激光扫描共聚焦显微镜是生物、医药、化学等科学领域成像研究的一个重要工具,也是目前大型仪器平台不可或缺的成像设备。LSM900 with Airyscan2是目前市面上较新的一款多功能、快速、高分辨率的激光扫描共聚焦显微镜。对激光扫描共聚焦...激光扫描共聚焦显微镜是生物、医药、化学等科学领域成像研究的一个重要工具,也是目前大型仪器平台不可或缺的成像设备。LSM900 with Airyscan2是目前市面上较新的一款多功能、快速、高分辨率的激光扫描共聚焦显微镜。对激光扫描共聚焦显微镜的原理、其运行的环境要求、管理人员职责、开放共享管理以及设备维护保养注意事项几方面做详细地介绍,供相关管理人员与使用者参考。展开更多
Zeiss LSM 880 with Airyscan是一款多功能、高分辨率、高效率的激光扫描共聚焦显微镜(以下称激光共聚焦显微镜)。结合使用和管理的经验,本文对激光共聚焦显微镜的原理、LSM 880 with Airyscan的高级功能及日常维护管理等内容进行详细...Zeiss LSM 880 with Airyscan是一款多功能、高分辨率、高效率的激光扫描共聚焦显微镜(以下称激光共聚焦显微镜)。结合使用和管理的经验,本文对激光共聚焦显微镜的原理、LSM 880 with Airyscan的高级功能及日常维护管理等内容进行详细介绍。列举了Airyscan、长时间多视野拍摄、Experiment Designer、荧光漂白后恢复(FRAP)、光谱扫描/拆分、荧光能量共振转移(FRET)、Tile Scan和Line Scan快速成像技巧等高级功能,供管理人员或同行使用者借鉴参考。展开更多
Age-related macular degeneration is the leading cause of irreversible visual impairment in the elderly.It manifests in two forms,wet and dry.However,the mechanisms underlying spontaneous dry age-related macular degene...Age-related macular degeneration is the leading cause of irreversible visual impairment in the elderly.It manifests in two forms,wet and dry.However,the mechanisms underlying spontaneous dry age-related macular degeneration(SD-AMD)remain unclear.Herein,we constructed a single-cell retinal transcription atlas in aged non-human primates with SD-AMD.Retinal tissues affected by SD-AMD exhibited a more degenerative and dysfunctional transcriptomic landscape,with global activation of the oxidative stress response and apoptotic signaling pathway.We found two distinct Müller glia subtypes in normal aged and SD-AMD macaques,one exhibiting a photoreceptor-like transcriptome and the other exhibiting a typical Müller glia transcriptome.As SD-AMD progressed,the proportion of photoreceptor-like Müller glial cells decreased,and photoreceptor-function-associated genes were downregulated,indicating weaker Müller glia potential to transit into photoreceptor-like functional states.Microglial cells showed activated features,and the complement system was activated during disease pathogenesis.We also found that the disruption of iron homeostasis and ferroptosis could promote SD-AMD pathogenesis in neural cells.Further experimentation revealed that a ferroptosis inhibitor exerted a profound rescuing effect in SD-AMD mouse models.Based on these results,our study introduces a path toward understanding the pathogenesis of SD-AMD in a non-human primate model at single-cell resolution.展开更多
Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins(RSFPs),super-resolution optical fluctuation imaging(SOFI)and its variant photochromic stochast...Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins(RSFPs),super-resolution optical fluctuation imaging(SOFI)and its variant photochromic stochastic optical fluctuation imaging(pcSOFI)are valuable tools for wide field super-resolution(SR)imaging.Live-cell(pc)SOFI,which requires a small number of original frames to reconstruct an SR image,is prone to structural discontinuity artifacts and low spatial resolution.Herein,we developed a repeated synchronized on-and gradually off-switching SOFI(SOGO-SOFI)that maximized the photoswitching frequency of RSFPs by light modulation and required only 20 frames for high-quality reconstruction.Live-cell SOGO-SOFI imaging of the endoplasmic reticulum(ER)exhibited 10 times higher temporal resolution(100 fps)and fewer artifacts than pcSOFI.Moreover,a combination of SOGO-SOFI with Airyscan further increased the image contrast and the resolution of Airyscan by a factor of 1.5 from 140 nm to 91 nm.The capabilities of SOGO-SOFI were further demonstrated by dual-color imaging of nucleolar proteins in mammalian cells and deep imaging of ER structures in thick brain slices(20.6µm).展开更多
文摘激光扫描共聚焦显微镜是生物、医药、化学等科学领域成像研究的一个重要工具,也是目前大型仪器平台不可或缺的成像设备。LSM900 with Airyscan2是目前市面上较新的一款多功能、快速、高分辨率的激光扫描共聚焦显微镜。对激光扫描共聚焦显微镜的原理、其运行的环境要求、管理人员职责、开放共享管理以及设备维护保养注意事项几方面做详细地介绍,供相关管理人员与使用者参考。
基金supported by the National Key Research and Development Program of China(2017YFA0105804 and 2020YFA0112701).
文摘Age-related macular degeneration is the leading cause of irreversible visual impairment in the elderly.It manifests in two forms,wet and dry.However,the mechanisms underlying spontaneous dry age-related macular degeneration(SD-AMD)remain unclear.Herein,we constructed a single-cell retinal transcription atlas in aged non-human primates with SD-AMD.Retinal tissues affected by SD-AMD exhibited a more degenerative and dysfunctional transcriptomic landscape,with global activation of the oxidative stress response and apoptotic signaling pathway.We found two distinct Müller glia subtypes in normal aged and SD-AMD macaques,one exhibiting a photoreceptor-like transcriptome and the other exhibiting a typical Müller glia transcriptome.As SD-AMD progressed,the proportion of photoreceptor-like Müller glial cells decreased,and photoreceptor-function-associated genes were downregulated,indicating weaker Müller glia potential to transit into photoreceptor-like functional states.Microglial cells showed activated features,and the complement system was activated during disease pathogenesis.We also found that the disruption of iron homeostasis and ferroptosis could promote SD-AMD pathogenesis in neural cells.Further experimentation revealed that a ferroptosis inhibitor exerted a profound rescuing effect in SD-AMD mouse models.Based on these results,our study introduces a path toward understanding the pathogenesis of SD-AMD in a non-human primate model at single-cell resolution.
基金support for the LSM980 microscopy operationfunded by the National Key Research and Development Program of China(2017YFA0505300)+1 种基金the Natural Science Foundation of China(21927813,32027901,21778069,31870857)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB37040301).
文摘Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins(RSFPs),super-resolution optical fluctuation imaging(SOFI)and its variant photochromic stochastic optical fluctuation imaging(pcSOFI)are valuable tools for wide field super-resolution(SR)imaging.Live-cell(pc)SOFI,which requires a small number of original frames to reconstruct an SR image,is prone to structural discontinuity artifacts and low spatial resolution.Herein,we developed a repeated synchronized on-and gradually off-switching SOFI(SOGO-SOFI)that maximized the photoswitching frequency of RSFPs by light modulation and required only 20 frames for high-quality reconstruction.Live-cell SOGO-SOFI imaging of the endoplasmic reticulum(ER)exhibited 10 times higher temporal resolution(100 fps)and fewer artifacts than pcSOFI.Moreover,a combination of SOGO-SOFI with Airyscan further increased the image contrast and the resolution of Airyscan by a factor of 1.5 from 140 nm to 91 nm.The capabilities of SOGO-SOFI were further demonstrated by dual-color imaging of nucleolar proteins in mammalian cells and deep imaging of ER structures in thick brain slices(20.6µm).
