To maximize signal collection in nonlinear optical microscopy, non-descanned epi-detection is generally adopted for in vivo imaging. However, because of severe scattering in biological samples, most of the emitted fluorescence photons go beyond the collection angles of objectives and thus cannot be detected. Here, we propose an extended detection scheme to enhance the collection of scattered photons in nonlinear fluorescence microscopy using a silicon photomultiplier array ahead of the front apertures of objectives. We perform numerical simulations to demonstrate the enhanced fluorescence collection via extended epi-detection in the multi-photon fluorescence imaging of human skin and mouse brain through craniotomy windows and intact skulls. For example, with red fluorescence emission at a depth of 600 µm in human skin, the increased collection can be as much as about 150% with a 10×, 0.6-NA objective. We show that extended epi-detection is a generally applicable, feasible technique for use in nonlinear fluorescence microscopy to enhance signal detection.

为了在非线性光学显微镜中最大程度地收集信号，体内成像通常采用非反扫描的落射检测。但是，由于生物样品中的严重散射，大多数发射的荧光光子超出了物镜的收集角度，因此无法检测到。在这里，我们提出了一种扩展的检测方案，以在物镜前孔之前使用硅光电倍增管阵列来增强非线性荧光显微镜中散射光子的收集。我们进行数值模拟，以通过颅骨切开术窗口和完整的头骨通过人类皮肤和小鼠大脑的多光子荧光成像中的扩展光子检测来证明增强的荧光收集。例如，人体皮肤在600 µm深度处发出红色荧光，对于10倍，0.6 NA的物镜，增加的收集量可以高达150％。我们表明，扩展的落射检测是一种普遍适用的，可行的技术，用于非线性荧光显微镜以增强信号检测。