Evaluation of rapid volume changes of substrate‐adherent cells by conventional microscopy 3D imaging

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Abstract
Summary: Precise measurement of rapid volume changes of substrate‐adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate‐adherent cells based on phase‐contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom‐designed, side‐viewing chamber and subjected to hypotonic swelling. Side‐view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off‐line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (ΔVolume ≤ 14%), but our method has superior temporal resolution limited only by the time of single‐image acquisition, typically ∼100 ms. The advantages of using standard phase‐contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.