In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy

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 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-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

Intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model

“…To visualize the in vivo pulmonary microcirculation through the pulmonary imaging window, a custom-built video-rate laser-scanning confocal microscopy system was implemented as previously described [24][25][26][27][28]. Three laser modules with output wavelengths at 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt), and 640 nm (MLD640, Cobolt) were used as excitation light sources.…”

“…1(a) [30,31]. It was further modified to be integrated with the custom-built video-rate laser-scanning confocal microscopy platform [23][24][25][26][27][28]. The minimal negative suction pressure of 20~30 mmHg was applied via a suction tube to minimize the motion-induced interruption during the pulmonary imaging [30,31].…”

“…As described above, utilizing the customized video-rate laser-scanning confocal microscopy platform [23][24][25][26][27][28], the real-time dynamics of the individual cell could be directly observed in the lung in vivo. In order to visualize the individual circulating cells in the pulmonary capillary those known to be mostly erythrocytes, negative staining of them based on the intravenous injection of fluorescent dye (FITC-dextran, 40 kDa) was facilitated as shown in Fig.…”

“…To visualize and investigate pulmonary ESL in vivo, we utilized a custom-design videorate laser scanning confocal microscope and a pulmonary imaging window chamber, which enabled a real-time subcellular-resolution imaging of the rapidly flowing erythrocyte in vivo [23][24][25][26][27][28]. With fluorescence angiography, the track of erythrocytes flowing in capillary was successfully visualized in vivo, which revealed the exclusion zone representing the pulmonary ESL.…”

In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina

“…1A). [18][19][20] Continuous-wave laser module at 488 nm was used as an excitation sources for GFP. Raster scanning pattern of excitation laser was generated by a scanner system composed of a rotating polygonal mirror (MC-5; Lincoln Laser, Phoenix, AZ, USA) and a galvanometer based scanning mirror (6230H; Cambridge Technology, Bedford, MA, USA), and then delivered to the back aperture of an imaging lens.…”