© 2016 Nature America, Inc. All rights reserved. Cambridge, Massachusetts, USA. 8 6 of Pennsylvania, Philadelphia, Pennsylvania, USA. Cambridge, Massachusetts, USA. 1 the of swelling osmotic composite. by specimen–gel followed specimen homogenize properties to mechanical treatment proteolytic by achieved polyelectrolyte swellable a synthesized to gel anchored then and via interest protocol, of antibodies to original targeted our first were In tags lens. fluorophore objective ~300-nm an diffraction-limited using resolution ~70-nm yields expansion linear on For ~4× example, microscopes. imaging diffraction-limited conventional super-resolution enabling tissues, magnifies cally (ExM) expansion tissues, magnify an for approach to Recently, physically we RNA developed function. important proteins or compartments cellular with ciations asso pinpoint to required precision nanoscale the with tissues RNA to image in intact However, difficult states. it remained has and types cell of RNA, roles and for defining structural mapping for processing, RNA local understanding for key is organs and tissues, cells, of RNA throughout imaging Nanoscale-resolution importance to biology and medicine. specimens, such as intact brain tissue and other tissues of and location with diffraction-limited microscopes in thick E chain reaction) as well as multiplexed amplification ofsignals single-molecule (i.e., via hybridization E precision in both cultured cells and intact brain tissue. with high yield and specificityas well as single-molecule in situ a biological specimen. swellable polyelectrolyte gel synthesized throughout linker that enables expansion microscopy of biological settings. for definingcell types and states in normal and pathological nanoscale precision in intact tissues is of great interest T Edward S Boyden Evan R Daugharthy Fei Chen N Received Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. he he ability to image xpansion xpansion F xF ISH anoscale anoscale imaging of hybridization (F thus enables super-resolution imaging of

14

Ma 1 – ISH r 3 ch; , 10 (

, , Asmamaw T Wassie accepted in situ in E xF RN H ISH 1 RN ere, we present a strategy for – A A to be covalently attached to a 3 6 T ISH . Isotropic expansion was subsequently subsequently was expansion Isotropic . A A identity and location with ) ) separates ,

, hen, hen, postexpansion, fluorescent 9 18 7 RN , , Jae-Byum Chang 10 3

McGovern Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. ) ) imaging of May; These authors contributed equally to this work. Correspondence should be addressed to E.S.B. ( A. A. We developed a small-molecule

published RN As and supports RN 5

RN Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. online 1 A A F – A A can be performed 3 , ISH 10

4 2 , Allison , J Allison Cote

July 1 , readout. 3 . ExM isotropi ExM . RN , , Adam Marblestone

2016; A A structure

RN

doi

:10.1038/n A A with expansion microscopy 9 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, - - 7

Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. 4 , , Anubhav Sinha m FISH (smFISH) can be performed with multiple fluorophores fluorophores multiple with performed be can (smFISH) FISH delivered is mRNA of strand target a to tary complemen probes In of RNA a fluorescent set FISH, precision. which FISH, transcripts of RNA identification enables enables ExFISH, call we which procedure, Wegel. ExM to the this that show attached RNA covalently to be listed in in listed are reagents all Technologies; Life AcX, (Acryloyl- abbreviated here acid SE, X 6-((acryloyl)amino)hexanoic and Amine (MirusBio) Label-IT as such profiles, two these of each satisfy that exist reagents available Commercially moiety. acrylamide able polymeriz a and ester succinamide amine-reactive an contains the to reacts primarily group that alkylating an as well as blocks: amine an building containing two molecule from a reagent a synthesized we aim, this Toachieve postexpansion. molecules these of interrogation the molecules RNA securing enable would linker a small-molecule via gel ExM to the covalently directly that reasoned thus We a reliance on proteins for RNA retention ( of RNAs the strong proteolysis ExM precludes fixation, proteins during linking to crosslinked are covalently transcripts Although gel. for ExM the to directly strategy a determined first We E RESULTS functions. biological to a diversity of questions relating the structure and location of RNA for useful be will ExFISH spines. dendritic mRNAs to individual neural for localizing as well as RNAs (lncRNAs) noncoding long of structures nanoscale revealing for ExFISH of power the strate We demon tissues. brain mouse intact in FISH HCR-amplified and culture cell in smFISH support can ExFISH that show We mRNA. a to single targeted be to of fluorophores number a large (HCR) tion amplification tissues, such strategies, as hybridization chain reac to delivered a probes mRNA single via oligonucleotide eth.389 xF Here, we have developed a small-molecule linker that enables enables that linker small-molecule a developed have we Here, ISH 2 , 3 9 , design and validation of validation and , design , , George M Church upeetr Tbe 1 Table Supplementary 5 , 6 n bace DA amplification DNA branched and n a 5 ture methods , , Shahar Alon 2 Media Lab, Massachusetts Institute of Technology, N7 4 6 Department of Bioengineering, University

, of guanine and a molecule that that molecule a and guanine of 8 | [email protected] ADVANCE , , Arjun Raj 2 ). We named this molecule, molecule, this named We ). , 3 RN in situ in , , Shoh Asano A anchoring chemistry A anchoring Supplementary Fig. Supplementary 1

ONLINE with single-molecule single-molecule with 2 , 3 4 . Single-molecule Single-molecule . & ).

Articles PUBLICATION

7 , 8 2 , can allow allow can , , 3 4 , . . In intact

|

). ).  - - - -

© 2016 Nature America, Inc. All rights reserved. spots that were previously indistinguishable. For example, example, For indistinguishable. previously were that spots decrowding process expansion the with state, unexpanded the in ence arose from the high density of smFISH spots for these targets thousands) the in ( example, (for mRNAs expressed highly for the tens); however, more transcripts were detected after expansion in example, (for counts transcript low at unity near was detected and low- both for ( transcripts expansion high-copy-number with detectability transcript of ( after and cells. smFISH images, taken with probes delivered before ( cell, per ~10,000 to ~10 from ranging number copy with targets, (seven number copy varying of mRNAs targeting probes smFISH used through ( FISH interrogated be then could RNAs the expanded, thus was sample a Once swelling protocol. ExM osmotic original the in and performed as proteolysis, formation, gel by followed anchorable, gel RNAs its make to LabelX with treated be could 2 Fig. ( readout ( smFISH impede not does LabelX LabelX that group, polymerizable radical free- a with functionalized covalently be to RNA enables which ( units; pre-expansion in coding color by represented ( noted); factor expansion the by divided are bars scale blue units; expansion NEAT1 of (bottom) in envelope nuclear denotes line (white expansion before ( transcripts ( nucleus. in sites transcription highlighting region, in ( expansion. and digestion, gelation, after (right) hybridization via probed be can RNA Anchored (middle). gel ExM the to anchored be to RNA enables which (left), LabelX with treated are specimens biological ExFISH: ( (bottom). of guanines) position N7 the (e.g., bases its alkylating by anchorable gel make RNA to serves which (middle), LabelX form Figure  against smFISH for Supplementary Fig. Supplementary 3 a b

Articles Alk | To quantify RNA-transcript-anchoring yield after expansion, we ADVANCE ylation ofnucleicacid (see Online Methods). ( Methods). Online (see 1 ). We then designed a procedure in which a sample sample a which in procedure a designed then We ). Fig. 1 Fig. | Design and validation of ExFISH chemistry. ( chemistry. of ExFISH validation and Design n = 59 cells across all seven targets) in cultured HeLa HeLa cultured in targets) seven all across cells 59 = n NEAT1 Fig. 1 Fig. = 59 cells; each symbol represents one cell). ( cell). one represents symbol each cells; = 59

ONLINE b ). lncRNA in the nucleus of a HeLa cell. Magenta and green indicate probesets binding to different parts of 5 the parts different to binding probesets indicate green and Magenta cell. of a HeLa nucleus the in lncRNA d ) expansion to the same cells, showed no loss loss no showed cells, same the to expansion )

PUBLICATION LabelX ACTB and Linker i ) Insets showing a showing ) Insets , we were able to resolve individual individual resolve to able were we , Supplementary TableSupplementary 2 Guanine | Fig. 1 Fig.

Linker n Linker a ture methods e f ). The ratio of transcripts transcripts of ratio The ). d c i. 1 Fig. NEAT1 h ) ) 2 d a a Supplementary Supplementary ) As in ) As in cluster (boxed region of region (boxed cluster ) Acryloyl-X SE (top left) is reacted to Label-IT amine (top right) via NHS-ester chemistry to chemistry NHS-ester via right) (top amine Label-IT to reacted is left) (top SE ) Acryloyl-X ). We verified verified We ). f µ – m (3.3×); and ( and m (3.3×); ). ). This differ h f ) smFISH image of XIST long non-coding RNA (lncRNA) in the nucleus of an HEK293 cell cell HEK293 of an nucleus the in (lncRNA) RNA non-coding long of XIST image ) smFISH ). ( ). c c , using ExFISH. ( ExFISH. , using ) smFISH image of image ) smFISH g ) As in ) As in Fig. 1 4.5 Z =0

c µ f - ) m , using ExFISH. ( ExFISH. , using

i ) 200 nm (3.3×). nm ) 200 c g , revealed being still is that process a through subregions chromatin cific spe with association initial on depend may inactivating chromosome X the in role whose XIST, lncRNA the imaged We ogy. biol cell in roles structural Weto serve known lncRNAs imaged N of probes. of manuscript) because the salt required to support hybridization this of legends figure the in found be can factors expansion ~4×; versus ~3.3× (i.e., paper ExM original our in than lower is slightly factor expansion The distortion. ExFISH the bound to serve chemistry, were similar with they obtained since results, earlier These tissues). and cells in distortion percent few (a small linker similar a with ExFISH, in used as gel ExM protocols in which proteins are anchoredprotocol to the same ExM hydro original the in expanded and labeled were targets when observed that to similar numerically 4 Fig. process enables uniform expansionExFISH the ofDNA, the to nucleus the reacts ( also in Label-IT Since readout state. expanded RNA single-molecule supporting of ExFISH capable Thus, is bursting. transcriptional of account on mRNA ( nucleus the in foci ACTB d ) 10 ) 10 anoscale imaging of lnc imaging anoscale e h ) smFISH counts before versus after expansion for seven different different seven for expansion after versus before counts ) smFISH ) with smFISH (left) and ExFISH (right). Scale bars (white, in pre- in (white, bars Scale (right). ExFISH and (left) smFISH ) with ACTB µ mRNA puncta postexpansion even within transcriptional transcriptional within even postexpansion puncta mRNA . h iorp o EFS ( ExFISH of isotropy The ). m (expansion factor, 3.3×), inset 2 inset factor, 3.3×), m (expansion before expansion of a cultured HeLa cell. Inset shows zoomed- shows Inset cell. HeLa of a cultured expansion before 1 1 h . The pre-expansion image ( image pre-expansion The . ) smFISH image before expansion (top) and using ExFISH ExFISH using and (top) expansion before image ) smFISH e

4.5 Number of RNA molecules Z Fig. 1 Fig.

=0 after expansion 100,00 µ 10,000 m 1,00 10 10 h 0 0 0 c RN 10 versus versus A with A with Number 100 d upeetr Fg 5 Fig. Supplementary before expansio ), which can be dense with with dense be can which ), µ E m; ( m; Fig. 1 Fig. xF of RNA molecule 1,000 ISH f , ′ g (1–3, 756 nts) of nts) 756 (1–3, 9 ) ) 2 f , 1 ) shows two bright bright two shows ) i 0 , the distortion is is distortion the , 10,000 b µ n ) Workflow for ) Workflow Supplementary m (3.3×), m (3.3×), s 1 . In recent recent In . 100,000 GAPDH TFR EEF UB USF2 Top2 ACTB Z scale scale ) was was ) C 2 C A - - - © 2016 Nature America, Inc. All rights reserved. cycle-to-cycle consistency, we next demonstrated the capability capability the demonstrated next we consistency, cycle-to-cycle ( ratio signal-to-noise or ciency ( round to round from rounds, RNA spots five individual of locations the of the distortion no was there Across formamide). 100% example, (for talk cross cycle-to-cycle to minimize designed gent conditions wash strin under even observed was stability This cells). cultured on ( set 6 probe Fig. same the of application they were and robust by as to cycles assessed repeated multiple wash–stain ~3×), (i.e., samples non-re-embedded as factors sion expan similar exhibited samples re-embedded Such protocol. the throughout specimen expanded salt concentration changes in the stabilized additionally and imaging multiround for bilized This gels process expanded allowed to be immo polyacrylamide. charge-neutral in specimens expanded re-embedded we FISH, of cycles multiple facilitate to order In sets. probe multiple with readout RNA tiplexed mul for proposed been have that strategies facilitate could sion the decrowding of the environment local that results from expan gel to RNA and ExM the of covalent anchoring The combination S their and configurations. spatial complexes regulatory chromatin key defining in ful genome the of ( structure 3D clear became morphology ( puncta limited retention expansion, mRNA nuclear and gene regulation in role expression important an play hypothesized have researchers of ensembles of morphology described previously the to examine ExFISH used within both ( nearby as well as regions apparent globular the were puncta individual postexpansion, inactivation undergoing cells HEK of chromosomes X the to corresponding presumably regions, fluorescent globular in images (raw cell HeLa cultured a in targets RNA six against probes delivered serially with ExFISH showing ( background. the of s.d. the by divided brightness puncta of staining of rounds five the across ExFISH of of mean is plotted count; 1 round the to normalized ( colocalization. showing yellow), or magenta, blue, green, red, color, different a each (with images five the of overlay an shows panel right lower Methods); Online (see removal probe following of ( cell. HeLa cultured targeting ExFISH of image ( ExFISH. multiplexed Figure of multiplexed ExFISH by applying probes for for probes applying by ExFISH multiplexed of green; green; c uper-resolved, multiplexed imaging of imaging multiplexed uper-resolved, ) ExFISH RNA counts for each round, round, each for counts RNA ExFISH ) a a . ( . restainings repeated five showing , d , showing five rounds of smFISH staining against against staining smFISH of rounds five showing , USF2 2 ) Signal-to-noise ratio (SNR) (SNR) ratio Signal-to-noise ) ± | standard error; error; standard Serially hybridized and and hybridized Serially a , light blue. Scale bars (expanded coordinates): ( coordinates): (expanded bars Scale blue. light , , computed as the mean mean the as computed , NEAT1 Fig. 1h Fig. b e a presented in the form of bright, diffraction- bright, of form the in presented ) Composite image image Composite ) ) Boxed region region Boxed ) ) Widefield fluorescence fluorescence Widefield ) 17– n GAPDH Fig. 2 Fig. = 3 regions regions 3 = , i 1 ), but after expansion, the ring-shaped ring-shaped the expansion, after but ), 9 based upon sequential hybridization hybridization sequential upon based 1 in a in b 6 NEAT1

S

, mapping lncRNAs may be use be may lncRNAs mapping , i. 1h Fig. ), nor variance in detection effi detection in variance nor ),

upplementary Fig. 6 Fig. upplementary

Fig. 2c Fig.

lncRNAs ( lncRNAs i. 2 Fig. Fig. 1 Fig. , , i d ). Given the complex complex the Given ). ). Having validated the the validated Having ). RN g a ). Additionally, we Additionally, ). ; ; A with A with b a c

Supplementary Supplementary Normalized spot count Fig. 1 Fig. GAPDH 0.5 1.0

1 Round 1 ); colors are as follows: follows: as are colors ); 0 4 ring-shaped ring-shaped Round 2 a E 1 h ) 20 20 ) 11– 5 xF ), which which ), GAPDH . Before Before . Round 3 , , ISH 1 3 UBC µ , but but ,

m; ( m; Round 4 ------Round 5 b ,

) 10 10 ) NEAT1 molecule precision throughout a volume ~575 × 575 × 160 160 × 575 × ~575 volume a throughout precision molecule single- with transcripts multiple of visualization enable to slice brain Thy1–YFP a on microscope lightsheet available mercially which can be to scaled very fast imaging speeds bles super-resolution imaging on diffraction-limited microscopes, ( was tion observed FISH probes were HCR background omitted, amplifica minimal using the YFP protein to delineate neural morphology ( We applied ExFISH to samples of Thy1–YFP mouse brain tissue mens such as brain tissue on conventional microscopy hardware ExM allows for super-resolution imaging of facile thick 3D speci 3D MERFISH as such cycle, FISH per information more yield sets to probe designed apply computationally to straightforward it making as described, polymer here swellable the to RNA securing after expanded ples 6 Fig. Supplementary ( cell same the in localized and identified RNAs be to (e.g., (e.g., YFP) cleanly resolved into individual puncta ( transcripts overexpressed highly even with highlighted), mRNA of because ( ExM of magnification distinguished physical the be could transcripts individual that magnification using a confocal spinning-disk microscope revealed hippocampus and cortex layers of the specific throughout arrangement with characteristic ( cells YFP-fluorescing to localized well mRNA YFP with microscope, ( 1 lase ( mRNAs for YFP regions, 7 Fig. DNA HCR architecture amplifier HCR of technique described previously the applied we imaging, confocal using tissues intact in visualize to dim too signals yields smFISH Since LabelX. via protocol proExM the using AcX via gel polyacrylate the to anchored was protein YFP Endogenous µ m; and ( and m; d nanoscale imaging of imaging nanoscale

NEAT1 SNR

Round 1 10 15 0 5 Fig. 3 Fig. ). In samples containing mouse cortical and hippocampal hippocampal and cortical mouse containing samples In ). Gad1 , ,

USF2 Round 2 , blue; blue; , e ) 20 20 )

e Round 3 ) and ) and ) ( ) , , ACTB µ Fig. 3 Fig. n EEF2 Round 4 m. a ture methods Gad1 Round 5 Supplementary Fig. 8 Fig. Supplementary , orange; orange; , , , and d ) were easily visualized using a widefield widefield a using visualized easily were ) ). Thus, serial FISH is applicable to sam to applicable is FISH serial Thus, ). mRNA localized to a population of cells of cells a to population mRNA localized 18– 2 e 2 Fig. 3 Fig. EEF2 . Examining brain specimens at high high at specimens brain Examining . RN 2 0 GAPDH 5 . , in particular the next-generation next-generation the particular in , A in mouse brain tissue brain mouse A in

in series, enabling six individual six individual enabling in series, | c ADVANCE 6 ) and glutamic acid decarboxy acid glutamic ) and (schematic in (schematic , yellow; yellow; , Fig. 3 Fig. 9 , and RNA was anchored anchored was RNA and , ). ). Given that ena ExM

ACTB ONLINE f , with YFP and and YFP with , 2 , purple; purple; , 3 , , we used a com Supplementary Supplementary

Articles PUBLICATION Fig. 3 Fig. 2 Fig. Fig. Fig. 3a UBC f ). ). When , Gad1 e

and µ , | b 2

m 1 1 ). ).  ------, .

© 2016 Nature America, Inc. All rights reserved. sets targeting the same transcript with different initiators. initiators. different with transcript same the probe targeting of pairs sets delivered we tissues, expanded single-molecule in for visualization rates false-negative and assess to false-positive order In the is positives. false this excludes it as independence); bound lower probe a (assuming efficiency detection as interpreted is colocalization 50% a culture; cell in assessed when labeled doubly be will targeted mRNAs of thus probe different colors sets bearing RNAsdifferent with individual to is label accuracy detection ing ( signal 9 Fig. in thick just 3 h (~6 × 10 ( ( noted): factor expansion the in images Raw location. puncta colocalized depicts ( examples. representative colocalized showing tissue red, protein; YFP (green, brain mouse blue, missense magenta, mRNA; YFP cyan, protein; YFP (red, region of boxed the of distribution of Figure  two of signals fluorescent amplified in resulted scheme This e

Articles , g c a | HCR amplifies a target-binding event into a bright fluorescent fluorescent into a event bright a target-binding HCR amplifies a i (ii) (i) ADVANCE ) maximum-intensity projection (MIP) 27 27 (MIP) projection ) maximum-intensity . . ( Dlg4 c and and 3 ) Widefield fluorescence showing HCR-ExFISH of YFP mRNA in the sample of sample the in mRNA of YFP HCR-ExFISH showing fluorescence ) Widefield | Supplementary Fig. 7 Fig. Supplementary Dlg4 missense odd). The processed image (ii) shows zero colocalized spots (magenta). ( (magenta). spots colocalized zero shows (ii) image processed The odd). missense Nanoscale imaging of RNA in mammalian brain. ( brain. mammalian in of RNA imaging Nanoscale Supplementary Video 1 Video Supplementary

probe in Thy1–YFP mouse tissue (green, YFP protein). The raw image (i) uses alternating probes in two colors (red, (red, colors two in probes alternating uses (i) image raw The protein). YFP (green, tissue mouse Thy1–YFP in probe ONLINE Gad1 versus Thy1–YFP mRNAs. ( mRNAs. Thy1–YFP versus

PUBLICATION k Dlg4 ) As in ) As in 10 h d b (ii) (i) voxels in colors; three a ) 500 ) 500 puncta (magenta) overlaid on YFP (green). ( (green). YFP on overlaid (magenta) puncta j ). A stringent method for assess for method stringent A ). , but with HCR-ExFISH of HCR-ExFISH with , but | µ

Actb n m; ( m; 24 a ). , ture methods 2 even; and blue, blue, and even; 5 b , , which shows that 50–80% – e µ ) 500 ) 500 f m thick (pre-expanded units); ( units); (pre-expanded m thick ) Confocal image of mouse hippocampal tissue from from tissue hippocampal of mouse image ) Confocal S µ upplementary Figure Figure upplementary m (expansion factor 2.9×); ( 2.9×); factor m (expansion i Supplementary Supplementary e Actb a ) Widefield fluorescence image of Thy1–YFP mouse brain. ( brain. mouse of Thy1–YFP image fluorescence ) Widefield Camk2a 5 0 odd in (i); colocalized spots in magenta (ii)). ( (ii)). magenta in spots colocalized (i); in odd . Gad1 (~70%) (~70%) mRNA showing transcripts in dendritic spines and processes. ( processes. and spines dendritic in transcripts showing mRNA mRNA). ( mRNA). -

1 j 0 ) Dendrites with with ) Dendrites . Scale bars (white, in pre-expansion units; blue scale bars are divided by divided are bars scale blue units; pre-expansion in (white, bars . Scale g color, 15,866/27,504 spots; spots; color, 15,866/27,504 other with colocalized color one in probes of 58% of average (an present was transcript a ( when contrast, In mCherry). spots, spots, (0/1,209 colocalization exhibited reversed; 1 per 48 amplified, puncta (1 per 61 samples, and we found a low but nonzero spatial density of dim, yet (mCherry, ( probe rate. We a a missense against set probe delivered of false-positive Delivering measure a gives efficiency. also transcript nonexistent a hybridization against sets probe the of measure a giving ( target same the from colors different , Actb h g ) Confocal image (i) and processed image (ii) of HCR-ExFISH using a using of HCR-ExFISH (ii) image processed and (i) image ) Confocal , f j , b ) 50 ) 50 k . . ( ) MIPs ~1.6 ~1.6 ) MIPs ), a large fraction of the puncta exhibited colocalization colocalization exhibited puncta the of fraction large a ), d Dlg4 µ ) As in ) As in m (2.9×), inset 10 10 inset m (2.9×), Supplementary Table 3 reversed, reversed, h Dlg4 ) As in ) As in c , but for for , but µ mRNA localized to spines (arrows). (i), (ii), two two (ii), (i), (arrows). spines to localized mRNA m m thick. µ e m , showing single RNA puncta. Inset, one plane plane one Inset, puncta. RNA single , showing g , but for HCR-ExFISH targeting targeting HCR-ExFISH for , but 3 , , mCherry). Essentially zero of these puncta Fig. 3 Fig. Gad1 f j µ (i) (ii) m; ( m; µ mRNA. ( mRNA. Fig. 3 Fig. i g ) Confocal image of hippocampal of hippocampal image ) Confocal m ) as well as a nonexistent transcript transcript nonexistent a as well as ) g 3 – in unexpanded coordinates, i h ) 10 ) 10 ), using Thy1–YFP mouse brain and b e ) Composite of ) Composite ) Postexpansion widefield image widefield ) Postexpansion µ m (3×); ( m (3×); Supplementary Table 3 Supplementary Supplementary Fig. 10 Fig. Supplementary Dlg4 Dlg4 k (i) (ii) i – reversed; 4/1,540 4/1,540 reversed; j , k k Actb missense even; even; missense ) Magenta channel channel ) Magenta ) ) 2 b – d in Thy1–YFP Thy1–YFP in µ , highlighting , highlighting m (3×). m (3×). Dlg4

), ), ), ),

© 2016 Nature America, Inc. All rights reserved. ehd lk lgthe microscopy lightsheet like diffraction-limited methods fast with ExM combining strategies Thus, components nonanchored of washout of because part in and dilution volumetric the of because part in refraction, of index in homogeneous and transparent them makes samples Expanding resolution even on microscopes. conventional diffraction-limited detec SNP tion or readout transcriptomic for strategies other and fluorescent (RCA), amplification rolling-circle transcription, as such reverse samples in expanded performed be to reactions enzymatic support also may gel ExM the to RNA of anchoring covalent more The information. exponentially transcript yields cycle additional each which in ing, strategies coding specific to according designed probes using that implying tion, loca and identity RNA of readout multiplexed and steps zation hybridi probe and washing repeated including their smFISH, support to serial for enough robust was implications anchoring The direct roles. has biological structure emergent whose lncRNAs NEAT1, and XIST as such single-molecule structures and RNA resolution, precision nanoscale with visualize, enabled to This us localization. and counts RNA accurate more for of RNAs and decrowding yield excellent finding expansion, after Weas HCR amplification. RNA before versus validated retention RNAs to be probed through FISH single-molecule labeling as well expansion microscopy. The resulting procedure, ExFISH, enables for anchored covalently be to RNA enables that commer precursors, from cial synthesized easily reagent, novel a present We DISCUSSION degeneration. to development to plasticity from may be for useful a diversity of questions in ranging neuroscience the ability to map mRNAs at synapses number, throughout copy neuronal low arbors have can mRNAs and synapses of thousands of tens have can neurons that Given cycles. subsequent between enable multiple cycles of HCR by disassembling the HCR polymer displace ment strand mediated toe-hold using disassembled be can that hairpins HCR modified developed we readout, HCR plexed multi Tofacilitate processes. dendritic fine in as well as spines dendritic of subset sparse a in detected be indeed could mRNAs that individual revealed ples Fig. 10 and giving a lower bound on transcript detection ( puncta that were colocalized, thus suppressing false-positive errors 61%; spots; 8,799/14,440 Camk2a ( 73% of efficiency tion detec high a suggesting spots) 5,174/9,795 (53%; colocalization enriched dendritically be to known is which and PSD-95 protein scaffolding postsynaptic prominent the probed We segmentation. or identification for accurate resolution nanoscale require which spines, dendritic as such compartments cellular within position above. described studies single-molecule amplified non the to comparable efficiency, detection 75% a of indicative both worlds’ performance metrics: the voxel sizes of classical classical of sizes voxel the metrics: performance worlds’ both ExM, ExM, being a physical form of magnification, enables nanoscale We used two-color HCR ExFISH against mRNAs to image their 2 8 2 , within intact samples. intact within , 6 ). Focusing on individual dendrites in these expanded sam ( Supplementary Supplementary Fig. 11 , finding a detection efficiency of 78% (colocalization, (colocalization, 78% of efficiency detection a finding , 17– 1 9 would support combinatorial multiplex combinatorial support would Fig. 3 Fig. Supplementary Fig. Supplementary 10 Dlg4 i ). We also probed the mRNA for for mRNA the probed Wealso ). Dlg4 ). These modified HCR amplifiers ( in in situ Fig. 3 Fig. mRNA, which encodes the the encodes which mRNA, 7 2 . We obtained a degree of degree a We . obtained 3 sequencing (FISSEQ) sequencing may result in ‘best of of ‘best in result may j ) ) and Camk2a ). ). We onfocused Supplementary ( Fig. 3 Fig. 2 k 1 7 ------) . ,

into the sample must be considered. be must sample the into reagents of diffusion and efficiency reaction although methods, HRP amplification) as well as nonenzymatic (e.g., branched DNA) RCA (e.g., enzymatic including well, as possible be may into strategies fluorophores amplification Other bulky samples. other and these of delivery better ble ena may state expanded The amplification. signal for need the dots quantum as such fluorophores brighter example, For possible. be may signals brighter achieving of methods Other precision. experiment per transcripts more = of imaging 500/3.5 of size the facilitates amplicons of effective density packing lower The nm. an ~150 have RNAs, between distance 500 nm size in the of samplepostexpanded would, because of amplicon the greater HCR An 6. and 5 refs. of protocols the follow strategies HCR molecule single- optimized developing is lab Pierce the tissues; intact in of RNAvisualization here applied to enable nanoscale-resolution size of the self-assembled amplification product of HCR, which we ing of of RNAs enables another key advantage: rates reducing the effective acquisition voxel microscopes fast diffraction-limited increasingly the but methods super-resolution to to the ExM gel. F.C. and A.T.W. conceived and developed the LabelX reagent. F.C., A.T.W., E.R.D., A.M., G.M.C., and E.S.B. conceived RNA-tethering strategies A and Joyce Wertheimer. J.-B.C. was supported by a Simons Postdoctoral Fellowship. 1R24MH106075, IARPA D16PC00008, the Open Philanthropy Project, and Jeremy 2R01DA029639, NIH Director’s Transformative Award 1R01MH103910, NIH Intelligence Project, NIH Director’s Pioneer Award 1DP1NS087724, NIH Award, NSF CBET 1053233, MIT Media Lab Consortium, the MIT Synthetic E.S.B. acknowledges support by the New York Stem Cell Foundation–Robertson AR and AC acknowledge support from NIH/NHLBI grant 1U01HL129998. Fellowship. F.C. acknowledges the NSF Fellowship and Poitras Fellowship. and NSF GRF grant DGE1144152. A.T.W. acknowledges the Hertz Foundation supported by NIH CEGS grant P50 HG005550, NIH CEGS grant 1 RM1 HG008525, and H.T. Choi and N. Pierce for advice and consultation on HCR. E.R.D. is S. Olenych from Carl Zeiss Microscopy for providing the microscopy filters, to acknowledge W. Salmon for assistance with the Zeiss Z.1 lightsheet, Imaging at the Whitehead Institute for Biomedical Research. We would like Lightsheet imaging was performed in the W.M. Keck Facility for Biological Ackno online version of the pape Note: Any Information Supplementary and Source Data files are available in the v and are Methods references any in available the associated M insights andthroughout biology medicine. new reveal may machinery trafficking and processing RNA with More broadly, visualizing RNAs within cells and their relationship anddysfunction. function circuit neural underlying mechanisms brain in intact key to circuits, an integrative of understanding the organization synaptic and connectivity neuronal of acterization of neuronal cell types profiling transcriptomic for used be could method this schemes, coding multiplexed straightforward with coupled that, anticipate and translation of axonal RNAs remains under investigation content specific synapses as a function of local synaptic activity In fields. the brain, for example, RNA is known to to be trafficked UTHOR e ethods ExFISH may find many uses in neuroscience and other biological r s i 3 o 0 w n or bottlebrush fluorophores bottlebrush or

ledgments CONTRI 3

o 3 , it is known to be locally translated f

t h e B

p UTIONS n a p a ture methods e r . r . in situ 2 9 as well as for the super-resolved char , although the results here shown shown here results the although ,

2 | 8 ADVANCE , tyramide amplification , tyramide 3 1 could, in principle, obviate obviate principle, in could,

7 ONLINE , 34 , 3 1 5 9 , and the presence with nanoscale nanoscale with 1

Articles PUBLICATION . . The decrowd 3 2 and intron

2 o

3 2

n 6 , , and

. We

l i | n

 e - - -

© 2016 Nature America, Inc. All rights reserved. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.htm R online version of t The authors declare competing financial interests: details are available in the COM and E.S.B. wrote the paper, and all authors contributed edits and revisions. lightsheet imaging and analysis. E.S.B. supervised the project. F.C., A.T.W., A.S., A.J.C. performed experiments. A.S. performed data analysis. S. Asano performed experiments. A.J.C. and A.R. provided FISH reagents and guidance on usage, and HCR strategies. F.C., A.T.W., and E.S.B. designed, and F.C. and A.T.W. performed re-embedding. F.C., A.T.W., and E.R.D. conceived and developed reversible F.C., A.T.W., J.-B.C., and S. Alon developed ExM gel stabilization by  eprints and permissions information is available online at

Articles

|

ADVANCE P 717–726 (2009). 717–726 paraspeckles. of structure the for essential is RNA NEAT1 microscopy. super-resolution using proteins and RNA of detection multicolor (2002). 233–278 inactivation.chromosome X of mechanism the stabilization. RNA Xist by mediated (2013). 1237973 chromosome.X the across spread to architecture proteins.fluorescent and retention. protein formalin-fixed, paraffin-embedded tissues. (2012). 453–466 imaging.high-resolution and sequencing deep by revealed Nano ACS durability.greater cost, lower gain, higher reaction: chain hybridization expression. mRNA of imaging Methods Nat. probes. labeled singly multiple using molecules mRNA individualImaging future. and present transcripts RNA microscopy. Clemson, C.M. Clemson, Simultaneous S. Nakagawa, & T.Hirose,T., Kawaguchi, M., Mito, and RNA Xist B.Panning, & Nusinow,D.A. S., Mlynarczyk-Evans, K., Plath, is inactivationchromosome X R. Jaenisch, & J. Dausman, B.,Panning, J.M. Engreitz, T.J.Chozinski, P.W.Tillberg, Wang,F. I.J. Cajigas, Next-generationN.A. Pierce, V.A.& Beck, H.M.T.,Choi, H.M.T.Choi, S. Tyagi, & Oudenaarden,A. van S.A., P.,Rifkin, Bogaard, den van A., Raj, Fluorescence Singer,R.H. & J.M. Levsky, single of Visualization Singer,R.H. & K. Fogarty,F.S.,Fay, A.M., Femino, Expansion imaging. Optical E.S. P.W.Boyden, Tillberg,F., & Chen, ETING F IN et al. et

8 ONLINE A , 4284–4294 (2014). 4284–4294 , Science Methods et al. et NCI

et al. et he pape et al. et 5 RNAscope: a novel RNAscope:a et al. et et al. et et al. et , 877–879 (2008). 877–879 , in situ in A L The local transcriptome in the synaptic neuropil synaptic the in transcriptome local The Programmable

Nat. Biotechnol. Nat. Expansion microscopy of biological specimens with specimens biological of microscopy Expansion

PUBLICATION INTERESTS The Xist lncRNA exploits three-dimensionalgenome exploits lncRNA Xist The An architectural role for a nuclear noncoding RNA: noncodingnuclear a for role architectural An J. Cell Sci. Cell J.

34 Expansion microscopy with conventional antibodiesconventional with microscopy Expansion l r 98 . . . 7 , 158–165 (2015). 158–165 , Science , 543–548 (2015). 543–548 , Nat. Methods Nat. Nat. Biotechnol. Nat.

11 2 80 in situ in 6 in situ in

| doi:10.1038/nbt.362 , 2833–2838 (2003). 2833–2838 ,

, 585–590 (1998). 585–590 , Cell n a

13 ture methods amplification for multiplexed for amplification RNA analysisplatformfor RNA 90 in situ in J. Mol. Diagn. Mol. J. , 485–488 (2016). 485–488 , , 907–916 (1997). 907–916 ,

2 Annu. Rev. Genet. Rev. Annu. hybridization: past, hybridization: 8 , 1208–1212 (2010). 1208–1212 , Science

http://www. 5 in situ in 14

(2016). 341 Mol. Cell Mol. , 22–29 (2012).22–29 , Neuron ,

3

6 33 7 nature. ,

4 , ,

16. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 36. 35. 34. 33. 32. 31. 30. 29. 28.

interactions reveals folding principles of the human genome.human the of principles folding revealsinteractions displacement reactions.displacement resolution. single-molecule and single-cell at travelsingly into dendrites. microscopy. illumination plane selective by embryos live inside deep sectioning Optical brain. GFP.of variants spectral multiple (2012). 21301–21306 probes. FISH Oligopaint with genomes cells. single in profiling RNA multiplexed highly resolved, Spatially imaging. (2014). 360–361 situ in labeling. combinatorial and imaging (2009). 289–293 Nat. Rev. Neurosci. Rev. Nat. repair.andmaintenance assembly, system nervous in synthesis protein 343 translatability.its regulating for mechanism a reveals neurons neurons. in proteins of degradation (2011). 877–884 retrotransposons.element ID via targeteddendritically be dendrites. on sites postsynaptic activated near selectively localize to Arc IEG the for mRNA the causes bristles. DNA-tipped with labels. press). the (in clearing. and embeddinghydrogel tissue and reaction chain cells. Science Lieberman-Aiden, E. Lieberman-Aiden, Lee, J.H. Lee, strand- using nanotechnology DNA Dynamic G. Seelig, & D.Y.Zhang, M.N. Cabili, Batish,M.,vanden Bogaard, P., Kramer, F.R. Tyagi,& NeuronalS. mRNAs E.H.K. Stelzer, & J. Wittbrodt,F., Bene, Del Swoger,J., J., Huisken, E.S. Lein, G. Feng, B.J. Beliveau, RNA X. Zhuang, & S. Wang, J.R., Moffitt, Boettiger,A.N., K.H., Chen, Single-cell L. Cai, & M. Ahmad,Zhiyentayev,T.,A.F., Coskun, E., Lubeck, super-resolution by biology systems Single-cell L. Cai, & E. Lubeck, Jung, H., Yoon, B.C. & Holt, C.E. Axonal mRNA localization and local and localization mRNA Axonal C.E. Holt, & B.C.Yoon, H., Jung, Single Singer,R.H. & B. Wu, A.R., Buxbaum, and synthesis Compartmentalized E.M. Schuman, & O. Steward, Buckley,P.T. Worley,P.F.activation & Synaptic G.L. Lyford, C.S., Wallace,O., Steward, M.F. Fouz, M. Bruchez, S. Shah, Ke, R. Ke, , 419–422 (2014). 419–422 , Science Nat. Methods Nat. RNA profiling by sequential hybridization. sequential by profiling RNA Nature et al. et

Science 343 et al. et et al. et et al. et et al. et et al. et , 1360–1363 (2014). 1360–1363 ,

Science et al. et et al. et

In situ In et al. et 34 445

Imaging neuronal subsets in transgenic mice expressing micetransgenic in subsets neuronalImaging Single-molecule RNA detection at depth via hybridization via depth at detection RNA Single-molecule et al. et Highly multiplexed subcellular RNA sequencing RNA subcellular multiplexed Highly 2 Genome-wide atlas of gene expression in the adult mouse adult the in expressiongene of atlas Genome-wide Bright fluorescent nanotags from bottlebrush bottlebrush from nanotags fluorescent Bright 8 8 Localization and abundance analysis of human lncRNAs human of analysis abundance and Localization Semiconductor nanocrystals as fluorescent biologicalfluorescent as nanocrystalsSemiconductor 1 , aaa6090 (2015). aaa6090 , , 168–176 (2007). 168–176 ,

Cytoplasmic intron sequence-retaining transcripts can transcriptssequence-retaining intron Cytoplasmic

, 2013–2016 (1998). 2013–2016 ,

13 sequencing for RNA analysis in preserved tissue and tissue preserved in analysis RNA for sequencing 1 Versatile design and synthesis platform for visualizing for platform synthesis and design Versatile 3 et al. et 0 0 , 308–324 (2012). 308–324 , , 857–860 (2013). 857–860 , 5 Nat. Chem. Nat. , 1007–1009 (2004). 1007–1009 , Comprehensive mapping of long-rangeof mappingComprehensive ACS Cent. Sci. Cent. ACS Proc.Natl. Acad. Sci.USA Neuron Neuron

3 Nat. Methods Nat. , 103–113 (2011). 103–113 , Neuron Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc.

21 1

2 , 431–438 (2015). 431–438 , 8 , 741–751 (1998). 741–751 , , 41–51 (2000). 41–51 ,

β Genome Biol. Genome 4 -actin mRNA detection in detection mRNA -actin 0 , 347–359 (2003). 347–359 , Nat. Methods Nat.

9 1 , 743–748 (2012). 743–748 , 09 , 4645–4650, (2012). Neuron

1 Development 6 Science , 20 (2015). 20 ,

Science 11 in situ in

1

, 69 09

, , 32 .

6

, © 2016 Nature America, Inc. All rights reserved. with LabelX diluted to a final Label-IT Amine concentration concentration Amine Label-IT final a to diluted LabelX with For range. and this in concentrations Amine with observed Label-IT was quality staining in difference no otherwise ( some staining in smFISH resulted dimmer what concentrations higher mg/mL; of concentration 0.006–0.02 Amine Label-IT a in resulted that used were LabelX of ranges cells, For PBS. 1× with washes two by followed overnight °C 37 at 7.7) pH MOPS mM (20 buffer MOPS in tion concentra final desired a to diluted LabelX with incubated and were with 1× twice washed PBS, once with 20 mM MOPS pH 7.7, slices. brain and cells cultured of treatment LabelX performed. was or of blinding samples No technology. randomization, exclusion, a was to demonstrate goal the since was performed, estimate size sample- No weeks. 6–8 range age the in mouse male YFP)16Jrs) used in mouse The use. until PBS in °C at 4 stored and VT1000S) (Leica (50 Slices mM glycine. 100 containing PBS to moving before day, one for °C 4 at paraformaldehyde 4% in left out, dissected were Brains dehyde. paraformal 4% ice-cold with transcardially perfused and rane isoflu with were anesthetized Mice reagents. from nuclease-free prepared PBS 1× in up made were below National solutions All Laboratory Animals. of the Use and with Care the for accordance Guide Health in of Institutes were and Care Animal on approved by the Massachusetts Institute of Technology Committee perfusion. Mouse use. until environment desiccated a in °C) (–20 frozen stored was subsequently LabelX room with shaking. temperature at overnight mg/mL) (1 Reagent Modifying Amine Label-IT of 10 LabelX, pare at 1 mg/ml and stored frozen in a desiccated environment. To pre Solution Reconstitution Mirus provided the in resuspended was LLC) Bio, (Mirus Reagent Modifying Amine Label-IT ronment. envi desiccated a in frozen stored and aliquoted, mg/mL, 10 of in DMSO anhydrous at a was concentration resuspended Fisher) Thermo- AcX; abbreviated here ester, succinimidyl acid, xanoic LabelX. of Preparation use. until at °C 4 ethanol 70% in stored then were cells Fixed PBS. 1× with twice washed and min, 10 for 10% formalin with fixed DPBS once (Cellgro), with were washed cells Cultured tools. new testing for lines cell common are they because used here were and stringency of levels standard their to contamination mycoplasma for tested and by manufacturer the authenticated 1% were Cells and (BioWhittaker). (Cellgro), pyruvate sodium (FBS) penicillin–streptomycin serum 1% bovine fetal (Invitrogen), 10% with supplemented (Cellgro) medium D10 in Scientific) (Thermo Coverglass Chambered II Lab-Tek Nunc on cultured were (Invitrogen) cells HEK293-FT fixation. and culture Cell in found be can pliers and sup numbers part with A of and table reagents chemicals all ONLINE doi: cells fixed cells, in experiments other all For mg/mL. 0.02 of 10.1038/nmeth.3899 upeetr Fgrs 1 Figures Supplementary Figure Figure

METHODS 3 and related analyses was a Thy1–YFP (Tg(Thy1– µ L of AcX (10 mg/mL) was reacted with 100 100 with reacted was mg/mL) (10 AcX of L µ All methods for animal care and use were were use and care animal for methods All m and 200 200 and m Supplementary Table 1 Supplementary Acryloyl-X, SE (6-((acryloyl)amino)he SE Acryloyl-X, HeLa (ATCC CCL-2) cells and and cells CCL-2) (ATCC HeLa µ – m) were sliced on a vibratome vibratome a on sliced were m) 3 Supplementary Fig. 12 Fig. Supplementary , fixed cells were incubated incubated were cells fixed , . Fixed cells cells Fixed iue 1 Figure ), but but ), µ L e ------

fluorophores, and subsequently purified by HPLC as described described as HPLC by purified subsequently and fluorophores, PBS for another 30 min. Imaging was performed in 1× PBS. 1× in performed was Imaging min. 30 another for PBS washed twice were with wash buffer,samples 30 min per wash, and hybridization, washed once with Following 1× °C. 37 at night over SSC) 2× sulfate, dextran 10% formamide, (10% buffer tion temperature and with hybridized RNA FISH probes in hybridiza room at min 30 for SSC) 2× formamide, (10% buffer wash with smFISH staining after expansion. described. previously as out carried was expansion gels were in stored 1× and use PBS until digestion, After (~1.5×). buffer digestion osmolarity high the in slightly expand gels The X-100, Triton out at overnight 37 was 500 mM carried °C. and NaCl), 0.5% digestion EDTA, mM 1 8), (pH Tris mM (50 buffer digestion in 8 units/mL to 1:100 diluted BioLabs) K England (New Proteinase with digested were slices brain and cells cultured Gelled negligible). was APS/TEMED of autofluorescence slight the amplification, HCR with (since, protocol in ExM as original the performed was gelation LabelX, with treated slices brain gelling For hours. two for incubator °C 60 a to transferred was chamber humidified the gelation, Toinitiate gas. nitrogen with was purged chamber chamber. fied Subsequently, the humidified VA-044plus to solution a humidi monomer the and transferred 200 in mer to solution of a concentration and 0.5% (w/w) final degassed mono the to but added was smFISH), of magnitude context the in in appreciable small was that autofluorescence in resulted protocol ExM original the of (TEMED) (APS)/tetramethylethylenediamine of persulfate the ammonium LabelX, a concentrated stock of VA-044 (25% w/w, with chosen instead treated cells cultured gelling For use. before °C 4 to cooled frozen in and aliquots, thawed before use. Monomer solution was (w/w) acryla 0.15% (w/w) mide, 2.5% acrylate, sodium (w/w) 8.625% NaCl, M 2 Gelation, digestion, and expansion. in found be can numbers sion previously TOP2A Stellaris with Quasar 570 dye. Probe sets against ACTB PBS. 1× in performed was Imaging twice with wash washed buffer, 30 min were per wash, and washed once samples with 1× PBS. hybridization, Following °C. 37 at overnight SSC) 2× sulfate, dextran hybridization 10% formamide, (10% in buffer probes FISH RNA with hybridized and SSC) 2× formamide, (10% buffer wash with once washed briefly were expansion. before cells cultured fixed in smFISH temperature. at h room >6 for PBS in AcX mg/mL 0.05 with pH at were 7.7) treated For slices 37 °C YFP retention, overnight. from in fragmentation formaldehyde MOPS postfixation) buffer (20 mM MOPS increased and thickness increased their to mg/mL (due 0.1 of concentration Amine Label-IT final a to diluted MOPS pH 7.7 for 30 min and subsequently incubated with LabelX Amine Label-IT final a to mg/mL. 0.006 of concentration diluted LabelX with treated were smFISH probe sets targeting the human transcripts for for transcripts human the targeting sets probe smFISH mM 20 with incubated were above, prepared as slices, Brain , GAPDH µ , and full-length full-length and , l aliquots for 15 min. Cells were briefly incubated with with incubated briefly were Cells min. 15 for aliquots l 3 7 . Oligonucleotide sequences . for sequences Oligonucleotide probe sets and acces , XIST N , and 5 , N NEAT1 ′ -methylenebisacrylamide) was mixed, mixed, was -methylenebisacrylamide) ′ portion of Supplementary Table 4 Supplementary were synthesized, conjugated to to conjugated synthesized, were Expanded gels were incubated Monomer solution (1× PBS, NEAT1 1 because APS/TEMED APS/TEMED because were ordered from UBC n a ture methods , EEF2 Fixed cells cells Fixed . , TFRC USF2 ------, ,

© 2016 Nature America, Inc. All rights reserved. immediately before use. before immediately prepared were Coverslips again. dried then and ethanol, 100% with washed then dried, was coverslip the reagent, bind-silane the diluted bind-silane reagent. After a brief wash with the diluted were with washed ddH 200 ddH of mL 1.8 ethanol, of mL 8 into diluted was reagent 5 Briefly, polymerization. free-radical in form groups onto of the to surface acryloyl glass per that incorporates 24-well plates were treated with bind-silane, a silanization reagent Bind-silane treatment of coverslips. °C. at at h 37 6 formamide 100% temperature. For formamide stripping, samples were treated with U/ 0.5 at I DNAse with treated were samples I, DNAse For formamide. 100% or I DNAse with treatment via wash. two per min 60 formamide), (10% with buffer wash with washes replaced were washes posthybridization except gels, performed with exact conditions as above described for expanded gels. of re-embedded Staining is container moved to a 37 for°C incubator gelation for h. 1.5 gels before moving into a container and purged with nitrogen. The coverslip or glass-bottom plate with a coverslip placed onbelow) top(see of bind-silane-treated a the of top on placed are Gels wipe. the gels and the gels are dried with light wicking from a laboratory from removed is solution Excess step. this during size gel in tion 5 and mM Tris TEMED, pH 10.5 0.05% for 20 APS, min on a 0.05% shaker. Therewith is an ~30% reduc methylenebisacrylamide steel blade.stainless Cut a gels with were incubated thickness in 3% mm acrylamide, 0.15% ~1 to manually cut and water in stabilize the gels in the expanded state. Briefly: gels were expanded staining in cells, expanded gels were to in re-embeded acrylamide Re-embedding of expanded gels in acrylamide matrix. ( and measurements length before expansion after the between difference absolute the as defined was error Measurement script. Matlab custom a via expansion after of pairs points and corresponding the of expansion before points Hom at found be can instructions and code source (complete lab Raj the by developed code Matlab spot-counting custom a via counted Turboregthe plugin using FIJI points of expansion after isotropy. expansion of Analysis 2 Figures for performed was sea ( online available and lab Raj by the developed code a using counted and localized then were Spots generated. were maximum-intensity Subsequently, background subtraction algorithm (FIJI) using a before or were rolling-ball processed after expansion first cells. tured cul on performed smFISH of analysis and processing Image n a Probes were removed for multiple rounds of hybridization hybridization of rounds multiple for removed were Probes https: ture methods s.upenn.edu/StarSear µ e L of acetic acid. Coverslips and glass-bottom 24-well plates plates 24-well and glass-bottom Coverslips acid. L of acetic ). Length measurements were performed among all pairs pairs all among performed were measurements Length ). //bitbucket.org/arju – 4 Widefield images of smFISH staining performed performed staining smFISH of images Widefield , , 6 , and and , TOP2A Figures 1c Figures 8. 2 O, followed by 100% followed ethanol, by ch/launch.htm were rigidly aligned via two control control two via aligned rigidly were nrajlaboratory/rajla – Re-embeded staining of gels was was of gels staining Re-embeded e e and smFISH images before and and before images smFISH Z Coverslips and glass-bottom -projections of these images images of these -projections 3 2a 9 . Spots were localized and . Spots were localized Supplementary Fig. 5c Fig. Supplementary 3 l – 8 ). This image analysis analysis image This ). with a 200 pixel radius. c and and µ bimagetools/wiki/ L for 6 h at room room at h 6 for L µ Supplementary Supplementary L of bind-silane bind-silane of L http://rajlab. For serial 2 O and and O N , N ). ′ - - - -

fluorophores) at 3 3 at fluorophores) 647 Alexa and 546 initi To (Alexa stocks hairpin min. HCR amplification, 30 ate for 20) Tween 0.1% SSC, 5× sulfate, dextran (10% buffer amplification with preincubated were samples ized least 6 h at room temperature. Before HCR amplification, hybrid at for PBS 1× with incubated were at samples Subsequently, h °C. 37 2 for PBS 1× with incubated and wash, per min 30 buffer, wash with twice washed were samples hybridization, Following (20% formamide, 10% dextran sulfate, 2× SSC) overnight at 37 °C. buffer hybridization in probes FISH HCR-initiator-tagged with hybridized and temperature room at min 30 for SSC) forma 2× (20% mide, buffer wash with incubated were samples Gelled amplification. reaction chain hybridization with FISH RNA in channels. color different amplification for allow to initiators HCR different assigned were probes and even- odd-tiled For probe-sets, (AA). two-color spacer 2-base a via sequences tiled to appended were initiators HCR bases. 3–7 by spaced oligos 22-mer with targets mRNA of in found Table4 be can targets mRNA for numbers HCR-FISH. for design Probe 0.05 × SSC. The expansion factor can be controlled with the salt salt the with controlled be can factor expansion The SSC. × 0.05 filter. emission 685/40 with laser nm 640 a with excited were amplicons 647 Alexa filter. emission 546 HCR amplicons were excited with a 561 nm laser with 607/36 was excited with a 488 nm laser, with 525/40 emission filter. Alexa Fig. 10 Supplementary ( objective NAwater 1.15 × 40 a with system focal Yokogawa)on (CSU-X1 disk an spinning Andor performed con software. AR a NIS-Elements by and controlled (Andor), (Lumencor), camera engine sCMOS Zyla light 5.5 X SPECTRA a objective, air NA 0.2 × 4 a with microscope epifluorescence Ti-E Nikon a on quantify expansion factors of tissue slices, specimens were imaged ( expansion after and before sections brain of slices. brain expanded of Imaging and Atto Cy5-4040C-000. 647N, 594, FITC/TXRED-2X-B-NTE; Alexa LF561-B-000; 570, Quasar GFP-1828A-NTE-ZERO; 488, Alexa used: were NY) Rochester, (Semrock, cubes filter lowing cultured cells, a 60 × 1.4 NA oil-immersion objective was used. water-immersion objective was used. For all other experiments with Supplementary Figures 3 For software. AR NIS-Elements by controlled light engine (Lumencor), and a 5.5 Zyla sCMOS camera (Andor), X SPECTRA a with microscope epifluorescence NikonTi-E a on imaged were cells cultured expanded and LabelX-treated as well ExFISH. using cells cultured of Imaging wash. per h 1 with twice 20) Tween 0.1% SSC, (5× SSCT 5× with washed were gels fication, for ampli 3 After buffer h at temperature. room in amplification samples were then incubated with HCR hairpins diluted to 60 nM Gelled min. 30 for temperature room at cool to leaving and s 90 es ee xadd n ih he wse, 5 i ec of each min 15 washes, three with in expanded were Gels was tissue brain expanded of imaging confocal Postexpansion For imaging smFISH with probes fluorophores,labeled the fol . Probes were designed for HCR-FISH by tiling the CDS CDS the tiling by HCR-FISH for designed were Probes . µ M were snap-cooled by heating to 95 °C for for °C 95 to heating by snap-cooled were M ) ) on body. a Nikon GFP microscope TI-E – 5 , a 40 × 1.15 numerical aperture (NA) Probe sequences and accession accession and sequences Probe For epifluorescence imaging imaging For epifluorescence Both cultured cells as as cells cultured Both doi: 10.1038/nmeth.3899 Fig. 3a Fig. Figure 1c Figure Supplementary Supplementary Fig. 3f Fig. – e ) and to to and ) – , d k and and and and ------

© 2016 Nature America, Inc. All rights reserved. rajlabimagetools/wiki/Hom at found be can tions by developed code the Raj lab; complete source and code instruc RNA puncta were detected via a custom processing. 3D spot-counting Matlab spot-detection before artifacts movement suppress in scheme minimization) slices. in analysis Two-color Imaris. Bitplane and and Software ZEN Vision4D, Arivis FIJI, with Zeiss processed and the merged subsequently with taken were sets data Tiled for simultaneously. 59007m channels fluorescence two capture to and used were GFP for cameras sCMOS 59001m 5.5m Two 647. PCO.Edge Alexa and 546 Alexa Chroma a as a T560lpxr and Chroma a dichroic clean and included separate response to used fluorescence the filters Optical nm). (638 mW 75 and nm) 561 and nm (488 mW 50 of output maximum the with 5%, to set were transmissions laser individual The nm. 638 and nm, 561 nm, 488 were nm excitation for 469 used and laserlines The axially. laterally nm collection. 227 of size data voxel a with for pixels, 1,057 used were objective lightsheets The image volume dimensions water-immersion of a single tile were 1,400 Both × 1,400 × 20× NA). a by (1.0 detected fluores signal the and NA), cence 0.1 (5×, objectives freely illumination the two by on generated were mounted Lightsheets lightsheet. and Z.1 the of glue stage rotating super using custom-made holder a on plastic fixed was sample the Briefly, microscope. imaging. before gels the to encase to solidify allowed and gel the around pipetted excess all expansion, with plates (2% removed. low-melt agarose w/w) was If liquid needed, following 6-well glass-bottom imaging in placed during were gels drift against gels stabilize To the stability. hybridization for salt enough giving still concentration; we found that 0.05× SSC gives 3× expansion, while doi: Lightsheet imaging was performed on a Zeiss Z.1 lightsheet lightsheet Z.1 Zeiss a on performed was imaging Lightsheet 10.1038/nmeth.3899 https://bitbucket Z (three optical sections) was used to to used was sections) optical (three e . A sliding window averaging (or (or averaging window sliding A .org/arjunrajlaborat ory/ - -

fication, aliquots of ataliquots amplifiers fication, these 3 ampli HCR To initiate displacement. strand for toe-hold bp 6 a bears B2H1T where sequence), for below (see B2H2 and B2H1T amplifiers, HCR of pair a Wedesigned hairpins. amplifier able upon commences the of amplification addition two HCR metast HCR reversal via toe-hold-mediated strand displacement. the in the in radius pixel 3 a within were centroids their if colocalized be to determined were spots 39. 38. 37. below for sequence) at 200 nM in 5× SSCT. 5× in nM at 200 sequence) for below reversal was initiated by the addition of a displacement strand (see SSC, 0.1% Tween 20) twice with 1 h per wash. Subsequently, HCR perature. After amplification, gels were washed with 5× SSCT (5× pins diluted to 60 nM in buffer amplification for 3 h at room tem hair HCR with incubated then were samples Gelled min. 30 for to 95 °C for at to heating 90 s cool temperature room and leaving CAAATAG g GA g ATCCA AT A Spot centroids were extracted from both color channels, and and channels, color both from extracted were centroids Spot B2H1T: Displacement strand: Displacement /5 B2H2: gg TTTTAGTTAGGA TA T TGATGAGGAT TGATGAT T CTAT

c registrationintensity.onbased analysis. usingmultiple singly labeled probes. Thévenaz, P., Ruttimann, U.E. & Unser, M. A pyramid approach to subpixelpyramidapproachto Unser,A Thévenaz,P.,&M. Ruttimann,U.E. J. Schindelin, Tyagi,Detection& S.Raj,A. individual of endogenous transcriptsRNA g ′ gg - TGAG CTCA C A z dimension. gg lexa g ACT TAAACC Nat. Methods Nat. TTTACT c g g T 4-1/ CCTC 546-C12/ TCCATCCTC et al. et A g CT Fiji: an open-source platform for biological-imagefor platform open-source an Fiji: g

gg CC 9 , 676–682 (2012). 676–682 , g AT AT IEEE Trans.Process.IEEEImage g x , g g ATT y dimensions and a 2 pixel radius radius pixel 2 a and dimensions ATT MethodsEnzymol. TAAATCCTCATCAATCAT g g TAAATCCTCATCAATC g AT AT µ g M were snap-cooled M by were snap-cooled g A A gg gg

4 7 2 ATTTAC n c ATTTAC

, 365–386, (2010). a 7 GAGGATG ture methods , 27–41 (1998).27–41 , HCR g g in situin A A g g - - - -