CARS
Vibrational microscopy is an imaging method for visualizing several key molecular compounds in biological materials that neither fluoresce nor have a SHG sensitive non-centrosymmetric configuration. Contrast is derived from the molecular vibrational signatures that manifest themselves in the Raman spectrum. Many vibrational bands are excellent labels for classes of endogenous molecules. The C-H stretching region of the spectrum, for instance, is an excellent indicator of lipids, a crucial tissue component. Figure 1 shows CARS lipid signals taken in our labs from three breast cancer cell lines with increasing malignancy, clearly showing the different lipid distribution as a function of cancerous state.
Figure 1: CARS images (100 x 100 μm) of live nonmalignant (MCF-12A, left), mildly malignant (MCF-7, middle), and malignant (MBA-MB-231, right) breast cancer cells. The bright spots are the lipid droplets. Note that the number of lipid droplet correlates inversely with the malignancy of the cells. This observation is in concert with depletion of lipid pools in breast cancer tissue as seen with NMR and DOS/I. The laser was set to the 2845 cm-1 symmetric CH2 stretching vibration of lipids.
CARS microscopy is a nonlinear Raman technique with 3D resolution that enables rapid acquisition of vibrationally sensitive images. It has been demonstrated that CARS clearly resolves lipid structures in cells and in tissue in vivo with high sensitivity. The CARS technique has also been used to measure cellular hydration maps and image protein densities in single cells. Similar to TPEF and SHG, CARS is efficiently generated with ultrashort near-infrared pulses, and can be combined with other nonlinear imaging modalities.
