Laser-scanning confocal microscopy based 3d intravital imaging

Abstract

Sectioning microscopy such as confocal microscopy has been used to visualize various biological tissues. By detecting fluorescence signals only from focal plane, confocal microscopy can provide multiple sectioning images at each imaging depth. Those images can be used to reconstruct 3D structure of biological tissues. Confocal microscopy can also provide cellular-level repetitive visualization in vivo. In this work, I optimized visualization techniques for several biological tissues using 4-channel confocal microscopy in conjunction with either of optical clearing technique or dorsal skinfold chamber implantation. Optical clearing enhances imaging depth by reducing light scattering within biological tissues. I optimized optical clearing procedures for cellular-level 3D visualization of mouse ear skin and lymph node. Based on en-hancement at maximum achievable imaging depth and detected fluorescence signal intensity, I examined clearing efficacy of various OCAs. I could visualize cell distribution and vascular/lymphatic network in whole cortex of intact lymph node. Hair follicles in dorsal skin of mouse was also clearly visualized.In addition, I established rapid in vivo efficacy monitoring method for drug candidates through a repetitive longitudinal visualization of same imaging site in cellular-level by utilizing a dorsal skinfold chamber implanted cancer xenograft mouse model. Efficacy of anti-cancer therapeutic candidates either block angiogenesis or induce apoptosis of cancer cells were examined.