Coherent Raman Scattering Microscopy in Biology and Medicine

Abstract

Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take.

Raman spectroscopy: techniques and applications in the life sciences

“…for stimulated Raman loss (SRL) in the pump field [22,23]. These intensity differences are typically small compared with I p and I s .…”

“…They were able to measure the 992 cm −1 SRG band of liquid benzene with high spectral resolution, free of the artifacts seen in CARS due to non-resonant background see Figure 4(c) . Extension of this idea to high-frequency modulation of pulsed laser beams was shown in the past decade by Xie and colleagues, who have demonstrated sufficient SRG/SRL detection sensitivity to measure microscopic biological samples (either SRL/SRG of the pump/Stokes fields can be used for generating image contrast by SRS, and provide similar sensitivity to one another [22]). This will be discussed further in Section 4.4.…”

“…However the choice can change the relative background contributions to the SRS signal, for example the two-photon absorption will generally be more prevalent in the SRL (pump beam) signal [49]. Further detailed discussions of instrumentation and noise considerations can be found in Min et al [23] and Zhang et al [22].…”

“…This increase in signal and imaging speed comes at the expense of diverse spectral information, often acquiring signal at only one or a few wavenumbers. [20][21][22][23]. The low Raman scattering signal also limits the technique's ability to detect small concentrations of molecules.…”

Effect of scattering on coherent anti-Stokes Raman scattering (CARS) signals

“…Coherent anti-Stokes Raman scattering (CARS) microscopy is a nonlinear, label-free imaging technique that has matured into a reliable tool for visualizing lipids, proteins and other endogenous compounds in biological tissues and cells based on their spatially-dependent third-order polarization [1][2][3]. In the CARS process, a pair of incoming beams (named "pump" and "Stokes") are exploited to coherently and resonantly excite selected vibrational levels of a population of molecules.…”