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
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
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).
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.
