Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous
Raman microscopy provides specific vibrational signatures of chemical bonds, but is often
hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging
technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is
significantly greater than that of spontaneous Raman scattering, and is further enhanced
by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage
over previous coherent Raman techniques in that it offers
background-free and easily
interpretable chemical contrast. We show a variety of biomedical applications, such as
differentiating distributions of omega-3 fatty acids and saturated lipids in living cells,
imaging of brain and skin tissues based on intrinsic lipid contrast.
Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.