Development of GRIN lens based confocal endomicroscopy for intravital imaging of deep tissue

Abstract

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for studying chronic disease such as Crohn’s disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro gradient index (GRIN) lens based side-view endomicroscope integrated into a custom-built video-rate confocal microscopy system, minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi was successfully performed. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, a repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse was demonstrated, which revealed the continuous homeostatic renewal of the small intestinal epithelium. Meanwhile, a direct observation of a deep tissues such as a solid tumor, skin dermal layer and deeper brain with a live mouse over an extended duration of time has been exceedingly desirable to understand the complicated physiological processes and contribute a new insight in various microenvironment. However, applications of the conventional laser scanning confocal microscope for a live mouse have been mostly limited to the superficial parts of the tissues due to a light scattering problem of the inhomogeneous tissues. In this work, minimally-invasive in vivo cellular-level confocal endomicroscopic visualizations of microvasculature and individual fluorescent cells deeply placed inside a solid tumor, deeper skin and brain were achieved, by utilizing GRIN lenses based side-view endomicroscope integrated into a small diameter hypodermal needle. And then, a repetitive and longitudinal visualization of breast cancer cells expressing green fluorescence protein, 4T1-GFP in the growing whole breast tumor was established.