Embed Size (px)
Citation preview
ChemicalScience
EDGE ARTICLE
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article OnlineView Journal
DNA-barcoded la
aWyss Institute for Biologically Inspired E
Massachusetts, USA. E-mail: [email protected] of Systems Biology, Harvard MecNew Chemistry Unit and Chemistry & Phy
Centre for Advanced Scientic Research (JNCdProgram of Biological and Biomedical Sc
Massachusetts, USAeDepartment of Physics and Center for Nan
80539 Munich, GermanyfMax Planck Institute of Biochemistry, 8215
† Electronic supplementary informa10.1039/c6sc05420j
‡ These authors contributed equally to th
Cite this: DOI: 10.1039/c6sc05420j
Received 11th December 2016Accepted 28th January 2017
DOI: 10.1039/c6sc05420j
rsc.li/chemical-science
This journal is © The Royal Society of
beling probes for highlymultiplexed Exchange-PAINT imaging†
Sarit S. Agasti,‡abc Yu Wang,‡abd Florian Schueder,abef Aishwarya Sukumar,a
Ralf Jungmann*abef and Peng Yin*ab
Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical
diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge
for traditional super-resolution methods is their limited multiplexing capability, which prevents
a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently
developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by
sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach
holds great promise for the bioimaging community, its widespread application has been hampered by
the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for
the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using
a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule
binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to
over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized
conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed
cells.
Introduction
Fluorescence microscopy has become a standard method for insitu characterization of molecular details in both biological andclinical samples. Compared to complementary characterizationmethods such as electron microscopy,1 uorescence imagingallows the efficient and specic detection of targets like proteinsor nucleic acids using affinity labeling reagents such as anti-bodies.2 However, the spatial resolution of conventional uo-rescence microscopy is limited, by the diffraction limit of light,to �200 nm. Large efforts have been devoted to overcome thislimitation, resulting in a number of so-called super-resolutionmethods that can nowadays readily achieve sub-20 nm resolu-tion in cells.3 Most super-resolution microscopy techniques,
ngineering, Harvard University, Boston,
rd.edu; [email protected]
dical School, Boston, Massachusetts, USA
sics of Materials Unit, Jawaharlal Nehru
ASR), Bangalore, India
ience, Harvard Medical School, Boston,
oscience, Ludwig Maximilian University,
2 Martinsried near Munich, Germany
tion (ESI) available. See DOI:
is work.
Chemistry 2017
such as Structured Illumination Microscopy (SIM),4 StimulatedEmission Depletion (STED) microscopy,5 (uorescent) Photo-Activated Localization Microscopy ((f)PALM)6,7 and (direct)Stochastic Optical Reconstruction Microscopy ((d)STORM),8,9 tothis date rely on target labeling using static or xed uorescenttags. This labeling is usually achieved via either geneticallyencoded fusion proteins (PALM) or immunolabeling using dye-conjugated antibodies (STED, STORM). While these super-resolution approaches have already enabled new biologicalndings, some limitations persist. Two of the major limitationsof single-molecule localization-based techniques such as PALMor STORM are the hard-to-control photophysical properties ofuorophores and the limited photon budget of xed targetlabels.
A different approach to create “blinking” target molecules isimplemented in the so-called Points Accumulation in NanoscaleTopography (PAINT) technique.10 In this technique, uorescentlylabeled ligands freely diffuse in solution and bind either staticallyor transiently to targets of interest.10,11 This binding is detectedas an apparent “blinking” of the target molecule or structure ofinterest. This enables the decoupling of blinking from the photo-physical dye switching properties and thus alleviates one issueof STORM or PALM. However, the binding of diffusing ligandsto their targets is achieved by electrostatic or hydrophobic in-teractions and is thus hard to program for different target speciesin a single cell, thus preventing easy-to-implement multiple-xed detection. DNA-PAINT,12–17 a variation of PAINT, achieves
Chem. Sci.
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
stochastic switching of uorescence signals between the ON- andOFF-states by the repetitive, transient binding of short uo-rescently labeled oligonucleotides (“imager” strands) to comple-mentary “docking” strands that are conjugated to targets (Fig. 1a).Upon binding of an imager strand, its uorescence emission isdetected and subsequently localized with nanometer precision.Importantly, the transient binding properties of these short DNAstrands enable the facile removal of imager strands. Hence,orthogonal imager strands can be used to sequentially visualizemultiple targets of interest. This so-called Exchange-PAINT15
approach in principle enables the spectrally-unlimitedmultiplexedsuper-resolution imaging of potentially hundreds of target mole-cules in the same sample, in a simpler and more straightforwardfashion than other multiplexing approaches,18–22 such as thosebased on sequential immunostaining, imaging, and dye bleachingor inactivation.
The original Exchange-PAINT study demonstrated sequen-tial 4-color imaging of cellular protein targets labeled withDNA-modied antibodies using different imager strands conju-gated with a single-color dye. While successful, this labelingapproach was based on biotinylated primary antibodies incombination with streptavidin and biotinylated docking strandsto form an ‘antibody-streptavidin-DNA’ sandwich. This labelingprocedure leads to two disadvantages; on one hand, the ‘linkage-error’, that is, the distance between the true target and labeledDNA docking site, is increased due to the addition of streptavi-din, which ultimately leads to a localization offset from the truetarget position.23 On the other hand, the large sizes of thesecomplexes leads to steric hindrance in the labeling process,which impedes the achievable labeling density and efficiency.Both of these effects can reduce the achievable spatial resolution.
Here, we introduce a general framework for labelingprotein targets using DNA-PAINT docking strands, which aredirectly coupled to various labeling probes, thus addressingthe aforementioned issues. First, we design and evaluate theperformance and orthogonality of 52 DNA sequences forExchange-PAINT. Next, we directly conjugate DNA oligonu-cleotides to antibodies, avoiding the biotin–streptavidinsandwich, and then extend the platform to small-sizedbinders, including nanobodies and small molecules, tofurther enhance the achievable labeling density and spatialaccuracy. Finally, we successfully use our labeling platformto demonstrate nine-target super-resolution imaging in xedbiological samples.
Results and discussionDesign of >50 orthogonal imager strands and DNA origamicrosstalk assay
To extend the multiplexing capabilities of Exchange-PAINT, wedesigned 37 sequences in addition to the previously published15 strands,15 to theoretically enable 52-plex super-resolutionimaging. We started with strand design using the “CANADA”soware,24 employing the following conditions: the length ofthe docking site is 9 base pairs (bp), the GC-content is 40%(3 out of these 9), and there should be no sequence homologywith more than 3 bases. To ensure the experimental
Chem. Sci.
orthogonality of the designed sequences and to test theirperformances in DNA-PAINT (e.g. achievable resolution), weconducted a series of 52 in vitro experiments (Fig. 1). Wedesigned 52 unique barcoded DNA origami structures. Fig. 1bshows an example of one of these “barcodes”. In the schematicrepresentation of the structure (Fig. 1b, right), each hexagonrepresents a potential DNA-PAINT docking site. The le-handside of the origami structure features a 6-bit barcode, which isunique for each of the 52 origami structures. The barcodestaple strands are universally extended with the DNA-PAINTsequence P1 for all structures. The right-hand side of eachorigami structure carries a geometric pattern, created withunique docking strand sequences for each of the 52 barcodedorigami structures, i.e. docking strands Pi, with i ˛ [2, 52]. Thecrosstalk experiment was then conducted as follows. Weprepared 52 samples, all of which contained 52 barcoded DNAorigami structures and imager strand species P1 in solution.Furthermore, each unique sample additionally contained oneorthogonal DNA-PAINT imager sequence out of the remaining52 imagers in the sequence pool (i.e. either P2, P3, P4, and soon). As an example, the result for the experiment with theimager sequence P40 is depicted in Fig. 1c. If there is nocrosstalk, the motive on the right side of the structure is onlyvisible for one origami species with the 6-bit barcode forsequence P40. For the remaining structure, only the respective6-bit barcode is visible, imaged with P1. A larger view image isshown in Fig. 1d, underlining the fact that there is indeed nocrosstalk between the imager strand P40 and all remainingsequences. This experiment was repeated 51 times for theremaining sequences and resulted in no detectable crosstalkof all 52 imager strands (see ESI Table 1† for the DNA origamisequences, ESI Table 2† for the imager sequences, ESI Fig. 1†for a schematic overview of all 52 barcoded DNA origamistructures, and ESI Fig. 2–52† for the respective crosstalkimaging rounds).
Synthesis of direct DNA-antibody conjugates for DNA-PAINTimaging
To translate this large multiplexing capability in situ, we nextdescribe the synthesis of barcoded DNA-antibody conjugates forDNA-PAINT imaging. Antibodies (Abs) are the most widely usedlabeling probes. High specicity and affinity for target antigenscoupled with the large repertoire of commercially availableantibodies make them an integral component in life scienceresearch.
They are routinely used in diverse immunoassay applica-tions, including immunouorescence (IF) imaging and immu-nohistochemistry (IHC). These attributes prompted us to rstadopt the antibody-labeling platform for DNA-PAINT imagingand develop a general DNA conjugation approach that buildsupon the vast array of available antibodies to complement thehigh multiplexing capability of Exchange-PAINT.
There are a few criteria to be considered for selecting antibodyconjugation methods for DNA-PAINT. Firstly, the conjugationchemistry should be versatile such that it is applicable tovarious antibody isotypes. Secondly, the method should work for
This journal is © The Royal Society of Chemistry 2017
Fig. 1 Crosstalk experiment to check the orthogonality of 52 docking sequences. (a) DNA origami carries single-stranded extensions (dockingstrands), which can transiently bind fluorescently labeled oligonucleotides (imagers) in solution. (b) Rectangular origami with modified extendedstaples (left side); a schematic representation of the structure is located on the right side; each hexagon represents a staple position that can beextended for DNA-PAINT imaging. Each origami contains a unique 6-bit barcode, addressable with the sequence P1 (left side), and single-stranded extensions that will act as docking sites for the imagers to be tested (P2–P52). Together, these extensions form a mirrored “F” shape(right side). (c) Crosstalk check for sequence P40. The upper row shows schematic representations of the barcode structures for each sequence.The bottom row shows the experimental data. The mirrored “F” appears only next to the barcode for the P40 sequence. This showsthe orthogonality of the P40 sequence to all other sequences. (d) Overview image of the crosstalk experiment for P40. Scale bars: 50 nm(c), 200 nm (d).
Edge Article Chemical Science
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
commercially available antibodies. Hence, conjugation techniquesinvolving technically involved genetic engineering of antibodies,such as unnatural amino acid incorporation, are not favored.Lastly, the method should be simple, economical, high yield andeasily accessible to researchers. Based on these criteria, we chose toconjugate thiol-modied DNA oligonucleotides to lysine residueson antibodies via SM(PEG)2 (PEGylated succinimidyl 4-(N-mal-eimidomethyl) cyclohexane-1-carboxylate) crosslinkers (Fig. 2a),and optimized the protocol for DNA-PAINT imaging. In thisstrategy, the small ‘footprint’ of SM(PEG)2 ensuresminimum sterichindrance for antigen binding while placing the DNA label in closeproximity to the antibody in order to achieve high-resolution. Inaddition, the use of the PEG spacer helps to reduce nonspecic
This journal is © The Royal Society of Chemistry 2017
binding.25 For conjugation, a phosphate-buffered solution ofantibody was rst incubated with SM(PEG)2 crosslinkers. In thisstep, the N-hydroxysuccinimide (NHS) ester group of SM(PEG)2reacts with the amine groups present on the lysine residues andanchors the maleimide group on the antibody surface. Aerremoving the excess cross-linker using size-exclusion chromatog-raphy, maleimide-functionalized antibodies were reacted withthiol-modied DNA oligonucleotides to form stable DNA-antibodyconjugates. The antibody conjugates were puried using a molec-ular weight cut-off lter (100 kDa). The successful conjugation ofDNA strands to antibodies was veried using matrix-assistedlaser desorption/ionization-mass spectrometry (MALDI-MS). Wehave optimized the protocols to yield conjugates with close to
Chem. Sci.
Fig. 2 Antibody-DNA conjugation method and super-resolution imaging with a DNA-conjugated secondary antibody. (a) Synthesis scheme forDNA-conjugated antibody preparation. Note that SM(PEG)2 is depicted here as NHS-EG2-Mal. (b) Labeling strategy for the DNA-conjugatedsecondary antibody. (c) Secondary antibody-based DNA-PAINT super-resolution imaging of microtubules inside a fixed BSC-1 cell. Zoomingin of the highlighted area shows the resolution improvement compared to the diffraction-limited micrographs of the same area. Thecross-sectional histogram of a hollow microtubule structure clearly shows two distinct lines with a separation of �40 nm. This is in goodagreement with earlier reports.26
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
1 DNA label/Ab (ESI Fig. 53† and the corresponding calculationbased on the MALDI mass shi). Limiting the number ofconjugated DNA oligonucleotides per antibody has twoadvantages: rst, it helps to decrease nonspecic binding,which is potentially mediated by interactions of conjugatedDNA with other cellular compartments; secondly it reduces theprobability that lysine residues in the antigen recognition sitesare labeled with DNA, which could otherwise decrease theantigen binding affinity. It should be noted that even thoughonly about one DNA oligonucleotide is conjugated per Ab on
Chem. Sci.
average, a close to 100% label readout could still be achieved byDNA-PAINT, as imager strands are continuously targeting thedocking strand on the antibody, leading to high imaging effi-ciency. This is in contrast to traditional imaging methods,which use uorophore-conjugated antibodies, where photo-bleaching of a low density of uorophores can lead to a loss oftarget visualization due to insufficient sampling.
A detailed step-by-step description of the preparation ofDNA-antibody conjugates and subsequent characterization canbe found in ESI Protocols 1 and 2.†
This journal is © The Royal Society of Chemistry 2017
Fig. 3 DNA-PAINT imaging with a DNA-conjugated primary antibody. (a) Labeling schemewith a DNA-conjugated primary antibody. (b) Primaryantibody-based DNA-PAINT imaging of microtubules inside a fixed BSC-1 cell. (c) Primary antibody-based DNA-PAINT imaging of Tom20 inmitochondria. Tom20 localizes to the mitochondrial membrane, which is clearly resolved. Scale bars: 5 mm.
Edge Article Chemical Science
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
DNA-PAINT imaging with DNA-conjugated secondaryantibodies
To test the super-resolution imaging capabilities of the directlyconjugated antibody probes, we rst used a DNA-conjugatedsecondary antibody and performed single-color DNA-PAINTimaging of the microtubule network in BSC-1 cells. Among thevarious brous cytoskeleton protein networks, microtubuleswere selected as a model system for evaluation of the imagingperformance due to their well-dened structure, shape andimportantly their nanoscale, subdiffraction dimensions (diam-eter �25 nm).23 To perform DNA-PAINT imaging, at rst wexed the microtubule network in the BSC-1 cells using meth-anol and stained it with primary antibodies against alpha-tubulin followed by the DNA-conjugated secondary antibody(Fig. 2b). Next, DNA-PAINT imaging was performed usingATTO655-conjugated imager strands and using highly inclinedand laminated optical sheet (HILO) illumination. Aerwards,a super-resolved DNA-PAINT image was reconstructed usinga custom spot-nding and 2D-Gaussian tting algorithm. Inaddition, ducial-based dri correction was performed usinggold nanoparticles to compensate for any sample movementduring image acquisition.
As shown in Fig. 2c, the resulting DNA-PAINT image showsa signicant resolution increase compared to the diffraction-limited representation. The increased resolution could be easilyobserved by visualizing a dense region of the microtubulenetwork where individual microtubule laments could be
This journal is © The Royal Society of Chemistry 2017
clearly distinguished, which are impossible to distinguish inthe standard diffraction-limited micrograph. More importantly,when a single microtubule ber was magnied, DNA-PAINT wasable to resolve the hollow tubular structure.26 This underlinesa substantial improvement of the labeling density and size overpreviously published DNA-PAINT cell data,15 where biotin–streptavidin-mediated DNA conjugated antibodies failed toresolve this hollow tubular structure. To semi-quantitativelyassess the achievable resolution, we measured the cross-sectional prole of localizations of a “hollow” microtubulestructure. As depicted in Fig. 2c, the cross-sectional prolesshow two well-resolved peaks with a separation of �40 nmbetween them, which is in good agreement with the previousreports.27 We also tested our direct DNA-conjugated antibodiesfor dual-color super-resolution imaging (ESI Fig. 54†). Here, weco-stained Tom20, a mitochondrial outer membrane protein,and HSP60, a mitochondrial matrix protein in xed HeLa cells.The image was taken using ATTO655- and Cy3B-conjugatedimager strands for Tom20 and HSP60, respectively. This dual-color DNA-PAINT image shows Tom20 localizing on the outermitochondrial membrane, while HSP60 localizes on the insideof the mitochondria.
DNA-PAINT imaging with DNA-conjugated primary antibodies
Although secondary antibodies are widely used as indirect im-munostaining approaches, they are not the ideal choice forhighly multiplexed super-resolution imaging for two primary
Chem. Sci.
Fig. 4 Synthesis of DNA-conjugated nanobodies for DNA-PAINT imaging. (a) Synthesis scheme for DNA-conjugated nanobody preparation.(b) Labeling scheme using the DNA-conjugated nanobody. (c, d) Nanobody-based DNA-PAINT super-resolution imaging of the mitochondrialnetwork inside a fixed HeLa cell. A comparison of the diffraction-limited image (c) to the DNA-PAINT image (d) underlines the achievedresolution increase. Scale bars: 5 mm.
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
reasons, one of which is the limited availability of primaryantibodies from different species. Additionally, due to theincreased size of the primary-secondary antibody sandwich, theresulting larger ‘linkage-error’ could lead to lower spatialaccuracy. Therefore, we next turned to more direct immuno-staining approaches, involving only primary antibodies.
To test primary antibody-based DNA-PAINT imaging,we used two model systems: microtubules and mitochondria.The microtubule network was stained with DNA-conjugatedprimary antibodies against alpha-tubulin, whereas the mito-chondria were stained for Tom20 (Fig. 3a). Fig. 3b and c showthe resulting DNA-PAINT images of directly-labeled microtu-bules and mitochondria structures, respectively. As canbe seen in Fig. 3b, individual microtubules are clearly visible
Chem. Sci.
in the super-resolved image, similar to the image obtainedusing secondary antibody-based staining. On the otherhand, as shown in Fig. 3c, the DNA-PAINT imaging revealed theouter mitochondrial membrane localization of the Tom20protein.
DNA-PAINT imaging with DNA-conjugated nanobodies
IgG antibodies, typically used in immunouorescence studies,are �150 kDa in MW and �10 nm in size. Although the largecommercially available repertoire of antibodies is advantageousfor their use in highly multiplexed imaging, their rather largesizes are ultimately a concern when highly accurate localizationof the target is necessary or when high density labeling23,28 isrequired for molecular counting. To address this issue, we used
This journal is © The Royal Society of Chemistry 2017
Fig. 5 Conjugation of DNA oligos to phalloidin for actin imaging with DNA-PAINT. (a) Synthesis scheme for DNA-conjugated phalloidin.(b) Labeling strategy for phalloidin using the DNA-phalloidin conjugate. (c) Resulting DNA-PAINT image of the actin network inside a fixed HeLacell. Zooming in to the highlighted area (green) highlights the achievable resolution. A Gaussian distribution was fitted to the cross-sectionalhistogram of an actin fiber (selected from the highlighted red region). FWHM of the distribution: �12 nm.
Edge Article Chemical Science
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
antibody-like affinity molecules with smaller sizes, includingnanobodies and high affinity small molecule binders. Nano-bodies are derived from heavy chain-only antibodies generatedby camelids.29 They are small in size (�1.5 nm � 2.5 nm), only
This journal is © The Royal Society of Chemistry 2017
�13 kDa in MW and have high affinity for their target mole-cule.23,28 Previous reports have demonstrated the enhancedresolving power of nanobodies for super-resolution imaging ofmicrotubules.23,30
Chem. Sci.
Fig. 6 Secondary antibody-based labeling for multiplexing with Exchange-PAINT. (a) Schematic representation of Exchange-PAINT. Targetproteins (T1/T8) are labeled with DNA (D1/D8)-conjugated secondary antibodies using an indirect immunostaining approach. ComplementaryATTO655-dye-labeled DNA strands (I1/I8) are sequentially applied to the sample. Post-acquisition, a washing buffer with reduced ionic strengthwas used to efficiently remove the imagers. Eight imaging rounds were performed using orthogonal imager strands with the same dye. (b) Eight-target DNA-PAINT image of fixed HeLa cells acquired in eight sequential rounds. Scale bars: 5 mm.
Chem. Sci. This journal is © The Royal Society of Chemistry 2017
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
Fig. 7 Primary antibody-based labeling for multiplexing with Exchange-PAINT. (a) Labeling strategy for primary antibody-based imaging. Thetarget proteins (T1/Tn) were labeled with DNA (D1/Dn)-conjugated primary antibodies using a direct immunostaining approach. Comple-mentary imager strands (labeled with ATTO655) were sequentially introduced to the sample for super-resolution imaging as before.Post-acquisition, a washing buffer with reduced ionic strengthwas introduced to remove all imagers. Nine imaging roundswere performed usingorthogonal imager strands conjugated to the same dye. (b) Nine-target super-resolution image of proteins in fixed HeLa cells acquired using ninerounds of Exchange-PAINT.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.
Edge Article Chemical Science
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
We began with optimizing the conjugation chemistry forDNA-labeled nanobodies. We used a cycloaddition reactionbetween 1,2,4,5-tetrazine (Tz) and trans-cyclooctene (TCO) tocouple DNA-PAINT docking strands to a model anti-GFPnanobody (Fig. 4a). The strain-promoted [4+2] cycloadditionreaction between Tz and TCO is fast with a rate constant of up to106 (Ms)�1, quantitative and can proceed in physiologicalconditions, which helps to rapidly and efficiently conjugateDNA while preserving the functionality of the nanobodies.31 Inbrief, a TCO-NHS ester was used to react with the primary aminegroups of lysine residues on nanobodies in PBS (pH ¼ 8) for3 hours. Simultaneously, amine-modied DNA-PAINT dockingstrands were reacted with Tz and subsequently puried usingHPLC. The TCO-modied nanobodies were then coupled withthe Tz-modied DNA-PAINT docking strands during a reactionin PBS (pH ¼ 7.4) for 3 hours. As for the case of antibodyconjugation discussed above, we have optimized the protocolsto yield conjugates with close to 1 DNA label/nanobody (see ESIFig. 55† and the corresponding calculation based on the MALDImass shi).
Next, we tested the performance of our DNA-conjugatednanobodies for DNA-PAINT super-resolution imaging in HeLacells expressing the mitochondria-green uorescent protein(GFP). HeLa cells were transfected with a baculoviral vectorcontaining mitochondrial leader sequence-fused GFP (Bac-Mam2.0),32 and the expression of GFP was detected aer 2 daysof transfection (Fig. 4b and c). The transfected cells were stainedwith DNA-conjugated anti-GFP nanobodies aer PFA xation.The DNA-PAINT image (Fig. 4d) shows a specic signal anda clear resolution increase when resolving the mitochondrialstructures. The shape of the mitochondria as detected withDNA-PAINT (Fig. 4d) correlated well with their correspondingGFP signals detected using conventional uorescence micros-copy (Fig. 4c). We note that some mitochondria in Fig. 4c didnot show up in Fig. 4d. These “missing” mitochondria wereactually out-of-focus when imaged in HILO mode, and hencedid not generate enough localization events for super-resolu-tion image reconstruction.
A step-by-step protocol for nanobody-DNA conjugation isdescribed in ESI Protocol 3.†
DNA-PAINT imaging with small molecule binders
Small molecule binders represent another important class oftargeting reagents for high-density protein labeling. To test thecompatibility of DNA-PAINT imaging with small moleculeprobes, we selected phalloidin, a bicyclic heptapeptide, toselectively target F-actin.33 Actin laments are usually present inhigh density in cells with individual ber diameters as small as5–10 nm.34 Imaging of the actin cytoskeleton structure usingDNA-conjugated phalloidin probes will not only allow investi-gation of the compatibility of small molecule probes withDNA-PAINT, but also demonstrate the benet of employinga smaller targeting agent to resolve high density sub-10 nmstructures using DNA-based imaging.
To create DNA-conjugated phalloidin probes, we used the Tzand TCO-based conjugation method (Fig. 5a), similar to the
Chem. Sci.
method described for the nanobodies. Here, a TCO-NHS esterwas rst reacted with amine-modied phalloidin molecules toform a phalloidin-TCO conjugate. Aer HPLC purication, theTCO-phalloidin conjugates were incubated with Tz-modiedDNA-PAINT docking strands to form DNA-phalloidin conju-gates, whose identity was then veried using MALDI-MS spec-troscopy (see ESI Fig. 56†).
We tested the performance of our DNA-phalloidin probes inHeLa cells. To preserve the cytoskeleton ultrastructure, we xedHeLa cells using 0.1% glutaraldehyde along with 3% PFA.Staining of the actin laments was achieved by incubating cellswith 1 mM of DNA-phalloidin probes (Fig. 5b). Aer removingexcess probes, DNA-PAINT imaging was performed usingATTO655-labeled imager strands. Fig. 5c shows the super-resolved DNA-PAINT image of actin cytoskeletons, where theindividual actin laments are well resolved and clearly visible.For a more “quantitative” determination of the imaging reso-lution, we measured the cross-section of a single lament(Fig. 5c), yielding an apparent lament width of �12 nm(FWHM), a dimension which is consistent with earlier reports.35
A step-by-step protocol for Phalloidin-DNA conjugation isdescribed in ESI Protocol 4.†
Highly multiplexed Exchange-PAINT imaging using a pool oforthogonally labeled antibodies
Protein interaction networks mediate cellular responses tovarious environmental stimuli. It is increasingly evident thatthe spatial heterogeneity of protein distribution in cells leads tointracellular functionality differences among distinct compart-ments and intercellular variance among cells located indifferent regions. Mapping the heterogeneity of proteinnetworks is challenging for three reasons: (1) the locationinformation of proteins needs to be well preserved; (2)comprehensive studies probing multiple protein targets need tobe performed in order to understand the whole network; (3)high spatial accuracy is required to achieve subcellularmapping, rendering conventional diffraction-limited uores-cence imaging unsuitable.
The development of Exchange-PAINT imaging enableshighly multiplexed super-resolution detection in single cellsand is hence desirable for protein network mapping directlyin situ. By synergistically combining optimized DNA probedesign and improved DNA-antibody conjugation, we here reportthe thus far unprecedented nine-target super-resolutionimaging in biological samples.
Given that indirect immunostaining approaches are mostwidely used and present a cost-effective method for labelingprotein targets, we rst tested the multiplexed super-resolutionimaging using DNA-conjugated secondary antibodies. Here, westained xed HeLa cells with phalloidin and primary antibodiesfrom seven different species followed by DNA-conjugatedsecondary antibodies from the donkey species. Seven rounds ofprobe exchange were performed to image all eight targets. Theresults showed that eight cellular structures could be clearlyvisualized with Exchange-PAINT, and there was minimal-to-nocrosstalk of signals among the different antibodies (Fig. 6). It
This journal is © The Royal Society of Chemistry 2017
Edge Article Chemical Science
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
can be seen that paxillin localized at the tip of the actin la-ments, which is consistent with the fact that paxillin is an actinregulation protein in focal adhesions.36 The nuclear porecomplex signal was present specically in the nucleus whichwas indicated by DAPI staining of the nucleus.
The use of secondary antibodies for multiplexed detection,however, is limited by the availability of primary antibodies fromdifferent species. Therefore, we next used directly DNA-labeledprimary antibodies and small molecule binders, and achievednine-target super-resolution visualization (Fig. 7). Nuclearprotein Ki67 signals were mostly located in the nucleus whileLamin and Nuclear Pore Complex (NPC) marked the nuclearmembrane. Clathrin signals indicated the distribution of coated-vesicles in the cytoplasm. We note that the super-resolutionsignal in the reconstructed images obtained using primary anti-bodies was lower compared to the signal obtained by indirectlabeling using secondary antibodies, which is expected due to thelack of signal amplication in the primary antibody only case. Weanticipate that this fact can be improved by increasing theimaging time to obtain more localization events. This, again, isunique to Exchange-PAINT, due to its resistance to photo-bleaching and replenishable imaging probes.
Detailed information regarding the primary and secondaryantibodies and imager sequences can be found in ESI Tables 3–7.†The immunostaining protocols with PFA, PFA and glutaralde-hyde, and methanol are detailed in ESI Protocols 5–7.†
Conclusion
In summary, we have developed a versatile labeling platform forthe conjugation of DNA oligonucleotides to various labelingprobes for DNA-PAINT and Exchange-PAINT with high labelingdensity, spatial accuracy and achievable resolution. We alsodemonstrated the use of our labeled probes for highly multi-plexed imaging in biological samples with nanoscale resolu-tion. The conjugation method is efficient and simple toimplement, and should be easily adopted in common biologicallabs. We anticipate that the conjugation methods developed herecan make Exchange-PAINT accessible to a broader scienticcommunity, and will consequently be used to solve more complexbiological questions.
Acknowledgements
This work was supported by grants from the NIH (1R01EB018659-01 and 1-U01-MH106011-01) NSF (CCF-1317291), and ONR(N00014-13-1-0593, N00014-14-1-0610, and N00014-16-1-2182) toP. Y.. S. S. A. acknowledges the support from theWellcome Trust-DBT India Alliance Intermediate Fellowship (IA/I/16/1/502368).R. J. acknowledges the support from the DFG through an EmmyNoether Fellowship (DFG JU 2957/1-1), the ERC through an ERCStarting Grant (MolMap, Grant agreement number 680241) andthe Max Planck Society. F. S. acknowledges the support from theDFG through the SFB 1032 (Nanoagents for the spatiotemporalcontrol of molecular and cellular reactions). Competing nancialinterest: The authors have led a patent application. P. Y. and
This journal is © The Royal Society of Chemistry 2017
R. J. are the co-founders of Ultivue, Inc., a start-up company withinterests in commercializing the reported technology.
References
1 M. Knoll and E. Ruska, Das elektronenmikroskop, Z. Phys.,1932, 78, 318–339.
2 A. H. Coons, H. J. Creech and R. N. Jones, Immunologicalproperties of an antibody containing a uorescent group,Exp. Biol. Med., 1941, 47, 200–202.
3 S. W. Hell, et al., The 2015 super-resolution microscopyroadmap, J. Phys. D: Appl. Phys., 2015, 48, 443001.
4 M. G. Gustafsson, Surpassing the lateral resolution limit bya factor of two using structured illumination microscopy,J. Microsc., 2000, 198, 82–87.
5 S. W. Hell and J. Wichmann, Breaking the diffractionresolution limit by stimulated emission: stimulated-emission-depletion uorescence microscopy, Opt. Lett.,1994, 19, 780–782.
6 S. T. Hess, T. P. K. Girirajan and M. D. Mason, Ultra-highresolution imaging by uorescence photoactivationlocalization microscopy, Biophys. J., 2006, 91, 4258–4272.
7 E. Betzig, et al., Imaging intracellular uorescent proteins atnanometer resolution, Science, 2006, 313, 1642–1645.
8 M. Heilemann, et al., Subdiffraction-resolution uorescenceimaging with conventional uorescent probes, Angew.Chem., Int. Ed., 2008, 47, 6172–6176.
9 M. J. Rust, M. Bates and X. Zhuang, Sub-diffraction-limitimaging by stochastic optical reconstruction microscopy(STORM), Nat. Methods, 2006, 3, 793–795.
10 A. Sharonov and R. M. Hochstrasser, Wide-eldsubdiffraction imaging by accumulated binding ofdiffusing probes, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,18911–18916.
11 G. Giannone, et al., Dynamic superresolution imaging ofendogenous proteins on living cells at ultra-high density,Biophys. J., 2010, 99, 1303–1310.
12 R. Jungmann, et al., Single-molecule kinetics and super-resolution microscopy by uorescence imaging oftransient binding on DNA origami, Nano Lett., 2010, 10,4756–4761.
13 C. Lin, et al., Submicrometre geometrically encodeduorescent barcodes self-assembled from DNA, Nat. Chem.,2012, 4, 832–839.
14 R. Iinuma, et al., Polyhedra self-assembled fromDNA tripodsand characterized with 3D DNA-PAINT, Science, 2014, 344,65–69.
15 R. Jungmann, et al., Multiplexed 3D cellular super-resolutionimaging with DNA-PAINT and Exchange-PAINT, Nat.Methods, 2014, 11, 313–318.
16 R. Jungmann, et al., Quantitative super-resolution imagingwith qPAINT, Nat. Methods, 2016, 13, 439–442.
17 M. Dai, R. Jungmann and P. Yin, Optical imaging ofindividual biomolecules in densely packed clusters, Nat.Nanotechnol., 2016, 11, 798–807.
18 D. Y. Duose, R. M. Schweller, W. N. Hittelman andM. R. Diehl, Multiplexed and reiterative uorescence
Chem. Sci.
Chemical Science Edge Article
Ope
n A
cces
s A
rtic
le. P
ublis
hed
on 3
0 Ja
nuar
y 20
17. D
ownl
oade
d on
28/
02/2
017
15:4
3:10
. T
his
artic
le is
lice
nsed
und
er a
Cre
ativ
e C
omm
ons
Attr
ibut
ion
3.0
Unp
orte
d L
icen
ce.
View Article Online
labeling via DNA circuitry, Bioconjugate Chem., 2010, 21,2327–2331.
19 R. M. Schweller, et al., Multiplexed in situimmunouorescence using dynamic DNA complexes,Angew. Chem., Int. Ed., 2012, 51, 9292–9296.
20 J. Tam, G. A. Cordier, J. S. Borbely, A. Sandoval Alvarez andM. Lakadamyali, Cross-talk-free multi-color STORM imagingusing a single uorophore, PLoS One, 2014, 9, e101772.
21 C. C. Valley, S. Liu, D. S. Lidke and K. A. Lidke, Sequentialsuperresolution imaging of multiple targets using a singleuorophore, PLoS One, 2015, 10, e0123941.
22 J. Yi, et al., madSTORM: a superresolution technique forlarge-scale multiplexing at single-molecule accuracy, Mol.Biol. Cell, 2016, 27, 3591–3600.
23 J. Ries, C. Kaplan, E. Platonova, H. Eghlidi and H. Ewers, Asimple, versatile method for GFP-based super-resolutionmicroscopy via nanobodies, Nat. Methods, 2012, 9, 582–584.
24 U. Feldkamp, CANADA: designing nucleic acid sequences fornanobiotechnology applications, J. Comput. Chem., 2010, 31,660–663.
25 Q. He, et al., The effect of PEGylationof mesoporous silicananoparticles on nonspecic binding of serum proteinsand cellular responses, Biomaterials, 2010, 31, 1085–1092.
26 G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates andX. Zhuang, Evaluation of uorophores for optimalperformance in localization-based super-resolutionimaging, Nat. Methods, 2011, 8, 1027–1036.
27 M. Bates, B. Huang, G. T. Dempsey and X. Zhuang,Multicolor super-resolution imaging with photo-switchableuorescent probes, Science, 2007, 317, 1749–1753.
Chem. Sci.
28 U. Rothbauer, et al., Targeting and tracing antigens in livecells with uorescent nanobodies, Nat. Methods, 2006, 3,887–889.
29 S. Muyldermans, et al., Camelid immunoglobulins andnanobody technology, Vet. Immunol. Immunopathol., 2009,128, 178–183.
30 M. Mikhaylova, et al., Resolving bundled microtubules usinganti-tubulin nanobodies, Nat. Commun., 2015, 6, 7933.
31 J. B. Haun, N. K. Devaraj, S. A. Hilderbrand, H. Lee andR. Weissleder, Bioorthogonal chemistry ampliesnanoparticle binding and enhances the sensitivity of celldetection, Nat. Nanotechnol., 2010, 5, 660–665.
32 T. A. Kost, J. P. Condreay, R. S. Ames, S. Rees andM. A. Romanos, Implementation of BacMam virus genedelivery technology in a drug discovery setting, DrugDiscovery Today, 2007, 12, 396–403.
33 E. Wulf, A. Deboben, F. A. Bautz, H. Faulstich andT. Wieland, Fluorescent phallotoxin, a tool for thevisualization of cellular actin, Proc. Natl. Acad. Sci. U. S. A.,1979, 76, 4498–4502.
34 E. Grazi, What is the diameter of the actin lament?, FEBSLett., 1997, 405, 249–252.
35 K. Xu, H. P. Babcock and X. Zhuang, Dual-objective STORMreveals three-dimensional lament organization in the actincytoskeleton, Nat. Methods, 2012, 9, 185–188.
36 C. E. Turner, Paxillin interactions, J. Cell Sci., 2000, 113(23),4139–4140.
This journal is © The Royal Society of Chemistry 2017
