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Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system.
Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin
are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living
cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular
protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used
Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to
simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and
morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation
from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear
optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique
approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular,
with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan
signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value
dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in
epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early
development of cancer in epithelial tissue.
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