The emerging second near-infrared (NIR-II, 1000–2300 nm) fluorescence imaging based on microscopy set-ups (NIR-II FLI MS) has been proven to enable real-time tracking of dynamic physiological processes and facilitate the reconstruction of clear two- and three-dimensional morphological images of living tissues, which can offer high signal-to-background ratio and imaging contrast of live tissues at the cellular level. Focusing on the significant role of NIR-II FLI MS in intravital imaging and bioanalysis, in this contribution, we begin with the performance trade-off between NIE-II fluorescence probes and microscopy set-ups, focused on specific requirements for fluorescent probes across different NIR-II FLI MS modalities and feasible strategies to enhance their optical performances of the different NIR-II probes. Subsequently, we summarize recent advances in NIR-II microscopy set-ups, with an emphasis on the operational principles and specific requirements of wide-field, confocal, and light-sheet systems for high-performance in vivo detection. The cutting-edged applications of these probes and set-ups for in vivo NIR-II FLI MS is also discussed in detail. Finally, we provide a comprehensive and interdisciplinary analysis of the enduring challenges and prospective directions within this field, aiming to inspire further development of novel NIR-II high-performance probes and microscopy technologies for advanced intravital detection and facilitate the transition of in vivo NIR-II FLI MS from “technology-driven” to “science-driven”.
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