Fluorescence In Vivo Endomicroscopy
terminal ileum and rectum [28]. Mucosal imaging with FIVE has also been demonstrated for diseases like gastrointestinal mucosa-associated lymphoid tissue (MALT) lymphoma, where results are in almost perfect agreement with histological find- ings [29]. Out of 26 cases reported with histology, only one case of deep tissue lymphoma was not diagnosed with FIVE, as a limitation of FIVE is that it cannot access structures deep in the tissue. FIVE has also been used for imaging graſt versus host disease (GvHD) [30], celiac disease [27], restorative proc- tolectomy [31], inflammatory bowel disease [32], and ulcerative colitis [33]. In a study focused on epithelial cell permeability and mucosal healing, FIVE was used for imaging small intesti- nal permeability using fluorescein. High intestinal permeabil- ity, confirmed through FIVE imaging, is highly correlated with altered lipid metabolism, heightened innate immune response, and junctional protein signaling [34]. FIVE imaging has also been used for imaging colorectal cancer, as it can detect pre- cursor colorectal polyps and predict intraepithelial neoplasia with 99.1% accuracy [35]. In a parallel study, Kiesslich evalu- ated intraepithelial neoplasia and colorectal cancer with acri- flavine and fluorescein and found FIVE to be rapid, accurate, and highly predictive of cancer [36]. Many other studies have also reported sensitivity, specificity, and accuracy of FIVE in colorectal cancer detection [14,15,37]. FIVE offers dynamic preclinical animal imaging, as one
Figure 2: Cellular detail captured in the gastrointestinal tract with FIVE. (A) Dog gastric mucosa stained with 0.5% acriflavine.
(B) Canine helicobacter
in mucus surrounding gastric villus. Images published with permission from Dr. M. Sharman, Faculty of Veterinary and Agricultural Science, University of Melbourne.
significant lines of investigation relating to epithelial cell turnover and gut barrier function. During the establishment of gastrointestinal applications of FIVE, small bright struc- tures were commonly observed in the single columnar epi- thelial cell layer. Tese were initially dismissed as artifacts, or “junk” tissue fragments absorbing fluorescein. However, investigators noted that single epithelial cells pool fluores- cein, burst, and are ejected from the epithelium (Figure 3). Further, it was noted that the surrounding epithelial cells joined basally, thus maintaining barrier function. Over just a few minutes, this was observed to happen repeatedly in vari- ous parts of the epithelium—a constant process of stunningly rapid and frequent cell shedding. Tis led to multiple inves- tigations of aberrations to this process in patients suffering inflammatory bowel disease (IBD) and molecular studies in animals which were then refined to human patients. It has since been quantified and found predictive of recurrent flares in patients with quiescent inflammatory conditions [27]. Tis reset of our understanding has clarified the dynamics of gut cell turnover and barrier function. Te surprising impact of these observations highlight the importance of observing cellular events in vivo. Kiesslich imaged intestinal mucosa aſter staining with acriflavine in 17 patients, obtaining 6,277 images from human
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can use brighter fluorophores with higher specificity and lower toxicity. A mouse study comparing FIVE with two-photon microscopy (2P) showed that both achieved 176 µm penetra- tion depth and could identify goblet cells [38]. In a similar study, cresyl violet-based staining was used to stain the ileum and colon of BL-6 mice [39]. Similarly, intestinal imaging in experimental pericardial tamponade in anesthetized Viet- namese mini pigs demonstrated injury to intestinal mucosa [40]. FIVE has also been used to image temporal progression of 2,4,6-trinitrobenzenesulfonic acid-induced acute colitis and its treatment with oral phosphatidylcholine (PC) in rats. Dam- age included serosal microcirculation and structural injury, including disruption of the capillary network [41]. Varga found similar
results, including changes in epithelial structure,
microvasculature, and inflammation [42]. Barrier function studies. Vargas reported a lack of tools
for evaluation of rectal mucosal barrier function and explored FIVE for assessing in vivo structural and functional barrier integrity [43]. Topical and i.v. fluorescent probes provided sub- cellular resolution of the mucosal surface and assessment of barrier function loss following topical application of a micro- bicidal agent. Injury due to the microbicides compromised mucosal barrier function of the rectum, increasing the risk for transmission of HIV and other pathogens. Tough H&E staining could not offer functional or temporal imaging, it did confirm the in vivo findings of barrier compromise. Tese reports suggest FIVE as an effective tool for detecting epithe- lial injury and barrier loss, offering real-time rectal mucosal evaluation. Pharynx and pulmonary imaging. Haxel examined vari-
ous regions of the anterior oropharynx in patients, collecting real-time in vivo and ex vivo cellular and subcellular imaging with topical acriflavine and i.v. fluorescein as contrast agents [44]. FIVE imaging provided details of the surface epithelium,
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