron neuron primary staining, SNOTRAP For o sird 2-(diphenylphosphino)- stirred a To δ Supplementary Figure 1e Figure Supplementary Supplementary Table 4 Table Supplementary ) −10.3; HRMS-ESI −10.3; ) After gelation, gels were recovered recovered were gels gelation, After Specimens, either before gelation 3 µ CN, CN, g/mL in PBT, for 4 h (RT), (RT), PBT, h in 4 for g/mL δ ) 7.42–7.38 (m, 9H), 9H), (m, 7.42–7.38 ) doi: + ( 10.1038/nbt.3625 m/z ) at 3 3 at ) , ): [M + H + [M ): g , h µ and goat goat and g/mL in in g/mL µ + m) m) ] ′ + - - - -

© 2016 Nature America, Inc. All rights reserved. for 10 min before withdrawal. Blunt 33G needles were used for all injections. injections. for all used were needles 33G Blunt withdrawal. before min for 10 place in left were syringe and needle the injection, each After Instruments). Kopf Instruments) and UMP3 micro-syringe injector pumps (World Precision (David arms micromanipulator stereotactic using nL/min 100−200 of rate a 500 then (deep locations two at brain the 3 into of infused was total A (Sigma). oil silicone with back-filled been had that (AAV8) were and centrifuged into loaded 10 anesthesia using coordinates stereotactic to target eight injection sites. Viruses procedures. Macaque gender. for regard without used were Mice old. months ~1–3 ages mice, Thy1-YFP 10 and mice, Emx1-Cre 4 ( macaque mulatta rhesus male adult One Animals. Laboratory of Use and and Care Animal Care the on for Guide Health of Institutes National the with Committee accordance in were Technology of Institute Massachusetts care. Animal above. described as performed was proExM and antibody) secondary 546 Fluor (Alexa anti-tubulin for staining Antibody min. 5 × 5 washed afterward and h 1 for RT at (PBS-Triton) dilu tion 1/500 in Scientific) (Thermo 488 Fluor streptavidin-Alexa with bated atv/v) RT(50%:50% for 1 incu h, in and acetonitrile-PBS-triton further then (250 probe then SNOTRAP with were incubated and PBS Cells using min 5 × 3 proteins. washed on group –SH free the block at to min v/v) 30 for (0.3% °C X-100 37 Triton containing PBS in Scientific) (Thermo (NEM) N-ethylmaleimide nM 300 with incubated were Neurons min. 15 for °C −20 doi: 10.1038/nbt.3625 ) weighing 12 kg was used for this study, as well as 1 C57BL/6 mouse, mouse, study, 1 as C57BL/6 for this well as used was kg 12 ) weighing All methods for animal care and use were approved by the the by approved were use and care animal for methods All Virus injections were performed with sevoflurane sevoflurane with performed were injections Virus µ L gas-tight syringes L (Hamilton)syringes gas-tight µ m superficial) at superficial) m µ L of virus virus of L Macaca Macaca µ M) M) - -

28. 27. 26. 25. 24. 23. 22. 21. 20. 0.1% 40- into cut with finally and solution, glycerol azide sodium 20% a in stored blocked, extracted, then was brain The swelling. and 4% paraformaldehyde. PBS with brain perfusion before administered was bital prevent to administered of pentobar overdose An injection. viral after 4 weeks also killed was animal The was dexamethasone mg 1

otne, .. Cucmn LS, pdc, .. Fybeg H Optimized H. Flyvbjerg, & J.A. Spudich, L.S., Churchman, K.I., Mortensen, open- accurate, fast, Localizer: J. Zhang, & R.K. Neely, S., Duwé, P.,Dedecker, control Computer N. Stuurman, & R. Vale, K., Hoover, N., Amodaj, A., Edelstein, high-speed for microscope imaging three-camera A R.H. Singer, & B.P. English, Vlfeat. B. Fulkerson, & A. Vedaldi, Lowe, D.G. Distinctive image features from scale-invariant keypoints. Registration. based Point and Image B-splineGrid D.-J. Kroon, A.E. Carpenter, Wachter, R.M. & Remington, S.J. Sensitivity of the yellow variant of green fluorescent source, and modular software package for superresolution microscopy. superresolution for package software modular and source, (2010). using microscopes of 2015). Photonics, and Optics for + Eng. single-molecule tracking and super-resolution imaging in living cells. 2010). Press, Vis. and-point-bas http://www.ma phenotypes. cell quantifying Opt. nitrate. and halides to protein Nat. Methods Nat. microscopy. super-resolution and tracking single-molecule for analysis localization

60 17 (eds. Mohseni, H., Agahi, M.H. & Razeghi, M.) 955008 (International Society , 91–110 (2004). 91–110 , , 126008 (2012). 126008 ,

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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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

Supplementary Figure 4

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

Nature Biotechnology: doi:10.1038/nbt.3625 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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625

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).

Nature Biotechnology: doi:10.1038/nbt.3625 Supplementary Table 1. Performance of selected FPs in proExM.

Protein Ex Em Molecular Brightness Addgene Reference max, max, brightness in proExM plasmid nm nm relative to cells, % of code EGFP, % live cells EBFP2 383 448 54 62±4 55243 1 mTagBFP2 399 454 98 65±9 55302 2 mTurquoise2 434 474 85 68±8 36207 3 mCerulean3 433 475 105 69±4 55421 4 ECFP 434 477 39 51±2 55344 5,6 mTFP1 462 492 165 70±7 55488 7 mEmerald 487 509 118 53±4 54112 8 EGFP 489 509 100 65±5 56436 9

mClover 505 515 128 61±4 56533 10 c EYFP 514 527 155 64±7 56592 11 mVenus 515 528 159 44±5 56615 12 mCitrine 516 529 177 54±7 56555 13 mOrange2 549 565 105 32±2 57962 14 LSSmOrange 437 572 71 42±3 37133 15 tdTomato 554 581 144 67±4 58102 16 mRuby2 559 600 130 90±7 55898 10 mCherry 587 610 48 72±3 55056 16 mKate2 588 633 76 37±3 NAa 17 mCardinal 604 659 50 36±3 56161 18 b iRFP 690 713 15 14±1 NA 19

amKate2 gene from Addgene plasmid 37132 was swapped with LSSmOrange gene in Addgene plasmid 37133. bcloned as N-terminus fusion with nuclear localization sequence. csince EYFP is particularly sensitive to the high Cl- used to shrink the gel20, retention of EYFP fluorescence was measured in fully expanded gel.

Nature Biotechnology: doi:10.1038/nbt.3625 Supplementary Table 2. Performance of selected secondary antibody dyes in proExM.

Dye Ex Em Brightness in Source max, max, proExM as % nm nm of post antibody stain DyLight405 400 421 28±5 Life Technologies CF405M 408 452 51±4 Biotium Alexa488 495 519 48±2 Life Technologies Alexa546 556 573 68±3 Life Technologies Alexa594 590 617 46±2 Life Technologies CF633 630 650 51±10 Biotium Alexa647 650 668 7±3 Life Technologies Atto647N 644 669 55±2 Sigma

Nature Biotechnology: doi:10.1038/nbt.3625 Supplementary Table 3. Performance of selected photoswitchable and photoactivatable FPs in proExM.

Protein Ex max, Em max, Molecular Brightness Addgene Reference nm nm brightness in proExM plasmid relative to cells, % of code EGFP, % live cells 490 507 68 21±3 57725 21 Dendra2 553 573 58 ND 506 519 143 45±9 57384 22 mEos2 573 584 92 ND 505 515 102 31±2 57326 23 mKikGR 580 591 53 ND PATagRFP 562 595 76 66±7 NAa 24

aPATagRFP gene from Addgene plasmid 31945 was swapped with LSSmOrange gene in Addgene plasmid 37133. ND, not determined.

Nature Biotechnology: doi:10.1038/nbt.3625 Supplementary Table 4. Primary antibodies used.

Target Host Clonality Manufacturer Catalog No. GFP chicken poly Abcam ab13970 GFP rabbit poly Life Technologies A11122 bassoon mouse mono Abcam ab82958 homer rabbit mono Abcam ab184955 homer rabbit poly Synaptic Systems 160 003 lamin A/C mouse mono Cell Signaling Technologies 4777S TOM20 rabbit poly Santa Cruz Biotech sc-11415 post-synaptic density 95 mouse mono Neuromab 73-028 glutamic acid decarboxylase rabbit poly Millipore AB1511 myelin basic protein rabbit poly Abcam ab40390 vimentin chicken poly Abcam ab24525 glial fibrillary acid protein mouse mono Santa Cruz Biotech sc-166458

Nature Biotechnology: doi:10.1038/nbt.3625