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 mm (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 ax-
ially. Shown in Fig. 4A is a three-dimensional
(3D) rendered image of the data set (an ani-
mated rendering is provided in movie S1). Zoom-
ing 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 CA1 slm,
we observed the postsynaptic protein Homer1
Fig. 3. ExM imaging of mammalian brain tissue. (A) Widefield fluorescence
(white) image of Thy1-YFP mouse brain slice. (B) Post-expansion widefield
image of sample (A). (C) RMS length measurement error for pre- versus
post-ExM images of brain slices (blue line, mean; shaded area, SD; n = 4
samples). (D and E) 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 and G) 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 mm; (B) 500 mm (physical size post-expansion 2.01 mm);
(D) 5 mm; (E) 5 mm (20.1 mm); (F) 2.5 mm; (G) 2.5 mm (10.0 mm); and (H) 250 nm
(1.00 mm).
