Development and application of custom-built real-time confocal microscopy for biomedical research

Abstract

Intravital fluorescence confocal microscopy has enabled directly in vivo observation of the dynamic 3D cel-lular-level visualization, providing invaluable information impossible to obtain by conventional ex vivo or in vitro experimental methods. In this study, a custom-built real-time laser-scanning confocal microscopy with sub-micron resolution, multicolor, and multi-modal imaging capacity was built and applied to various bio-medical researches.

At first, the effect of THz wave on the biological sample was analyzed due to the increase of interests in a potential biomedical application. Despite the importance of THz wave based applications, yet our knowledge about how THz wave irradiation could affect a live tissue in cellular level are very limited. The immune response in cellular-level was analyzed after pulsed THz wave irradiation generated from the compact FEL in live mouse ear skin in vivo.

In addition, the confocal microscopy system was modified to monitoring ICG, FDA approved biocompatible fluorophore. Many pharmacokinetic studies of ICG on a gross physiological scale were reported due to its clinical value of ICG

however, in vivo cellular-level real-time dynamics has not been well-known. By utilizing the NIR confocal microscopy system, the cellular-resolution ICG and liposomal ICG fluorescence images of the lymph node, the blood vessel in an ear skin, and the liver was successfully visualized in a live anesthetized mouse to investigate pharmacokinetic dynamics in vivo.

Finally, the capability of retinal imaging was achieved by changing the imaging optics of the confocal mi-croscopy platform. Relay imaging optics were carefully optimized for the purpose of retinal imaging in rodent model with an appropriate field of view. Using the system, various pathophysiological studies related with retina was performed, such as monitoring of gene delivery and CNV model, and visualization and tracking of a fluorescent cell.