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Multiphoton Imaging Can Be Used for Microscopic Examination of Intact Human Gastrointestinal Mucosa Ex Vivo

https://doi.org/10.1016/j.cgh.2007.10.008Get rights and content

Background & Aims: The ability to observe cellular and subcellular detail during routine endoscopy is a major goal in the development of new endoscopic imaging techniques. Multiphoton microscopy, which relies on nonlinear infrared optical processes, has the potential to identify cellular details by excitation of endogenous fluorescent molecules. We examined the feasibility of using multiphoton microscopy to characterize mucosal histology in the human gastrointestinal tract. Methods: A multiphoton microscope was used to determine the optimal excitation wavelength for examination of gastrointestinal mucosa. Fresh, unfixed, and unstained biopsy specimens obtained during routine endoscopy in human subjects then were examined by confocal microscopy and multiphoton microscopy. Multiphoton images also were compared with standard H&E images obtained from paired biopsy specimens. A prototype miniaturized multiphoton probe was used to examine intact rat colon. Results: Peak multiphoton autofluorescence intensity was detected in mucosa excited at 735 nm. Multiphoton microscopic examination of unstained biopsy specimens revealed improved cellular detail relative to either unstained or stained specimens examined by confocal imaging. Resolution of structures such as epithelial nuclei, goblet cells, and interstitial fibers and cells was comparable with what was obtained using standard H&E histology. Similar findings were observed when using a prototype miniaturized multiphoton probe. Conclusions: Multiphoton microscopy can be used to examine gastrointestinal mucosa at the cellular level, without the need for fluorescent dyes. The construction of a multiphoton endomicroscope therefore could provide a practical means of performing virtual biopsies during the course of routine endoscopy, with advantages over currently available endomicroscopy technologies.

Section snippets

Acquisition of Human Samples

Patients presenting to the Yale Gastrointestinal Procedure Center for elective outpatient endoscopy were recruited to participate in this study, which was approved by the Yale University Human Investigations Committee. Written informed consent was obtained before study participation. Inclusion criteria included age older than 18 years and the ability to provide informed consent. Exclusion criteria included a known or suspected bleeding disorder, international normalized ratio exceeding 1.4,

Optimal Multiphoton Excitation Wavelength for Mucosa

To determine the optimal multiphoton excitation wavelength for tissue autofluorescence and collagen SHG in gastrointestinal tissue, a femtosecond-pulsed, mode-locked Ti:S laser was used to examine fresh untreated segments of rat colon. The laser was tuned across a range of wavelengths between 715 and 790 nm. Fluorescence emission was collected in the 410- to 490-nm range, and the mean fluorescence intensity across the field of view was calculated. Figure 1 shows the relationship between

Discussion

Initial reports using confocal laser scanning microscopy to examine gastrointestinal mucosa ex vivo showed limited image detail in specimens that were not treated with fluorescent dyes.15, 16 Our results show that multiphoton laser microscopy provides the ability to detect cellular and subcellular details in unfixed, unstained gastrointestinal mucosa, with image quality that is superior to confocal imaging with or without fluorescent staining. Throughout the gastrointestinal tract, mucosal

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    Supported by National Institute of Health grants P30 DK34989 and R01 DK45710 (M.H.N.) and R01 CA116583, P41 EB01976, and P41 RR04224 (W.R.Z.). Also supported by a pilot grant from the Pentax Corporation (Montvale, NJ) to support the cost of processing biopsy specimens for H&E staining.

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