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

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.

Recognizing local artifacts in two-photon imaging of dendrites beneath blood vessels in vivo

Localized wavefront aberrations would introduce artifacts in biomedical imaging,which,however,are often neglected,as their compensations are at the cost of the field-of-view.Here,we show rarely reported local artifacts in two-photon imaging of dendrites beneath blood vessels in a mouse brain in vivo and interpret the phenomena via numerical simulations.The artifacts of divided parallel structures are found to be induced by coma and astigmatism,resulting from sample tilting and the cylinder shape of vasculatures,respectively.Different from that in single-photon microscopy,such artifacts in nonlinear microscopy show unique characteristics and should be recognized for proper interpretation of the images.

Improving signal-to-background ratio by orders of magnitude in high-speed volumetric imaging in vivo by robust Fourier light field microscopy

Fourier light field microscopy(FLFM)shows great potential in high-speed volumetric imaging of biodynamics.However,due to the inherent disadvantage of wide-field illumination,it suffers from intense background,arising from out of the depth-of-field signal and tissue scattered noise.The background will not only deteriorate the image contrast,making quantitative measurement difficult,but also introduce artifacts,especially in functional imaging of the neuronal network activity in vivo.Here,we propose the robust Fourier light field microscopy(RFLFM),which suppresses the background in FLFM by introducing structured illumination and computational reconstruction based on HiLo.The superior performance of RFLFM is verified by volumetric imaging of biological dynamics in larval zebrafish and mouse in vivo,at a volumetric imaging rate up to 33.3 Hz.The statistical results show that the fluorescence background can be significantly depressed,with the signal-to-background ratio improved by orders of magnitude and the whole image contrast improved by as much as~10.4 times.Moreover,we stress that,in functional imaging of neuronal network activity in turbid brain tissues,our system can avoid artifacts resulting from background fluctuations,while conventional light field microscopy fails.As a simple but powerful tool,we anticipate our technique to be widely adopted in robust,high-contrast,high-speed volumetric imaging.