The emergence of super-resolution(SR)fluorescence microscopy has rejuvenated the search for new cellular substructures.However,SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the...The emergence of super-resolution(SR)fluorescence microscopy has rejuvenated the search for new cellular substructures.However,SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment.Thus,we developed SR fluorescence-assisted diffraction computational tomography(SR-FACT),which combines label-free three-dimensional optical diffraction tomography(ODT)with two-dimensional fluorescence Hessian structured illumination microscopy.The ODT module is capable of resolving the mitochondria,lipid droplets,the nuclear membrane,chromosomes,the tubular endoplasmic reticulum,and lysosomes.Using dual-mode correlated live-cell imaging for a prolonged period of time,we observed novel subcellular structures named dark-vacuole bodies,the majority of which originate from densely populated perinuclear regions,and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane.This work demonstrates the unique capabilities of SR-FACT,which suggests its wide applicability in cell biology in general.展开更多
Despite the wide application of super-resolution(SR)microscopy in biological studies of cells,the technology is rarely used to monitor functional changes in live cells.By combining fast spinning disc-confocal structur...Despite the wide application of super-resolution(SR)microscopy in biological studies of cells,the technology is rarely used to monitor functional changes in live cells.By combining fast spinning disc-confocal structured illumination microscopy(SD-SIM)with loading of cytosolic fluorescent Ca2+indicators,we have developed an SR method for visualization of regional Ca2+dynamics and related cellular organelle morphology and dynamics,termed SR calcium lantern imaging.In COS-7 cells stimulated with ATP,we have identified various calcium macrodomains characterized by different types of Ca2+release from endoplasmic reticulum(ER)stores.Finally,we demonstrated various roles of mitochondria in mediating calcium signals from different sources;while mitochondria can globally potentiate the Ca2+entry associated with store release,mitochondria also locally control Ca2+release from the neighboring ER stores and assist in their refilling processes.展开更多
基金supported by grants from the National Natural Science Foundation of China(91750203,91854112,81925022,31521062,91850111,31901061,and 31327901)the National Science and Technology Major Project Programme(2016YFA0500400,2017YFC0110203,and SQ2016YFJC040028)+3 种基金the Beijing Natural Science Foundation(L172003,7152079,and 5194026)the National Postdoctoral Program for Innovative Talents(BX201800008)the China Postdoctoral Science Foundation(2019M650329)the High-performance Computing Platform of Peking University.
文摘The emergence of super-resolution(SR)fluorescence microscopy has rejuvenated the search for new cellular substructures.However,SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment.Thus,we developed SR fluorescence-assisted diffraction computational tomography(SR-FACT),which combines label-free three-dimensional optical diffraction tomography(ODT)with two-dimensional fluorescence Hessian structured illumination microscopy.The ODT module is capable of resolving the mitochondria,lipid droplets,the nuclear membrane,chromosomes,the tubular endoplasmic reticulum,and lysosomes.Using dual-mode correlated live-cell imaging for a prolonged period of time,we observed novel subcellular structures named dark-vacuole bodies,the majority of which originate from densely populated perinuclear regions,and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane.This work demonstrates the unique capabilities of SR-FACT,which suggests its wide applicability in cell biology in general.
基金Supported by the grants from the National Science and Technology Major Project Program(2016YFA0500400)the National Natural Science Foundation of China(81925022,91854112,31327901,31521062,31570839,91750203)and Beijing Natural Science Foundation(L172003,7182063).
文摘Despite the wide application of super-resolution(SR)microscopy in biological studies of cells,the technology is rarely used to monitor functional changes in live cells.By combining fast spinning disc-confocal structured illumination microscopy(SD-SIM)with loading of cytosolic fluorescent Ca2+indicators,we have developed an SR method for visualization of regional Ca2+dynamics and related cellular organelle morphology and dynamics,termed SR calcium lantern imaging.In COS-7 cells stimulated with ATP,we have identified various calcium macrodomains characterized by different types of Ca2+release from endoplasmic reticulum(ER)stores.Finally,we demonstrated various roles of mitochondria in mediating calcium signals from different sources;while mitochondria can globally potentiate the Ca2+entry associated with store release,mitochondria also locally control Ca2+release from the neighboring ER stores and assist in their refilling processes.
