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bioRxiv · 10.1101/2024.05.27.595975

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue

Shioka, I.·Morita, R.·Yagasaki, R.·Wuergezhen, D.·Yamashita, T.·Fujiwara, H.·Okuda, S.
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Abstract

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of embryonic skin as well as the role of collagens in maintaining its integrity. Furthermore, quantitative comparisons of stiffness distributions of preserved tissue samples unveiled the distinct impacts of conventional tissue preservation techniques on the tissue stiffness pattern. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/595975v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8a32f5org.highwire.dtl.DTLVardef@117a350org.highwire.dtl.DTLVardef@e2fb99org.highwire.dtl.DTLVardef@1f41d24_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Shioka, I., Morita, R., Yagasaki, R., Wuergezhen, D., Yamashita, T., Fujiwara, H., Okuda, S.. 2024-06-01. Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue. https://doi.org/10.1101/2024.05.27.595975

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