Intravital Endoscopic Small Intestine Imaging
A recent paper published in Biomedical Optics Express demonstrated a side-view endomicroscopy for in vivo longitudinal repetitive cellular-level visualization of small intestinal villi. (Villi are fingerlike protrusions of the intestine wall.) A high-resolution side-view endomicroscope fabricated by using miniaturized micro-GRIN(Gradient-index) lenses and a micro-mirror was integrated into a custom-built ultrafast laser-scanning confocal microscopy platform. Various types of fluorescent cells and microvasculature in the small intestinal villi were successfully visualized in a minimally-invasive manner in vivo. Furthermore, by utilizing a transgenic mouse, universally expressing Kaede, a photoconvertible protein, the repetitive cellular-level confocal endoscopic visualization of the same area in the small intestinal lumen of a single mouse was achieved.
For inflammatory bowel disease research, direct observation of cellular phenomena in the gastrointestinal tract of a mouse model has been a long-pursued goal. Especially, for chronic conditions such as Crohn’s disease, mostly affecting small intestine, longitudinal and repetitive cellular visualization and analysis of luminal surfaces under the natural in vivo environment is highly desirable; it can provide valuable information for investigating complex pathogenesis from a sequential viewpoint.
A research team led by Professor Pilhan Kim at the Graduate School of Nanoscience and Technology, KAIST developed a micro-GRIN lens-based video-rate confocal side-view endomicroscopy system, enabling minimally-invasive in vivo cellular level visualization of microvasculature and fluorescent cells in the mouse small intestine. The side-view endomicroscope was fabricated by stacking 2 mm diameter GRIN lenses – a coupling lens, a relay lens and a high NA imaging lens attached with an aluminum coated 90° micro-prism. The endomicroscope was integrated to a custom-built video-rate laser scanning confocal microscopy platform. By using various transgenic mice expressing green fluorescent protein (GFP), the distribution of mononuclear phagocytes (CX3CR1-GFP), T cells (CXCR6-GFP) and lymphatic vessels (Prox1-GFP), along with microvasculature (CD31-Alexa647) were successfully visualized in the small intestinal villi in vivo. Also, fluorescently labeled red blood cells (RBC), those were rapidly flowing through the capillary network in the small intestinal villi, were directly imaged in “real time.”
Finally, by using a transgenic mouse universally expressing the photoconvertible protein Kaede, the repetitive cellular-level visualization of the same area in the small intestinal lumen in a single mouse was demonstrated, revealing the continuous self-renewal of the small intestinal epithelium in vivo. Enterocytes, the most abundant type of cells in the epithelium of the villi, are replaced in every 3~5 days by the newly differentiated cells, those were originated from the stem cells existing in the crypt bases. Under the ultraviolet light irradiation (350 – 400 nm), Kaede irreversibly transmutes its fluorescent emission from green to red. Photoconverted Kaede expressing cells retain the red fluorescence, while all of the newly-generated cells express the original green fluorescent version of Kaede. On day 0, the normal-looking area was identified in the small intestine, and then, under 405 nm light, was delivered through the side-view endomicroscope to irreversibly photoconvert the Kaede protein inside the villi. After 2 days, newly originated green fluorescent Kaede expressing cells were observed in lamina propria; these were immune cells actively circulating the whole body for immune surveillance. After 4 days, most of enterocytes express original green-fluorescent Kaede, suggesting the renewal of the epithelium of the small intestinal villi. This technique can provide a new avenue through which to investigate the complex pathophysiology of diseases originated from the epithelium of intestinal tracts.
Pilhan Kim, his students Jinhyo Ahn, Kibaek Choe, Yoonha Hwang, Eunjoo Song and Ki Hean Kim, his student Taejun Wang at POSTECH were listed as the authors of this contribution published in Biomedical Optics Express in September of 2015 (Biomed. Opt. Express, 6(10):3963-3972, 2015 ).
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