The layered–resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy

Abstract

The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

Multiphoton microscopic imaging of adipose tissue based on second‐harmonic generation and two‐photon excited fluorescence

“…The TPEF image of the adipose cell in the adipose tissue is very different from that in the epithelial tissue. In the TPEF images of epithelial cells in the epithelial tissue, the nonfluorescent nuclei displayed darkly on the optical section are located in the cell center and surrounded by the fluorescent mitochondria and the clear cellular boundary can be seen (Zhuo et al 2007c). The special cell structure in the adipose tissue makes multiphoton microscopic imaging to be more suitable for investigating the adipose tissuerelated disease.…”

Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues

“…Several groups have used TPF spectroscopic analysis for in vivo or ex vivo tissue studies at one or a few excitation wavelengths. [5][6][7][8] Collection of the TPF emission spectrum as a function of excitation wavelength to generate a two-photon excitation-emission matrix (2P-EEM) can allow even greater sensitivity to distinguish fluorophores and disease states. EEM can also assist in choosing the optimal excitation/ emission wavelengths for the best performance of imaging analysis of specific tissue types.…”

Multiphoton microscopic imaging of human normal and cancerous oesophagus tissue

“…Recently, the multiphoton microscopy with high resolution and high sensitivity, based on two-photon excited fluorescence (TPEF) and second harmonic generation, is more advantageous for microstructures imaging of biotissues at cellular and subcellular level (Aparecida De Aro et al, 2012;Brown & McKee, 2003;Zipfel et al, 2003;Koehler et al, 2006;Zhuo et al, 2006;Zhuo et al, 2007;Chen et al, 2007;Ustione & Piston, 2011). The multiphoton microscopy has become a powerful tool for imaging biotissue samples without label and has been used to noninvasively evaluate and monitor morphological structure and functional state of epithelial tissues with layer structures (Georgakoudi et al, 2002;Skala et al, 2005;Liu et al, 2010).…”