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| Coherent Raman Scattering Microscopy and Its Recent Research Progress in in Vivo Imaging |
| LI Shu-qi, LUO Guo-quan, CHEN Yu, YU Bin, QU Jun-le, LIN Dan-ying* |
Key Laboratory of Optoelectronic Devices and Systems of the Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
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Abstract In vivo imaging technology has revolutionary significance, enabling real-time observation of living organisms and providing dynamic information directly related to biological processes. This is crucial for understanding disease mechanisms and evaluating treatment effects. Coherent Raman scattering (CRS) microscopy offers the advantage of specific molecular imaging in biological samples without the need for fluorescent probes that might interfere with biomolecular function, making it a promising tool in the field of in vivo imaging. However, CRS microscopy still faces challenges in practical applications in in vivo imaging, including photodamage, limited imaging depth, and motion artifacts. Recent advancements in related technology have led to significant breakthroughs, addressing these challenges by minimizing photodamage, extending imaging depth, eliminating or reducing motion artifacts, and enabling multimodal imaging. In vivo real-time imaging of human skin, brain, and spine in experimental animal models and tumors has driven substantial progress in CRS microscopy, both in in vivo imaging research and clinical applications. This paper offers a comprehensive review of the latest developments in CRS microscopy for in vivo imaging, providing an in-depth analysis of current challenges and their solutions to contribute to this technology's ongoing development and broader application. Common strategies to overcome photodamage involve reducing the thermal effects and chemical reactions induced by the laser in the sample, typically by limiting laser power and integration time. Several approaches have been explored to address the limitation of imaging depth, including imaging superficial tissues such as the skin or areas near the surface, combining optical windows, or directly imaging deeper tissues or organs exposed through minimally invasive surgery. Adaptive optics technology helps balance depth with non-invasive imaging, while endoscopic imaging provides an additional solution. To minimize or eliminate motion artifacts, it is crucial first to keep the organism stationary or reduce movement through appropriate anesthesia and fixation techniques. In addition, optical windows and real-time motion correction algorithms can be employed to mitigate further jitter caused by physiological activities like breathing and heartbeat in anesthetized samples. Increasing imaging speed is another way to reduce motion artifacts. Finally, combining CRS with other nonlinear optical microscopy techniques, such as two-photon excitation fluorescence and second-harmonic generation, enables multimodal imaging, providing richer information, enhancing the analysis of in vivo biological samples, and offering deeper insights into biological processes.
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Received: 2025-01-20
Accepted: 2025-03-04
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Corresponding Authors:
LIN Dan-ying
E-mail: dylin@szu.edu.cn
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