Expanded vacuum-stable gels for multiplexed high-resolution spatial histopathology

Abstract

Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.

Microscale visualization of cellular features in adult macaque visual cortex

“…Expansion microscopy (ExM) facilitates nanoscale imaging of biological structures with diffraction-limited microscopy in a wide variety of tissue preparations. [1][2][3][4][5][6][7][8][9][10][11][12][13] In brain tissue, ExM enables discrete visualization of hydrophilic biomolecules such as proteins, DNA, and mRNA, which are densely packed within and around hydrophobic myelin sheaths, by physically separating hydrophobic and hydrophilic tissue regions through isotropic expansion 3,7,12,13 . Detailed analyses of cellular features across large samples of brain tissue become feasible when ExM is combined with light sheet fluorescence microscopy (LSFM), which accelerates acquisition of high resolution image data in cleared tissues.…”

“…Detailed analyses of cellular features across large samples of brain tissue become feasible when ExM is combined with light sheet fluorescence microscopy (LSFM), which accelerates acquisition of high resolution image data in cleared tissues. [14][15][16][17] This combinatorial technique is particularly useful when investigating neuroanatomy and connectivity in large, gyrencephalic nonhuman primate and human brains, 5,[11][12][13][14] where prior studies are limited to sparsely labeled neural populations or subsets of cellular features within neural circuits. 18…”

Expansion Strategy-Driven Micron-Level Resolution Mass Spectrometry Imaging of Lipids in Mouse Brain Tissue

“…However, SIMS has its problems, including a tendency for the ions to break apart, which reduces sensitivity [10] . Other methods, such as multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC) [11–15] , have their own unique features but also have limitations, such as the need for costly tags and limits on what can be detected simultaneously. Furthermore, MIBI and IMC are specific methods aimed at spatial proteomics and are not suitable for analyzing metabolites and lipids.…”

“…However, the primary limitation of SIMS is severe ion fragmentation during analysis, which can diminish its sensitivity and potentially impact the accuracy of molecular identification [10] . Alternative techniques such as multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC) demonstrate remarkable resolution capabilities at the sub-micrometer level for high multiplex protein imaging [11-15] . However, a significant constraint is the requirement for expensive immuno-tags, which can elevate the overall cost of the procedures.…”

Filter-aided expansion proteomics

“…Proteins on the surface of selected tissue region could also be extracted using a fine liquid junction in line with LC-MS/MS hardware 12,13 . Tissue expansion technology has been recently introduced to MS-based proteomics [14][15][16][17] . The concept of expansion proteomics (ProteomEx) is based on the expansion microscopy approaches [18][19][20] , like protein-retention Expansion Microscopy 21 , appended with MS-based proteomics analysis.…”