In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina.However,optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcell...In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina.However,optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures.We developed an adaptive optics two-photon excitation fluorescence microscopy(AO-TPEFM)system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina.With accurate wavefront sensing and rapid aberration correction,AOTPEFM permits structural and functional imaging of the mouse retina with submicron resolution.Specifically,simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated.Moreover,the timelapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder.In addition,precise laser axotomy was achieved,and degeneration of retinal nerve fibres was studied.This highresolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye diseases as well as neurodegenerative disorders in the central nervous system.展开更多
Imaging of the brain in its native state at high spatial resolution poses major challenges to visualization techniques.Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provid...Imaging of the brain in its native state at high spatial resolution poses major challenges to visualization techniques.Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provides a minimally invasive tool for in vivo imaging of the cortex of mice without activating immune response and inducing brain injury.However,the imaging contrast and spatial resolution are severely compromised by the optical heterogeneity of the skull,limiting the imaging depth to the superficial layer.In this work,an optimized configuration of an adaptive optics two-photon microscope system and an improved wavefront sensing algorithm are proposed for accurate correction for the aberrations induced by the skull window and brain tissue.Using this system,we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 700μm below pia in living mice.In addition,we investigated microglia–plaque interaction in living brain of Alzheimer’s disease and demonstrated high-precision laser dendrotomy and single-spine ablation.展开更多
基金supported by the Hong Kong Research Grants Council through grants 662513,16103215,16148816,16102518,16149316,T13-607/12R,T13-706/11-1,AOE/M-09/12,T13-605/18W,C6002-17GF,C6001-19EF and N_HKUST603/19the Hong Kong University of Science and Technology(HKUST)through grant RPC10EG33+1 种基金the Innovation and Technology Commission through grant ITCPD/17-9the Health and Medical Research Fund through grant HMRF18SC17.
文摘In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina.However,optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures.We developed an adaptive optics two-photon excitation fluorescence microscopy(AO-TPEFM)system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina.With accurate wavefront sensing and rapid aberration correction,AOTPEFM permits structural and functional imaging of the mouse retina with submicron resolution.Specifically,simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated.Moreover,the timelapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder.In addition,precise laser axotomy was achieved,and degeneration of retinal nerve fibres was studied.This highresolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye diseases as well as neurodegenerative disorders in the central nervous system.
基金Hong Kong University of Science and Technology(RPC10EG33)Area of Excellence Scheme of the University Grants Committee(AOE/M-09/12,AoE/M-604/16)+1 种基金Innovation and Technology Commission(ITCPD/17-9)Research Grants Council,University Grants Committee(16102518,16103215,16148816,662513,C6001-19E,C6002-17GF,N_HKUST603/19,T13-605/18W,T13-607/12R,T13-706/11-1).
文摘Imaging of the brain in its native state at high spatial resolution poses major challenges to visualization techniques.Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provides a minimally invasive tool for in vivo imaging of the cortex of mice without activating immune response and inducing brain injury.However,the imaging contrast and spatial resolution are severely compromised by the optical heterogeneity of the skull,limiting the imaging depth to the superficial layer.In this work,an optimized configuration of an adaptive optics two-photon microscope system and an improved wavefront sensing algorithm are proposed for accurate correction for the aberrations induced by the skull window and brain tissue.Using this system,we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 700μm below pia in living mice.In addition,we investigated microglia–plaque interaction in living brain of Alzheimer’s disease and demonstrated high-precision laser dendrotomy and single-spine ablation.
