In vivo fluorescence imaging is an essential tool for many biomedical researches and clinical applications. It provides intuitive, direct evidence to help researchers understand complicated biological phenomena and clinicians to make better decisions. In this article, we will introduce important tissue optical properties that determine the performance of in vivo fluorescence images. Besides the biological specimen itself, imaging methods and fluorophores are the two key components that determine its performance and capabilities. Several types of imaging methods, suitable cameras, and the currently available fluorophores will be described. In the last section, we focus on the clinical applications, mainly image‐guided surgeries. The future development of in vivo fluorescence imaging tackles the challenge of deep tissue imaging, and short‐wave infrared fluorescence will be our new solution. Therefore, we will also comment on the recent developments of SWIR techniques in each topic discussed.
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