In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulate the photon propagation in the NIR region (to 2340 nm), confirm the positive contribution of moderate light absorption by water in intravital imaging and perfect the NIR-II window as 900–1880 nm, where 1400–1500 and 1700–1880 nm are defined as NIR-IIx and NIR-IIc regions, respectively. Moreover, 2080–2340 nm is newly proposed as the third near-infrared (NIR-III) window, which is believed to provide the best imaging quality. The wide-field fluorescence microscopy in the brain is performed around the NIR-IIx region, with excellent optical sectioning strength and the largest imaging depth of intravital NIR-II fluorescence microscopy to date. We also propose 1400 nm long-pass detection in off-peak NIR-II imaging whose performance exceeds that of NIR-IIb imaging, using bright fluorophores with short emission wavelength.

第二近红外窗口（NIR-II）中的体内荧光成像被认为是一种有前途的哺乳动物可视化技术。然而，NIR-II区域的定义和解释优异性能的机制仍需要完善。在这里，我们模拟了 NIR 区域（至 2340 nm）的光子传播，证实了水的适度光吸收在活体成像中的积极贡献，并将 NIR-II 窗口完善为 900–1880 nm，其中 1400–1500 和 1700– 1880 nm 分别定义为 NIR-IIx 和 NIR-IIc 区域。此外，新提出2080-2340 nm作为第三近红外（NIR-III）窗口，被认为可以提供最佳的成像质量。大脑中的宽视场荧光显微镜围绕 NIR-IIx 区域进行，具有出色的光学切片强度和迄今为止活体 NIR-II 荧光显微镜最大的成像深度。我们还建议在非峰值 NIR-II 成像中使用 1400 nm 长通检测，其性能超过 NIR-IIb 成像，使用具有短发射波长的明亮荧光团。