Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo

Abstract

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.

Figure 1

Determination of optimal excitation wavelength for two-photon autofluorescence imaging. Unfixed colonic mucosa was excited over a range of wavelengths as fluorescence emission intensity was measured. Peak fluorescence emission intensity in the 410-490 nm range was detected in specimens excited at 735 nm.

Figure 2

Multiphoton microscopy (MPM) is superior to confocal laser-scanning microscopy (CLSM) for imaging fresh colonic mucosa. (A) CLSM (63x) of fresh, unstained tissue demonstrated relatively homogenous autofluorescence with limited subcellular detail. (B) CLSM (63x) of tissue stained with 0.01% fluoroscein showed slightly enhanced cellular detail relative to unstained specimens. (C) MPM image (63x) of fresh, unstained tissue revealed increased cellular and subcellular detail in the 410-490 nm range (green), plus additional autofluorescence details at lower (blue) and higher (red) wavelength ranges.

Figure 3

Comparison of MPM and H&E light microscopic images of biopsies obtained during upper gastrointestinal endoscopy. (A) Esophagus, examined by MPM (63x), demonstrates typical arrangement of nonkeratinized, stratified squamous epithelium. Borders between cells, cell nuclei, and a single papilla are readily identifiable. (B) Esophagus, examined by H&E (40x), shows stratified squamous epithelium that corresponds to the MPM image. (C) Stomach, examined by MPM (63x), demonstrates individual fundic glands composed of cuboidal epithelial cells surrounding a central pit. The glands are separated by connective tissue bands seen via second harmonic generation (368 nm) in a lower range (350-380 nm bandpass). A sparse cellular infiltrate with a peak fluorescence in a higher range (510-560 nm) is seen within the interstitial matrix. (D) The corresponding H&E (20x), reveals a similar arrangement of fundic glands with surrounding lamina propria containing small vessels and occasional mononuclear cells. The basement membrane of cells appears as a thin pink band which corresponds to the thin blue band surrounding glands in the MPM images. (E) Duodenum, examined by MPM (63x), demonstrates columnar epithelial cells lining villi, punctuated by occasional goblet cells characterized by absence of fluorescence. Smaller cells within the lamina propria can also be seen. (F) Duodenum, examined by H&E (20x), shows the corresponding image of an intestinal villous, lined by a single layer of enterocytes with basally located nuclei and interspersed goblet cells with apically located clear mucous. The interstitium containing leukocytes and capillaries can also be seen.

Figure 4

Comparison of MPM and H&E light microscopic images of biopsies obtained during colonoscopy. (A) Terminal ileum, examined by MPM (63x), demonstrates columnar epithelial cells interspersed with goblet cells, lining a single villous. The nuclei are elongated and arranged along the basal surface of the cells. The lamina propria contains a cellular infiltrate that fluoresces at a longer wavelength range. A faint band of lower fluorescence lines the apical aspect of the epithelium, which most likely represents the microvilli that comprise the brush border. (B) Terminal ileum, examined by H&E (20x), shows findings that correlate readily with MPM images, although the interstitial space appears more dense on H&E. (C) Colon, examined by MPM (63x), demonstrates typical glandular pattern with central, round crypt openings. A dense interstitial space separates the glands and contains cellular infiltrate with autofluorescence at longer wavelengths. (D) Colon, examined by H&E (40x), shows cross sections of crypts that correlate well with MPM images. (E) Rectum, examined by MPM (63x), demonstrates features similar to colonic mucosa, including the presence of interspersed goblet cells. Additionally, a thin blue band surrounds individual glands, which likely represents the basement membrane and portions of myofibroblastic sheath. (F) Rectum, examined by H&E (20x), has a similar appearance to colon, with an increased number of goblet cells. The basement membrane is difficult to visualize at this magnification.

Figure 5. Comparison of MPM and H&E light microscopic images of pathologic gastrointestinal lesions

(A) Squamous carcinoma of the esophagus, examined by MPM (63x), demonstrates nonkeratinized, stratified squamous epithelium. In contrast to normal squamous epithelia, borders between cells are less distinct, cell nuclei are larger and more heterogenous in size, and the nuclear to cytoplasmic ratio is much greater. (B) Squamous carcinoma of the esophagus, examined by H&E (40x), shows features that corresponds to the MPM image. (C) Colonic adenoma, examined by MPM (63x), demonstrates glandular pattern similar to what was observed in normal colon, but with heterogeneous gland sizes, elongated and irregular nuclei, much sparser and less regular interstitial fibers, and little to no cellular infiltrate in the interstitium. (D) Colonic adenoma, examined by H&E (40x), correlates well with the MPM image.

Figure 6

Three-dimensional reconstruction of gastrointestinal mucosa examined by MPM. Serial optical sections of unfixed biopsies were obtained in order to create cross-sectional images in the x-y, y-z, and x-z planes. (A) Stomach, 63x. Serial images through a depth of 128 μm were collected to reveal cross-sections through several gastric glands. (B) Terminal ileum, 63x. Serial images through a depth of 102 μm were collected to reveal cross-sections through a single villous structure. (C) Rectum, 63x. Serial images through a depth of 104 μm were collected to reveal opposing sides of a typical rectal gland with its central crypt.

Figure 7

Rat colon imaged using a miniaturized multiphoton probe. (A) The 27x/0.7NA 3.2 mm Olympus “stick” objective lens has a field of view of 220 μm, and two-photon excitation was achieved using a Ti:S laser tuned to 740 nm, with average power 3 mW. (B) Multiphoton image of rat colon collected through the stick objective. As in the multiphoton images of Figure 4, mucosal epithelial cells of the circular colonic glands show peak autofluorescence in the 410-490 nm range (green), while cells in the interstitial space are detected at longer wavelengths (510-560 nm; red). Collagen second harmonic generation again appears as blue (detection at ~370 nm) and is in close proximity to the colonic glands.