Expansion microscopy

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Science  30 Jan 2015:
Vol. 347, Issue 6221, pp. 543-548
DOI: 10.1126/science.1260088
  • Fig. 1 Expansion microscopy (ExM) concept.

    (A) Schematic of (i) collapsed polyelectrolyte network, showing crosslinker (dot) and polymer chain (line), and (ii) expanded network after H2O dialysis. (B) 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.

  • 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 cells. (B) Post-expansion confocal image of sample (A). (C) RMS length measurement error of pre- versus post-ExM confocal images of cultured cells (blue line, mean; shaded area, standard deviation; n = 4 samples). (D) SR-SIM image of microtubules. (E) Post-expansion confocal image of the sample of (D). (F and G) 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) RMS length measurement error of ExM versus SR-SIM 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) SR-SIM image of clathrin-coated pits (CCPs) in HEK293 cells. (L) Post-expansion confocal image of the sample of (K). (M and N) Magnified views of a single CCP in the boxed regions of (K) and (L), respectively. (O) Scatterplot of radii of CCPs measured via ExM versus SR-SIM (n = 50 CCPs from 3 samples). Green line, y = x line; shaded green region, half-pixel width of digitization error about the y = x 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).

  • 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 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).

  • Fig. 4 Scalable 3D superresolution microscopy of mouse brain tissue.

    (A) 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 CA1 slm. (C) Volume rendering of dendritic branch in CA1 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.

Supplementary Materials

  • Expansion microscopy

    Fei Chen, Paul W. Tillberg, Edward S. Boyden

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figures S1 to S5
    • Tables S1 to S4
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    Images, Video, and Other Other Media

    Movie S1
    3D animation of large-scale rendering of hippocampal volume from Fig. 4A. First, the volume appears with YFP only (green), then staining for Bassoon (blue) and Homer1 (red) are added. Scale bars: 100 μm.
    Movie S2
    3D animation of rendered CA1 slm dendrites from Fig 4B. Scale bars: 10 μm.
    Movie S3
    3D animation of rendered dendritic branch of CA1 slm from Fig. 4C, showing YFP (green), Bassoon (blue), and Homer1 (red). Scale bars: 2.5 μm./dd>

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