Light penetration depth in biological tissue is limited by tissue scattering.Correcting scattering becomes particularly challenging in scenarios with limited photon availability and when access to the transmission sid...Light penetration depth in biological tissue is limited by tissue scattering.Correcting scattering becomes particularly challenging in scenarios with limited photon availability and when access to the transmission side of the scattering tissue is not possible.Here,we introduce,to our knowledge,a new two-photon microscopy system with Fourier-domain intensity coupling for scattering correction(2P-FOCUS).2P-FOCUS corrects scattering by intensity modulation in the Fourier domain,leveraging the nonlinearity of multiple-beam interference and twophoton excitation,eliminating the need for a guide star,iterative optimization,or measuring transmission or reflection matrices.2P-FOCUS uses random patterns to probe scattering properties,combined with a single-shot algorithm to rapidly generate the correction mask.2P-FOCUS can also correct scattering beyond the limitation of the memory effect by automatically customizing correction masks for each subregion in a large field-of-view.We provide several proof-of-principle demonstrations here,including focusing and imaging through a bone sample,and imaging neurons and cerebral blood vessels in the mouse brain ex vivo.2P-FOCUS significantly enhances two-photon fluorescence signals by several tens of folds compared to cases without scattering correction at the same excitation power.2P-FOCUS can also correct tissue scattering over a 230μm×230μm×510μm volume,which is beyond the memory effect range.2P-FOCUS is able to measure,calculate,and correct scattering within a few seconds,effectively delivering more light deep into the scattering tissue.2P-FOCUS could be broadly adopted for deep tissue imaging owing to its powerful combination of effectiveness,speed,and cost.展开更多
Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging r...Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging rates are typically low,limiting the ability to image whole cleared tissues and large surgical specimens.While innovations in TPFM technology,such as parallel-scanning,have drastically increased imaging speed,these improvements have typically focused on high frame rate,single field-of-view imaging rather than extending the area/volume imaging rate.In this work,we bridge the gap between high imaging speed and high area and volumetric imaging throughput by combining parallel scanning with tilted-plane strip-scanning using custom silicon photomultiplier(SiPM)tiled-array detectors.We demonstrate 200 MP/s with four spectral channels(800 MSpectra/s)and an effective area imaging speed of up to 52 mm^(2)∕s using four parallel beams.Custom detectors and lens array enable non-descanned imaging with minimal crosstalk combined with light collection efficiency comparable to a conventional single-point scanning TPFM.Finally,the low-cost of the custom detectors(∼$250 per channel)and the scalability of the detection optics allow for ease of spectral multiplexing.展开更多
文摘Light penetration depth in biological tissue is limited by tissue scattering.Correcting scattering becomes particularly challenging in scenarios with limited photon availability and when access to the transmission side of the scattering tissue is not possible.Here,we introduce,to our knowledge,a new two-photon microscopy system with Fourier-domain intensity coupling for scattering correction(2P-FOCUS).2P-FOCUS corrects scattering by intensity modulation in the Fourier domain,leveraging the nonlinearity of multiple-beam interference and twophoton excitation,eliminating the need for a guide star,iterative optimization,or measuring transmission or reflection matrices.2P-FOCUS uses random patterns to probe scattering properties,combined with a single-shot algorithm to rapidly generate the correction mask.2P-FOCUS can also correct scattering beyond the limitation of the memory effect by automatically customizing correction masks for each subregion in a large field-of-view.We provide several proof-of-principle demonstrations here,including focusing and imaging through a bone sample,and imaging neurons and cerebral blood vessels in the mouse brain ex vivo.2P-FOCUS significantly enhances two-photon fluorescence signals by several tens of folds compared to cases without scattering correction at the same excitation power.2P-FOCUS can also correct tissue scattering over a 230μm×230μm×510μm volume,which is beyond the memory effect range.2P-FOCUS is able to measure,calculate,and correct scattering within a few seconds,effectively delivering more light deep into the scattering tissue.2P-FOCUS could be broadly adopted for deep tissue imaging owing to its powerful combination of effectiveness,speed,and cost.
基金National Institute of Biomedical Imaging and Bioengineering(R21-EB032839)National Cancer Institute(R37-CA258376)。
文摘Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging rates are typically low,limiting the ability to image whole cleared tissues and large surgical specimens.While innovations in TPFM technology,such as parallel-scanning,have drastically increased imaging speed,these improvements have typically focused on high frame rate,single field-of-view imaging rather than extending the area/volume imaging rate.In this work,we bridge the gap between high imaging speed and high area and volumetric imaging throughput by combining parallel scanning with tilted-plane strip-scanning using custom silicon photomultiplier(SiPM)tiled-array detectors.We demonstrate 200 MP/s with four spectral channels(800 MSpectra/s)and an effective area imaging speed of up to 52 mm^(2)∕s using four parallel beams.Custom detectors and lens array enable non-descanned imaging with minimal crosstalk combined with light collection efficiency comparable to a conventional single-point scanning TPFM.Finally,the low-cost of the custom detectors(∼$250 per channel)and the scalability of the detection optics allow for ease of spectral multiplexing.
