Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia¶

Abstract

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 ؎ 39) relative to normal tissue (85 ؎ 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 ؎ 34 [dysplasia] vs 151 ؎ 44 [normal]) and 460 nm excitation (93 ؎ 35 [dysplasia] vs 137 ؎ 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17-40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Optical and Opto-Acoustic Interventional Imaging

“…Collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are predominant intrinsic tissue molecules that emit fluorescence upon excitation with UV or blue light. 42,63 Alterations in the biochemical composition of tissue during the early stages of carcinogenesis can be observed as a change in the fluorescence signals captured indicating cancer-associated tissue changes. Various wavelengths for both the excitation (300-480 nm) and emission (500-700 nm) of intrinsic fluorophores have been utilized (especially for auto-fluorescence spectroscopy), but typically blue light excitation (~450 nm) is preferred to UV light, due to the low UV transmittance of conventional optics.…”

Spectral imaging technology of epithelial tissue based on two-photon excited fluorescence and second-harmonic generation

“…In fact, during the forming of the epithelial carcinogenesis, the morphological structures of these three layers gradually develop some obvious alterations, such as increase in the keratinizing layer thickness, cellular nuclear enlargement in the epithelial cell layer and variation of collagen framework in the stromal layer [2][3][4][5]. The corresponding autofluorescence spectroscopy of different layers also shows apparent changes [6][7][8][9]. In biology and medicine, the spectroscopic measurement is combined with an optical microscope to generate images with spatially-resolved biochemical functional information, which is known as spectral imaging.…”

Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

“…Although these problems can be overcome with the use of fluorescent microscopy, the complication of inherent autofluorescence has to be dealt with. Autofluorescence has been noted in a variety of human and animal tissues and many investigators have suggested that this autofluorescence is inversely correlated with metabolic and proliferative activity, and hence the redox state of the tissue at fixation (Poot et al 1985, Shimazaki et al 1993, Drezek et al 2001. Interestingly, autofluorescence in the human hair follicle has recently been linked with the possible presence of melanocyte and keratinocytes stem cells (Wun et al 2005).…”