Fig. 1: The ExPRESSO workflow for multiplex tissue imaging at 3.7× resolution.

a The ExPRESSO workflow is compatible with (1) archival tissue sections, which are (2) reverse-cross-linked via heat-induced epitope retrieval, (3) embedded into a hydrogel, (4) denatured, (5) stained with isotope-labeled antibodies before (6) expansion and (7) compression in a desiccated environment. (8) ExPRESSO samples are compatible with the MIBIscope, IMC, and beyond. b Representative images of a human hippocampus section after (1) sectioning, (2) denaturing, (3) expanding, and (4) compressing onto gold and glass slides for MIBI (upper) and IMC (lower) analysis, respectively. Each grid is 2.5 mm. Scale bars indicate 10 mm in physical measurements. c Representative immunofluorescence images of a human hippocampus section stained for GLUT1 and nuclei in pre-expansion (left), post-expansion (middle), and post-ExPRESSO compression (right) states. Scale bars indicate pre-expansion dimensions based on a 3.7-fold expansion: 500 μm (top), 100 μm (bottom). d Comparison of MIBI (left and middle) and ExPRESSO-MIBI (right) human tonsil imaging in two consecutive sections. Representative MIBI images for CD3, CD20, CD21, Vimentin, Ki-67, Lamin A/C, and Histone H3. Scale bars indicate the pre-expansion dimensions based on a 3.7-fold expansion: 100 μm (main), 25 μm (middle and right, enlarged). e Expansion fold quantification. Top: Independent quantifications from four tissues, represented with means and standard deviation. Bottom: Schematic representation of quantification strategies: (1) linear: ratio of the longest line in the tissue and its perpendicular, (2) area: ratio of the areas before and after ExPRESSO, (3) segmentation: ratio of segmented cell sizes between unexpanded and ExPRESSO tonsil tissues. f Long-term signal retention in ExPRESSO samples. The same ExPRESSO tonsil was imaged by MIBI after stored for 1, 3, and 6 months. Top: total histone H3 and dsDNA counts from 5 fields of view (FOVs) per time point. Bottom: representative MIBI images at each time. Scale bars indicate the pre-expansion dimensions based on a 3.7-fold expansion: 100 μm. g MIBI measurement of Histone H3 and dsDNA counts (per pixel) in an ExPRESSO tonsil analyzed with a range of primary ion currents. h Representative MIBI images of unexpanded and ExPRESSO-treated tonsils at equivalent spatial resolutions, capped at 300 counts per pixel. Values indicate counts (per pixel). Scale bars indicate the pre-expansion dimensions based on a 3.7-fold expansion): 25 μm.