In vivo deep tissue fluorescence imaging of the murine small intestine and colon

Abstract

Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/-mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160µm)-on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies.

Deep tissue fluorescence imaging andin vivobiological applications

“…Our detection method has proven to be very efficient in harvesting photons, as indeed no microscope objective has the ability to collect photons from such a wide area. 25,26 However, we acknowledge that our detection geometry does not capture the epi-fluorescence photons, which comprise a fraction of the total fluorescence photons under conditions of deep-tissue two-photon excitation. The diagram in Fig.…”

“…25,26 Here we present a modified upright version of the previous model that is better adapted to image live small animals. The experimental system diagram is shown in Fig.…”

“…24 To solve the problem associated with the loss of fluorescence photons in conventional fluorescence microscopy (described above), we separated the excitation and detection optics to allow a more efficient collection of fluorescence and enhancement of the achievable imaging depth. 25,26 The excitation optics retain their conventional design, however, our detection method introduces several innovative approaches. Our experimental results on deep imaging of turbid samples with optical properties that simulate brain tissue in addition to in vivo and ex vivo imaging of various biological tissues attest to the advantages of this approach.…”