Expansion microscopy: enabling single cell analysis in intact biological systems

Abstract

There is a need for single cell analysis methods that enable the identification and localization of different kinds of biomolecules throughout cells and intact tissues, thereby allowing characterization and classification of individual cells and their relationships to each other within intact systems. Expansion microscopy (ExM) is a technology that physically magnifies tissues in an isotropic way, thereby achieving super-resolution microscopy on diffraction-limited microscopes, enabling rapid image acquisition and large field of view. As a result, ExM is well-positioned to integrate molecular content and cellular morphology, with the spatial precision sufficient to resolve individual biological building blocks, and the scale and accessibility required to deploy over extended 3-D objects like tissues and organs.

Keywords: FISH; expansion microscopy; genomics; morphology; multiplexing; single cell analysis; super-resolution microscopy.

Figure 1

Overview of expansion microscopy mechanism and process. (A) The biological specimen is chemically fixed, then treated with compounds to bind key biomolecules/labels of interest. A polyelectrolyte hydrogel is formed in situ, followed by proteolytic digestion and expansion in water. (B) Photograph of fixed mouse brain slice. (C) The specimen of B after expansion. (D) Expansion significantly reduces scattering of the sample, since the sample is mostly water. A 200 μm fixed brain slice is opaque primarily due to scattering (i). However, the post-ExM sample is transparent (ii). (E, F) Confocal image of microtubules in cultured HEK293 cells before (E) and after (F) expansion. (G) RMS length measurement error of pre- versus post-ExM confocal images of cultured cells (blue line, mean; shaded area, standard deviation; n = 4 samples). Scale bars: (B) and (C) 5 mm, in physical size units. (E) 20 μm; (F) 20 μm in biological units (physical size post-expansion, 81.6 μm). Panel A adapted from ref. [63] and [40]. Panels B–G adapted from ref. [33].

Figure 2

Nanoscale detection of proteins with ExM and iterative ExM (iExM). Pre- (A) versus post- (B) expansion confocal fluorescence images of Thy1-YFP mouse brain slice, stained with presynaptic (anti-Bassoon, blue) and postsynaptic (anti-Homer1, red) markers, in addition to antibody to GFP (green). (C) Epifluorescence image of cultured hippocampal neurons stained with antibodies against Homer1 (magenta), glutamate receptor 1 (GluR1, blue), and Bassoon (green), after ~13-fold expansion via iExM. (D) Confocal image of immunostained Emx1-Cre mouse hippocampus with neurons expressing membrane-bound fluorescent proteins (Brainbow AAVs) before expansion. Blue, EYFP; red, TagBFP; green, mTFP. (E) As in D, but expanded 4.5-fold. Inset shows a magnified image of a spine in the dotted box of E. (F–I) Confocal z-stack image of 20-fold-expanded mouse hippocampal circuitry with labeled EYFP (blue) and mCherry (green). (F) Maximum intensity projection of the stack shown in (G–I). Inset in F shows a demagnified view of the image of F with the same scale bar as D and E. Inset of I shows a magnified view of a spine in the dotted box of I. Scale bars (A) 2.5 μm; (B) 2.5 μm (physical size post-expansion 10.0μm); (C) 1μm; (D) and (E) 3μm, inset of E 1μm; (F) 1μm, inset 3μm; (G)–(I) 3μm, inset of I 0.5μm. Panels A–B adopted from ref. [33]; panels C–I adapted from ref. [38].

Figure 3

Nanoscale and multiplexed detection of RNA with ExFISH. (A) smFISH image of ACTB before expansion of a cultured HeLa cell. Inset shows zoomed-in region, highlighting transcription sites in nucleus. (B) As in A, using ExFISH. (C) smFISH counts before versus after expansion for seven different transcripts (n = 59 cells; each symbol represents one cell). (D) Composite image showing ExFISH with serially delivered probes against six RNA targets in a cultured HeLa cell; colors are as follows: NEAT1, blue; EEF2, orange; GAPDH, yellow; ACTB, purple; UBC, green; USF2, light blue. (E) Confocal image of hippocampal tissue showing colocalized Dlg4 puncta (magenta) overlaid on YFP (green) in Thy1-YFP mouse tissue. (F) Dendrites with Dlg4 mRNA localized to spines (arrows). (i), (ii), two representative examples. Scale bars: (A) and (B) 10 μm (expansion factor, 3.3×), inset 2 μm; (D) (expanded coordinates) 20 μm. (E) 10 μm (expansion factor, 3×; white bar, biological scale; blue bar, physical scale); (F) 2 μm (expansion factor, 3×). Adapted from ref. [49].

Figure 4

Imaging of human tissue types using pathology-optimized expansion microscopy. Images of various tissue types for both normal (left images) and cancerous (right images) tissues from human patients. Within each block of images for a given tissue × disease type, there are five images shown. The left-most of the five images shows a core from a tissue microarray (scale bar, 200 μm). The middle column within the five images shows two images, the top of which is a small field of view (scale bar, 10 μm), and the bottom of which zooms into the area outlined in the top image by a white box (scale bar, 2.5 μm). The right column within the five images shows the same fields of view as are shown in the middle column, but postexpansion (yellow scale bars: top images, 10–12.5 μm; bottom images, 2.5–3.1 μm). Physical size postexpansion: top images, 50 μm; bottom images, 12.5 μm; expansion factors 4.0–5.0× (there is some sample-to-sample variability in expansion factor; in practice it is easy to measure the expansion factor for a given specimen: simply take a low-magnification image before you expand, and another low-magnification image after you expand, and compute the ratio of an easily seen feature, pre-expansion vs. post-expansion); Blue, DAPI; green, vimentin; magenta, KRT19. Adapted from ref. [41].