Fig 1.
Schematics for quantitating morphological changes in correlative SRM and EM images.
After image registration, cell boundaries in both SRM (green) and SEM (white) images are defined, for example with custom Matlab scripts. The SRM image is also divided into 100 x 100 nm2 blocks. The difference in cell areas are then calculated and normalized to the contour length determined by counting the number of blocks (gray) along the cell boundary in the SRM image (green). As such, both the area difference and contour length have the units of nm2.
Fig 2.
Correlative SRM and SEM imaging.
(A) Workflow of imaging the same cell with correlative SRM and SEM; (B) Correlative SRM and SEM image of U2OS cells expressing mEos4 tagged tubulin. Magnified views of the boxed regions in the left image are shown to the right. Scale bars, 1 μm.
Fig 3.
Image registration and quantitation of cell distortion.
(A, B, C) Registered SRM and SEM image of U2OS cells expressing PAmCherry1 tagged with the C-terminal tail of HRas. Distortions in cell morphology were evident in multiple regions of the cell; (D, E) Magnified views of the boxed regions in (C); Gold particles are both fluorescent and electron dense and thus were seen in both the SRM and the SEM images (E, F); (F) Magnified view of the gold particles in (E); (G) Defining cell boundaries in the SRM (green line) and the SEM (white line) image; (H) Area difference between the cells boundaries in the two images in (G); (I) Total area of the blocks that fell on the boundary of the SRM image (‘boundary blocks’) were used to represent the contour length in units of nm2 and to normalize the area difference. Scale bars, 1 μm in (A, B, D and E), 2 μm in (C), and 100 nm in (F).
Fig 4.
Cell distortion under different fixation and drying conditions.
In sample either dried with CPD or HMDS, larger cell distortions were observed with OsO4 post-fixation (A). The resulting images were then quantitatively analyzed for distortion (B) as described earlier. Effects of different primary fixation, drying and post-fixation conditions on cell distortion were examined. For each condition, images of 16–20 cells from 4–5 experiments were analyzed. Error bars represent standard deviations. Scale bars, 2 μm in (A).
Fig 5.
Effects of OsO4 post-fixation on cell morphology.
(A) STORM image of a cell stained with WGA conjugated to Alexa Fluor 647; (B) STORM image of the same cell after post-fixation with 1% OsO4 and re-staining with WGA-AF 647; (C) Overlaid image of (A) and (B); (D) Magnified view of the boxed region in (C); Cell shrinkage (D), distortion of protrusions (E), and discontinued membrane features (F) after OsO4 post-fixation were observed; (G-I) Quantitation of morphological changes after OsO4 post-fixation. In the absence of a continuous cell boundary after post-fixation, the morphological changes were quantitated by comparing line profiles of short segments at the cell boundary, where features were present in both images (G). In both line profiles, the ‘forefront’ of the cell boundary was defined as the pixel position at the half maximum intensities (H), and the distortion was then calculated as the distance difference between the two positions; (I) Average cell distortion after 1% OsO4 post-fixation (n = 20). The error bar represents standard deviation. Scale bars, 5 μm in (A, B and C), 1 μm in (D, E) and 2 μm in (F).
Fig 6.
Growing cells on fibronectin-coated coverslips reduced morphological changes by OsO4 post-fixation.
The cells were grown on non-coated coverslips (left) or those coated with 5 5 μg/mL (middle) or 10 μg/mL (right) fibronectin, fixed in an aldehyde buffer, then processed either directly (gray bars) or after OsO4 post-fixation (black bars) for SEM. The cell distortion index was reduced from 1.29 to 0.78 and 0.72 after coating with 5 or 10 μg/mL fibronectin, respectively (black bars). For samples only fixed with the aldehyde buffer (3.7% paraformaldehyde plus 0.1% glutaraldehyde, gray bars), morphology preservation was not evidently improved by fibronectin coating.