Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix

Abstract

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.

Label-free optical imaging of cell function and collagen structure for cell-based therapies

“…The longer wavelengths of light and optical sectioning properties of multiphoton excitation also reduce photobleaching. While collagen fluorescence can be imaged by either single- or multi-photon excitation by light-sheet scattering microscopy [ 30 ], and confocal microscopy [ 31 , 32 ], the fluorescence emission of collagen overlaps with other tissue chromophores including NAD(P)H, flavins, elastin, and lipofuscin. Therefore, SHG imaging provides better visualization of fibrillar collagen architecture than autofluorescence imaging.…”