Visualizing extracellular matrix and sensing fibroblasts metabolism in human dermis by nonlinear spectral imaging

Abstract

The purpose of this work was to demonstrate the use of the nonlinear spectral imaging technique with the image-guided spectral analysis function and the extracting channels function in visualizing the extracellular maxtrix (ECM) structures and sensing fibroblasts metabolism within fresh and untreated human dermis. A Zeiss LSM 510 META laser scanning microscopy and a femtosecond Ti: sapphire laser was used to obtain the nonlinear spectral images of human dermis. The nonlinear spectral imaging technique was useful in obtaining the biomorphology and biochemistry information from human dermis. By combining the image-guided spectral analysis function, intrinsic components were identified and their corresponding emission spectra can be analyzed. By integrating the extracting channels function, the information of ultrastructure of the ECM can be quantitatively obtained. The nonlinear spectral imaging technique has been demonstrated to be an effective technique for a detailed analysis and qualitative visualization of the ECM structure, and sensing of cell metabolism within fresh and untreated human dermis. Specifically, the ultrastructure of the ECM can be revealed, and collagen fibril spacing, elastic fiber diameters, the ration of collagen content over elastin can be quantitatively determined. It was found that the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) signals excited at 830 nm provide the information for a good estimation of cellular redox ratio and a parameter (NF), the ration of NADH over FAD fluorescence signal, is used for sensing fibroblasts metabolism. With the advent of the clinical portability of typical multiphoton microscopy, the technique has the potential to be applied for in vivo studies and clinical examinations.