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 fluores...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 600μm 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.展开更多
基金Project supported by the National Natural Science Foundation of China(Nos.61831014 and 61771287)the Tsinghua University Initiative Scientific Research Program,China(No.20193080076)the Graduate Education Innovation Grants,Tsinghua University,China(No.201905J003)。
文摘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 600μm 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.
