HiExM enables gel formation and expansion in a 96-well cell culture plate.

a. Schematic representation of HiExM devices showing the key features highlighted in color. b. Example devices used in 96-well cell culture plates. The device on the left is inserted into the well plate. c. Brightfield image of the conical post-tip shows the pattern of grooves that mediate fluid retention. d. Fluid retention at the conical post-tip of the device. Silhouettes taken by an optical comparator of the profile of a single post suspended above a surface (left) and in contact with a surface (right) show a fluid droplet interacting with the device. Upon device insertion, the gel solution fills the space under the conical post tip, forming the toroid gel. e-h. Schematic of HiExM gel deposition and expansion workflow. e. The device is immersed in a shallow reservoir of gel solution. f. Upon removal, the tip of each device post retains a small volume of gel solution. g. Gel solution is deposited by the device into the centers of each well of the cell culture plate. Brightfield image (right) shows gel geometry and size prior to expansion. Note that gels deposited in HiExM cover ∼1.1 mm2 of the cell culture surface to accommodate for expansion, and do not include cells outside the gel footprint. h. Polymerization and expansion are carried out with the device in place. Brightfield image (right) shows gel geometry and size after expansion.

HiExM is compatible with nanoscale expansion and automated image acquisition of human cells with minimal distortion.

a. Expanded gels in a 96 well plate imaged at 5x with an inset showing an individual gel. b. Imaging fields for pre-expansion and post-expansion data acquisition. Prior to expansion, 61 fields were imaged at 63x around the center of the gel. After expansion, 12 fields were imaged at 20x magnification. Max projections for each field were then stitched together and manually inspected to identify fields in the pre-expansion and post-expansion image sets containing matching cells. Arrows show an example of a field representing the same pair of cells before and after expansion. c. Representative registered fluorescence image of microtubules before (left) and after (right) expansion showing the increase in resolution conferred by expansion. d. Representative error curve calculated using non-rigid registration on 43 independent fields of view. The shaded region denotes one standard error of the mean. e. Error is not dependent on the location of a given field of view within the gel. Each data point represents the average percent error (up to 40 μm measurement length post-expansion) for a given field of view as determined by an NRR analysis. Six wells were analyzed in seven to ten fields of view. The distance from the center of each field of view to the center of the well was measured manually in FIJI using the stitched pre-expansion image set. f. Stacked-bar histogram showing the distribution of average error among gels across three plates (average = 3.63% +/-1.39). Average error percentages for each well plate: 3.73 +/-0.879, 3.89 +/-1.65, and 3.43 +/-1.44. g. Expansion factor is not dependent on the location of a given field of view within the gel. Images taken before and after expansion of the same cell or group of cells were used to measure the ratio of length scales in the two images. h. Stacked-bar histogram showing the distribution of expansion factors in 58 gels across three plates. Expansion factors ranged from 3.5-5.1x across gels with an average of 4.16 +/-0.394. Average expansion factors for each well plate: 3.78 +/-0.171, 4.26 +/-0.171, and 4.53 +/-0.321.

HiExM shows altered nuclear morphology of cardiomyocytes treated with low doses of doxorubicin.

a. hiPSC CMs imaged post-expansion in HiExM. b. Example images of hiPSC CM nuclei after dox treatment for 24 hrs. before (top) and after (bottom) expansion. *Scale bar represents biological scale assuming 4x expansion. c. Example images of hiPSC CM nuclei taken before (top) and after (bottom) expansion. d. Heatmaps of nuclei in b showing pixel groupings for subsequent analysis. These heatmaps are generated from the image mask after dilation, meaning that the outer edge represents a contour just beyond the nuclear periphery. e. Relative Hoechst intensity plotted as a function of pixel position relative to the edge of the dilated mask in pre- (top) and post- (bottom) expansion nuclei. Different curves represent dox concentrations, where Ctrl = DMSO control. Shaded regions represent SEM for n = 56, 71, 64, 92, and 62 nuclei in the pre-expansion case and n = 4 replicates in the expanded case. f. Insets of plots in e highlighting the nuclear periphery. g. Rate-of-change analysis of curves for pre- (left) and post- (right) expansion data. Outer edge values were taken as the maximum values of the derivatives for each condition, and inner edge values were taken as the minimum within the domain 0 - 0.4. h. Curvature analysis for pre- (left) and post- (right) expansion data. Values represent the minima of the second derivative for curves within the domain 0 – 0.4. i. Average Hoechst intensities plotted as gradients for each dox concentration. Scale bar represents 10 percent distance from the edge of the nucleus to the center. Error bars represent SEM as in e. * denotes significance from an independent two- sample t-test (p < 0.05).