White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imagi...White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imaging in animal models in vivo.The third harmonic generation(THG)signal can be generated from white matter,but the bottom part of the white matter layer generates weak THG due to its high scattering.Here,we demonstrate an in vivo labeling and imaging technology,capable of visualizing the white matter layer in the mouse brain,combining°uorescence labeling with MitoTracker Red and three-photon°uorescence(3PF)microscopy excited at the 1700 nm window.3PF signals are several times higher than THG signals,resulting in deeper imaging of the white matter layer with the former.Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.展开更多
Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditiona...Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.展开更多
Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the in...Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the instantaneous bloodflow velocity distribution.This is mainly due to the lack of measurement technology with high spatial and temporal resolution.Here,we tackle this problem with our recently developed dual-wavelength line-scan third-harmonic generation(THG)imaging technology.Simultaneous acquisition of dual-wavelength THG line-scanning signals enables measurement of bloodflow velocities at two radially symmetric positions in both venules and arterioles in mouse brain in vivo.Our results clearly show that the instantaneous bloodflow velocity is not symmetric under general conditions.展开更多
文摘超分辨结构光照明显微成像技术(super-resolution structured illumination microscopy,SR-SIM)具有时间分辨率高、光漂白和光毒性低和对荧光探针的要求少等优点,适用于活细胞的长时程超分辨成像.采用二维晶格结构光作为照明光,可以实现更快的成像速度和更低的光毒性,但同时也增加了系统的复杂性.为了解决此问题,本文提出了一种基于数字微镜器件的快速超分辨晶格结构光照明显微成像方法(digital micromirror device-based lattice SIM,DMD-Lattice-SIM),通过同步分时触发DMD和sCMOS相机的方式实现二维正交晶格结构光的产生,且只需要采集5幅相移原始图像即可重构出超分辨图像,相比于传统SR-SIM需要9幅相移原始图像的方法,图像采集时间减少了约44.4%.同时,在基于空域和频域联合的SIM重构算法(joint space and frequency reconstruction method-SIM,JSFR-SIM)的基础上,本文还发展了用于Lattice-SIM的JSFR超分辨图像重构方法(Lattice-JSFR-SIM),先在频域对原始图像进行预滤波处理;然后,在空域对滤波后的图像进行超分辨重构处理.与传统频域图像重构处理对比,该方法在512×512像素数的成像视场下重构时间减少了约55.6%,对于实现活细胞实时超分辨成像具有重要意义和应用价值.
基金funded by the National Natural Science Foundation of China(62075135,61975126)Shenzhen Key Laboratory of Photonics and Biophotonics(ZDSYS20210623092006020).
文摘White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imaging in animal models in vivo.The third harmonic generation(THG)signal can be generated from white matter,but the bottom part of the white matter layer generates weak THG due to its high scattering.Here,we demonstrate an in vivo labeling and imaging technology,capable of visualizing the white matter layer in the mouse brain,combining°uorescence labeling with MitoTracker Red and three-photon°uorescence(3PF)microscopy excited at the 1700 nm window.3PF signals are several times higher than THG signals,resulting in deeper imaging of the white matter layer with the former.Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.
基金funded by the National Natural Science Foundation of China(62125504,61827825,and 31901059)Zhejiang Provincial Ten Thousand Plan for Young Top Talents(2020R52001)Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.
基金funded by the National Natural Science Foundation of China(Grant/Award Numbers 62075135 and 61975126)the Science and Technology Innovation Commission of Shenzhen(Grant/Award Numbers JCYJ20190808174819083 and JCYJ20190808175201640)Shenzhen Science and Technology Planning Project(ZDSYS 20210623092006020).
文摘Measurement of bloodflow velocity is key to understanding physiology and pathology in vivo.While most measurements are performed at the middle of the blood vessel,little research has been done on characterizing the instantaneous bloodflow velocity distribution.This is mainly due to the lack of measurement technology with high spatial and temporal resolution.Here,we tackle this problem with our recently developed dual-wavelength line-scan third-harmonic generation(THG)imaging technology.Simultaneous acquisition of dual-wavelength THG line-scanning signals enables measurement of bloodflow velocities at two radially symmetric positions in both venules and arterioles in mouse brain in vivo.Our results clearly show that the instantaneous bloodflow velocity is not symmetric under general conditions.
