**2.5 Applications of CARS spectroscopy and microscopy**

As one of noninvasive research tools with high sensitivity, specificity and resolution, CARS microscopy has attracted more and more attention and been widely used in physics, chemistry, biology, medicine and life science et al. The capabilities and availability of CARS microscopy has been further improved with the recent technique's advances. Many exciting results have been presented in many literatures.

Because of the label-free characteristic of CARS microscopy, it has been regarded in the biological research, especially in the unstained cells. The first CARS microscopy was used to obtain the structural image of epidermal cells of onion immersed in D2O [21]. The water diffusion in live dictyostelium cells was researched with a broad vibrational resonance centered at 3300 cm-1, which could not be observed with fluorescence microscopy [75]. These early experimental results have proved that the CARS microscopy is an effective complementary method of fluorescence microscopy. Since many cellular processes take place on a subsecond timescale, high temporal resolution is required. By improving the temporal resolution, it is possible to image the chromosome distribution during mitosis using the symmetric stretching vibration of the DNA phosphate backbone [76]. Because of the good detectability of lipids, the structural and functional images of various living cells were obtained with CH bond of lipid [75, 77-79]. The sensitivity of CARS microscopy is high enough to detect lipid vesicles with sizes smaller than 300 nm in diameter [79]. Compared with fluorescence microscopy, CARS microscopy allows long-term investigations of cell without photobleaching. Therefore it can be used to long-term track biological molecules, such as lipid droplets, in living cells [80]. Nan and associates used the CARS microscopy to study the growth and transport of lipid droplets in live cells [79]. By tuning to the CH2 lipid vibration, Cheng and his colleagues observed the apoptosis, and identified different stages in the apoptotic process [76]. Potma and his associates visualized the intracellular hydrodynamics with the CARS signal of the O-H stretching vibration of water [81].

On the basis of cell imaging, the CARS microscopy is used in the living animal's tissue imaging, in which the tissue's optical properties, such as absorbability and scattering, are of obvious concern. The method of epi-detection is a good solution in tissue imaging with CARS. CARS microscopy has been successfully used for imaging of nonstained axonal myelin in spinal tissues in vitro [82]. Both the forward and backward CARS signals from the tissue slab were detected. The lipid distributions in skin tissue of live animals have been observed [83]. These all preliminary experimental results show us a vast potential of CARS microscopy in biomedical imaging and early diagnosis of diseases.
