Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples

Abstract

Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy

“…LSFM-based analyses can be recommended as an elegant and efficient approach for comprehensive quantitative characterization of adipocyte morphology in future studies, probably also in other species. Of note, LSFM of 3DISCO cleared tissue samples can also be combined with other imaging modalities, such as histology, immunohistochemistry, or mass spectrometry imaging, and also works using paraffin-embedded tissue samples, e.g., from pathology archives [84]. The swift determinability of accurate numerical values of adipocyte numbers and (individual) volumes can undoubtedly significantly contribute to increase the effectiveness of a study, e.g., by correlation of quantitative morphological adipocyte parameters with transcript profiling-, proteomic-, lipidomic-, or metabolomic analysis data, or measurements of inflammatory cytokines and hormones analyzed in the same adipose tissue samples, or with systemic marker concentrations, or clinical outcomes.…”

Proteomics of spatially identified tissues in whole organs

“…Towards this goal, new technologies emerged for the molecular and spatial characterization on tissue slices (Alon et al, 2021;van den Brink et al, 2020;Chen et al, 2020;Liu et al, 2020;Mahdessian et al, 2021;Rodriques et al, 2019;Ståhl et al, 2016;Takei et al, 2021). In parallel, tissue clearing methods enabled imaging cellular even sub-cellular level details of protein/ molecule of interests in intact transparent organs and organisms (Yang et al, 2014;Ueda et al, 2020;Blutke et al, 2020;Richardson and Lichtman, 2015). As many pathologies start in yet unknown regions of organs, a method first to determine the whereabouts of initial tissue abnormalities in whole organs, then to reveal their complete molecular profiles would be essential in understanding and handling diseases at their early stages.…”

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies

“…Remarkably, LSFM analyses of 3DISCO-cleared specimens can be combined with additional (multimodal) histo-technical analyses. Subsequent to LSFM analysis, optically cleared samples can e.g., be re-embedded in paraffin (or other embedding media) and sectioned for subsequent standard histological as well as immunohistological analyses [134]. Conversely, it is also possible to clear and image (gill) samples that had previously been embedded in paraffin (following deparaffinization of the samples) [135].…”