Abstract
Volumetric observation is essential for understanding the details of complex biological phenomena. In this study, a bright-field microscope, which provides information on a specific 2D plane, and a holographic microscope, which provides information spread over 3D volumes, are integrated to acquire two complementary images simultaneously. The developed system was successfully applied to capture distinct T-cell adhesion dynamics on inflamed endothelial layers, including capture, rolling, crawling, transendothelial migration, and subendothelial migration.
Highlights
Volumetric observation is essential for understanding the details of complex biological phenomena
When the dynamics of cells are spread over a 3D volume, researchers typically separate the overall dynamics into several parts, which falls within the depth of focus, and investigate them part by part
When a test object is located in the middle of a coherent laser beam, the beam diffracted by the object interferes with the remaining beam, creating an interference pattern containing 3D volumetric information of the object
Summary
Volumetric observation is essential for understanding the details of complex biological phenomena. Numerical reconstruction of a hologram generates images at different planes by using the equation of light propagation. The use of the holography technique can solve the problem of information loss from cells that are not located in the focal plane caused by the shallow depth of focus of optical microscopy.
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