Person:

Xie, Xiaoliang

Raman microscopy is a quantitative, label-free, and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman scattering (SRS) microscopy have significantly increased the acquisition speed of Raman based label-free imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. On the basis of a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of cholesterol in the presence of interfering chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, hyperspectral imaging data in the C–H stretching region is obtained within a minute. We show that mammalian cell SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol, and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.

The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.