Enhanced collection of scattered photons in nonlinear fluorescence microscopy by extended epi-detection with a silicon photomultiplier array

Abstract

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.

摘要

为提高非线性荧光显微技术在活体成像中的信号探测能力, 人们常采用基于非退扫描的背向探测策略。然而, 由于生物样本中存在严重散射, 物镜前孔径处大部分荧光光子辐射角度超出物镜收集角范围, 因而无法被探测。本文提出一种将硅光电倍增管阵列放置在物镜前孔径处以增强非线性显微镜对散射光子探测能力的扩展探测方案。通过数值仿真说明了扩展探测在对人体皮肤和小鼠大脑(透过颅窗及完整颅骨)进行多光子荧光成像的信号增强情况。例如, 在人类皮肤600 µm成像深度处使用红色染料标记情况下, 使用10×, 0.6NA物镜进行成像, 扩展探测方案引入的信号增强可达约150%。本文论证了扩展探测是一种灵活、广泛适用于非线性荧光显微镜增强探测信号的技术。