Fig. 1. Expansion microscopy (ExM) concept.
) Schematic of (i) collapsed polyelectrolyte
network, showing crosslinker
(dot) and polymer chain (line), and (ii) expanded network after H2O
) Photograph of fixed mouse brain slice. (C
) Photograph, post-ExM, of the sample (B) under side illumination. (D
) Schematic of label that can be anchored to the gel at site of a biomolecule. (E
) Schematic of microtubules (green) and polymer network (orange). (F
) The label of (D), hybridized
to the oligo-bearing secondary antibody top (top gray shape) bound via the primary (bottom gray shape) to microtubules (purple), is incorporated into the gel (orange lines) via the methacryloyl group (orange dot) and remains after proteolysis (dotted lines). Scale bars, (B) and (C) 5 mm. Schematics are not to scale.
Diapers and more diapers
What's in a gel?
What are polyelectrolyte gels?
How does the addition of an initiator and accelerator aid in forming the polymer network?
What are the chemical components of the fluorescent tags?
Figure 1B is a coronal section of a mouse brain slice; Figure 1C is an expanded image of 1B using ExM. A widefield microscope was used by illuminating the side of the specimen.
Chen and colleagues describe key facets of ExM.
Figures (Ai) and (Aii) illustrate the expansion of the polymer network after water dialysis, whereas B and C show before and after ExM of a brain slice.
Panels D and F illustrate the concept behind engineering the fluorescently labeled tags, that will prove useful in visualizing components of neurons and cells, as will be seen in later figures of this groundbreaking paper.
We developed a fluorescent labeling strategy compatible with the proteolytic treatment and subsequent tissue expansion described above, to see whether fluorescence nanoscopy would be possible
. We designed a custom fluorescent label (Fig. 1D
) that can be incorporated directly into the polymer network and thus survives the proteolytic digestion of endogenous biomolecules. This label is trifunctional, comprising a methacryloyl group capable of participating in free radical polymerization
, a chemical fluorophore for visualization, and an oligonucleotide
that can hybridize to a complementary sequence attached to an affinity tag (such as a secondary antibody) (Fig. 1, E and F
). Thus, the fluorescent tag is targeted to a biomolecule of interest yet remains anchored covalently with high yield (table S1) to the polymer network
. The entire process of labeling, gelation, digestion, expansion, and imaging we call expansion microscopy (ExM)
Fig. 2. Expansion microscopy physically magnifies, with nanoscale isotropy.
We compared images acquired via conventional microscopy
(blue scale bars) versus images acquired post-expansion (orange scale bars). (A
) Confocal image of microtubules in HEK293
) Post-expansion confocal image of sample (A). (C
length measurement error of pre- versus post-ExM confocal images of cultured cells (blue line, mean; shaded area, standard deviation; n
= 4 samples). (D
image of microtubules. (E
) Post-expansion confocal image of the sample of (D). (F
) Magnified views of boxed regions of (D) and (E), respectively. (H
) Profiles of microtubule intensity taken along the blue and orange dotted lines in (F) and (G). (I
length measurement error of ExM versus SR
images (blue line, mean; shaded area, standard deviation; n
= 4 samples). (J
) Transverse profile of a representative microtubule (blue line), with Gaussian fit (black dotted line). (K
image of clathrin-coated pits (CCP
s) in HEK
293 cells. (L
) Post-expansion confocal image of the sample of (K). (M
) Magnified views of a single CCP
in the boxed regions of (K) and (L), respectively. (O
) Scatterplot of radii of CCP
s measured via ExM versus SR
= 50 CCP
s from 3 samples). Green line, y
line; shaded green region, half-pixel width of digitization error about the y
line. Scale bars for pre- versus post-ExM images, (A) 20 μm; (B) 20 μm (physical size post-expansion, 81.6 μm); (D) 2 μm; (E) 2 μm (9.1 μm); (F) 500 nm; (G) 500 nm (2.27 μm); (K) 2 μm; (L) 2 μm (8.82 μm); (M) 100 nm; (N) 100 nm (441 nm).
In the paper on ExM, isotropy refers to the physical expansion of an object in all directions.
For example, if a golf ball is physically blown up in all directions, then this is isotropy.
Here are some additional examples:
(1) Blowing up a regular balloon is isotropic—it expands in all directions, whereas blowing up a balloon for making balloon animals is not isotropic because it expands in the cross-sectional directions first, then fills out the length of the balloon.
(2) A lump of dough rising appears to be isotropic on the macroscale, but if you look closer there are air pockets that open up within the dough—that is an example of distortion (anisotropy) on smaller scales.
(3) Stretching a rubber band is not isotropic because it will stretch in one dimension while contracting in the other two.
What's in the pits?
Figure panels C, D, E, and I illustrate that the images obtained by ExM compared with superresolution imaging methods are similar qualitatively and quantitatively.
Note that the error is within the point-spread-function size of a special superresolution method, called superresolution structured illumination microscopy (SR-SIM).
We next compared pre-ExM conventional superresolution images to post-ExM confocal images. We labeled features traditionally used to characterize the performance of superresolution microscopes, including microtubules (4
) and clathrin coated pits (6
), and imaged them with a superresolution structured illumination microscope (SR
) pre-ExM, and a spinning disk confocal post-ExM
. Qualitatively (Fig. 2, D and E
), the images were similar, and quantitatively (Fig. 2I
), measurement errors were again on the order of 1% and well within the point spread function size of the SR
= 4 samples). Microtubule networks were more sharply resolved in ExM (Fig. 2G
) than with SR
). ExM resolved individual microtubules that could not be distinguished with SR
). Microtubules imaged with ExM presented a full-width at half-maximum (FWHM
) (Fig. 2J
) of 83.8 ± 5.68 nm (mean ± SD
= 24 microtubules from 3 samples). This FWHM
reflects the effective resolution of ExM convolved by the width of the labeled microtubule. To estimate the effective resolution of ExM, we deconvolved
[as in (7
)] our observed microtubule FWHM
by the known immunostained microtubule width [55 nm (6
)], conservatively ignoring the width of the trifunctional label, and obtained an effective resolution for ExM of ~60 nm. This conservative estimate is comparable with the diffraction-limited confocal resolution [~250-nm lateral resolution (8
)] divided by the expansion factor (~4.5).
We next applied ExM to fixed brain tissue. Slices of brain from Thy1-YFP-H
mice expressing cytosolic
yellow fluorescent protein (YFP
) under the Thy1 promoter in a subset of neurons (9
) were stained with a trifunctional label bearing Alexa 488, using primary antibodies to green fluorescent protein (GFP
) (which also bind YFP
). Slices expanded fourfold, similar to the expansion factor in cultured cells. We compared pre- versus post-ExM images taken on an epifluorescence microscope. As with cultured cells, the post-ExM image (Fig. 3B
) was registered to the pre-ExM image (Fig. 3A
) via a similarity transformation. The registered images closely matched, although some features moved in or out of the depth of field
because of the axial expansion
post-ExM. Quantitatively, post-ExM measurement errors (Fig. 3C
= 4 cortical slices) were 2 to 4%.
Fig. 3. ExM imaging of mammalian brain tissue.
) Widefield fluorescence
(white) image of Thy1-YFP
mouse brain slice. (B
) Post-expansion widefield image of sample (A). (C
length measurement error for pre- versus post-ExM images of brain slices (blue line, mean; shaded area, SD
= 4 samples). (D
) Confocal fluorescence images of boxed regions in (A) and (B), respectively, stained with presynaptic (anti-Bassoon, blue) and postsynaptic (anti-Homer1, red) markers, in addition to antibody to GFP
(green), pre- (D) versus post- (E) expansion. (F
) Details of boxed regions in (D) and (E), respectively. (H
) Single representative synapse highlighted in (G). (I
) Staining intensity for Bassoon (blue) and Homer1 (red) of the sample of (H) along white box long axis. Dotted black lines, Gaussian fits
. a.u., arbitrary units. (J
) Bassoon-Homer1 separation (n
= 277 synapses from four cortical slices). Scale bars for pre-versus post-ExM images, (A) 500 μm; (B) 500 μm (physical size post-expansion 2.01 mm); (D) 5 μm; (E) 5 μm (20.1 μm); (F) 2.5 μm; (G) 2.5 μm (10.0 μm); and (H) 250 nm (1.00 μm).
What's in an image of the brain?
For panels A through G and panel H, can you describe what part of the brain the authors are showing?
Consider browsing the Allen Institute’s Brain Atlas: http://developingmouse.brain-map.org/
How does the root-mean-square error length measurement percent compared with Figure 2C and 2I? Keep in mind the specimen types used in each figure.
Intensity line scans
Bassoon and Homer 1 label the pre- and postsynapse that express green fluorescent protein driven by the Thy1 promoter in transgenic mice.
Can you determine the importance of Bassoon and Homer 1’s function, and their role in neural function?
We synthesized trifunctional labels with different colors and oligonucleotides (supplementary materials, materials and methods) to enable multicolor ExM
. We obtained pre- (Fig. 3D
) versus post-ExM (Fig. 3E
) images of Thy1-YFP
-H mouse cortex
with ExM labels directed against YFP
, green) and the pre- and postsynaptic
scaffolding proteins Bassoon (Fig. 3E
, blue) and Homer1 (Fig. 3E
, red). In the pre-ExM image, Bassoon and Homer1 staining form overlapping spots at each synapse (Fig. 3F
), whereas the post-ExM image (Fig. 3G
) shows clearly distinguishable pre- and postsynaptic labeling. We quantified the distance between the Bassoon and Homer1 scaffolds, as measured with ExM. We fit the distributions
of Bassoon and Homer1 staining intensity, taken along the line perpendicular to the synaptic cleft (Fig. 3H
, boxed region), to Gaussians (Fig. 3I
). The Bassoon-Homer1 separation was 169 ± 32.6 nm (Fig. 3J
= 277 synapses from four cortical slices), similar to a previous study using stochastic optical reconstruction microscopy (STORM)
in the ventral cortex and olfactory bulb, which obtained ~150 nm separation (10
). We also imaged other antibody targets of interest in biology (fig. S4).
To explore whether expanded samples, scanned on fast diffraction-limited microscopes, could support scalable superresolution imaging, we imaged a volume of the adult Thy1-YFP-H mouse brain spanning 500 by 180 by 100 μm
(tissue slice thickness), with three labels (antibody to GFP
, green; antibody to Homer1, red; antibody to Bassoon, blue) (Fig. 4A
). The diffraction limit of our confocal spinning disk microscope (with 40×, 1.15 NA
, water immersion objective), divided by the expansion factor
, yields an estimated effective resolution of ~70 nm laterally and ~200 nm axially. Shown in Fig. 4A
is a three-dimensional (3D) rendered image of the data set (an animated rendering is provided in movie S1). Zooming into the raw data set, nanoscale features emerge (Fig. 4, B to D
). We performed a volume rendering of the YFP
-expressing neurons in a subset of CA1 stratum lacunosum moleculare (slm)
, revealing spine morphology (Fig. 4B
and movie S2). Focusing on a dendrite in CA
1 slm, we observed the postsynaptic protein Homer1 to be well localized to dendritic spine heads
, with the presynaptic
molecule Bassoon in apposition (Fig. 4C
and movie S3). Examination of a mossy fiber bouton in the hilus of the dentate gyrus reveals invaginations into the bouton by spiny excrescences of the opposing dendrite, as observed previously via electron microscopy (Fig. 4D)
). Thus, ExM enables multiscale imaging and visualization of nanoscale features, across length scales relevant to understanding neural circuits
Fig. 4. Scalable 3D superresolution microscopy of mouse brain tissue.
) Volume rendering of a portion of hippocampus showing neurons (expressing YFP
, shown in green) and synapses [marked with anti-Bassoon (blue) and antibody to Homer1 (red)]. (B
) Volume rendering of dendrites in CA
1 slm. (C
) Volume rendering of dendritic branch in CA
1 slm. (D
) Mossy fiber bouton in hilus of the dentate
gyrus. (i) to (iii), selected z
-slices. Scale bars, (A) 100 μm in each dimension; (B) 52.7 μm (x
); 42.5 μm (y
); and 35.2 μm (z
); (C) 13.5 μm (x
); 7.3 μm (y
); and 2.8 μm (z
); (D), (i) to (iii) 1 μm.
Synapses and dendrites
The physical magnification of ExM enables superresolution imaging with several fundamental new properties. The axial effective resolution is improved by the same factor as the lateral effective resolution. ExM can achieve superresolution with standard fluorophores, and on a diffraction-limited microscope
. Superresolution imaging is often performed within ~10 μm of the sample surface because of low signal-to-noise
, and refractive index mismatch
. We were able to perform three-color superresolution imaging of a large volume of brain tissue over an axial extent of 100 μm with a spinning disk confocal microscope. Because the ExM-processed sample is almost entirely water, eliminating scattering
, ExM may empower fast methods such as light-sheet microscopy (15
) to become superresolution methods. ExM potentially enables labels to be situated within a well-defined, in vitro–like environment, facilitating in situ analysis (16
). Because the sample is physically larger, any mechanical errors in post-expansion sectioning, or stage drift
, are divided by the expansion factor.
Materials and Methods
Figs. S1 to S5
Tables S1 to S4
Movies S1 to S3
References and Notes
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was funded by NIH
Director’s Pioneer Award 1DP1NS
087724 and NIH
Director’s Transformative Research Award 1R01MH
103910-01, the New York Stem Cell Foundation-Robertson Investigator Award, the MIT
Center for Brains, Minds, and Machines NSF CCF
-1231216, Jeremy and Joyce Wertheimer, Google, NSF CAREER
1053233, the MIT
Synthetic Intelligence Project, the MIT
Media Lab, the MIT
McGovern Institute, and the MIT
Neurotechnology Fund. F.C.
was funded by an NSF
Graduate Fellowship. P.W.T.
was funded by a Fannie and John Hertz Graduate Fellowship. Confocal imaging was performed in the W. M.
Keck Facility for Biological Imaging at the Whitehead Institute for Biomedical Research. Deltavision OMX SR
imaging was performed at the Koch Institute Swanson Biotechnology Center imaging core. We acknowledge W. Salmon and E. Vasile for assistance with confocal and SR
imaging. We acknowledge N. Pak for assistance with perfusions. We also acknowledge, for helpful discussions, B. Chow, A. Marblestone, G. Church, P. So, S. Manalis, J.-B. Chang, J. Enriquez, I. Gupta, M. Kardar, and A. Wissner-Gross. The authors have applied for a patent on the technology, assigned to MIT
Provisional Application 61943035). The order of co–first author names was determined by a coin toss. The imaging and other data reported in the paper are hosted by MIT