Characterization of third-degree burned skin by nonlinear microscopy technique

摘要

Nonlinear microscopy imaging technique enable take both images of collagen fibers in dermis through second harmonic generation (SHG) signal and elastic fibers by two-photon emission fluorescence microscopy (TPEFM). These techniques are the most commonly used technique for turbid and thick tissue imaging and also to image biological samples which presents highly ordered structural proteins without any exogenous label. The objective of this study is characterizing dermis of third-degree burned skin by TPEFM and SHG technique. The modelocked laser (Spectra Physics) source used in this study with pulse width of approximately 100 fs at 80 MHz was directed into a multiphoton microscope using a laser scanning unit (Olympus Fluoview 300), mounted on an inverted confocal system microscope (Olympus 1X81), with focusing objective (40x, NA = 1.30). The samples were obtained from Wistar rats, male, adult. One dorsum area was submitted to burn caused by vapour exposure. The biopsies obtained were cryosectioned in slices of 20 xm width. Selected area of interface between the injured and healthy subdermal burned skin were imaged by TPEFM and SHG technique. Two different autofluorescence signals are observed as a function of excitation wavelength. The autofluorescence observed at 760 nm and 690 nm suggest components of extracellular matrix at differents depths. In SHG images, collagen fibers are visible. According to the images obtained, these methodologies can be used to characterize dermis of burned tissue as its healing process with reduced out-of-plane photobleaching and phototoxicity.