Raman microspectroscopy, an alliance of Raman spectroscopy and confocal optical microscopy, is now widely used for label-free and molecular-level investigations of living cells. Here I review recent technical advances in our research group of the hardware as well as the software of Raman microspectroscopy of living cells. The advances in the hardware includes the developments of a multi-focus confocal Raman microspectrometer and a fast spiral raster scanning confocal Raman microspectrometer.These new techniques enable fast Raman imaging of living cells in several to several tens of second, an order of magnitude faster than existing commercial Raman microspectrometers. The new software utilizes the multivariate curve resolution-alternating least squares (MCR-ALS) method to obtain molecular component distribution imaging of living cells. Under appropriate model constraints, such as non-negativity and norm regularization for spectral profiles and their intensities, this new software provides physically interpretable spectral components without a priori knowledge on existing chemical components. These new software/hardware have been successfully applied to fast, automatic and label-free discrimination of living cells such as white blood cells and cardiac muscle cells.

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We developed an application of femtosecond-laser ablation to create a microscopic injury at selected sites of zebrafish heart. We analyzed the morphological and functional recovery of the zebrafish heart during regeneration. Our results might provide insight into the development of novel regenerative therapy.

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Full-field optical coherence tomography (FF-OCT)is suitable for rapid en-face imaging as it uses a 2D imaging device for planar interference and pixel processing of a sample plane. In this study, a red LED was applied in an FF-OCT system for skin imaging. A three-dimensional derm tissue image was obtained from the system to demonstrate the potential of LED in FF-OCT system. Noninvasive 3D skin imaging contributes to advance dermatology and cosmetology.

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Two-photon fluorescence microscopy (2PFM) features deep penetration and high resolution. However, the near infrared excitation of 2PFM results in inferior resolution to that of confocal imaging. To further enhance the resolution of 2PFM, here we propose super-resolution 2PFM. Resolution improvement is demonstrated in brain neurons imaging.

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We established the FRET fluorophore-labeling Dengue virus particles to visualize viral uncoating in living cells. In acceptor-bleaching imaging and single-particle tracking, the FRET signal decay was colocalized with autophagosomes. In addition, the autophagy inhibitors caused the reduction of viral uncoating process, suggesting that Dengue virus uncoating is correlated with autophagy.

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