In fluorescence microscopy,computational algorithms have been developed to suppress noise,enhance contrast,and even enable super-resolution(SR).However,the local quality of the images may vary on multiple scales,and t...In fluorescence microscopy,computational algorithms have been developed to suppress noise,enhance contrast,and even enable super-resolution(SR).However,the local quality of the images may vary on multiple scales,and these differences can lead to misconceptions.Current mapping methods fail to finely estimate the local quality,challenging to associate the SR scale content.Here,we develop a rolling Fourier ring correlation(rFRC)method to evaluate the reconstruction uncertainties down to SR scale.To visually pinpoint regions with low reliability,a filtered rFRC is combined with a modified resolution-scaled error map(RSM),offering a comprehensive and concise map for further examination.We demonstrate their performances on various SR imaging modalities,and the resulting quantitative maps enable better SR images integrated from different reconstructions.Overall,we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.展开更多
Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been a...Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been associated with hypomyelination.However,how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood.We used superresolution Hessian structured illumination microscopy(Hessian-SIM)and spinning discconfocal structured illumination microscopy(SD-SIM)to find that the wild-type TMEM106B protein is targeted to the plasma membrane,filopodia,and lysosomes in human oligodendrocytes.The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress.Most importantly,we detected reductions in the length and number of filopodia in cells expressing the D252N mutant.PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B,and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells.Therefore,interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes,which may be sustained by the delivery of these proteins from lysosomes via exocytosis.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 the National Natural Science Foundation of China(grant no.T2222009 to H.L.,grant no.32227802 to L.C.,grant no.81925022 to L.C.,grant no.92054301 to L.C.,grant no.62305083 to W.Z.,grant no.12174208 to P.L.,grant no.32301257 to S.Z.,grant no.32222022 to Y.J.,grant no.32071458 to H.M.)the National Key Research and Development Program of China(grant no.2022YFC3400600 to L.C.)+4 种基金the Natural Science Foundation of Heilongjiang Province(grant no.YQ2021F013 to H.L.)the Beijing Natural Science Foundation(grant no.Z20J00059 to L.C.)the Guangdong Major Project of Basic and Applied Basic Research(grant no.2020B0301030009 to P.L.)the China Postdoctoral Science Foundation(grant no.2023T160163 to W.Z.,grant no.2022M720971 to W.Z.)the Heilongjiang Provincial Postdoctoral Science Foundation(grant no.LBH-Z22027 to W.Z.).L.C.acknowledges support from the High-performance Computing Platform of Peking University。
文摘In fluorescence microscopy,computational algorithms have been developed to suppress noise,enhance contrast,and even enable super-resolution(SR).However,the local quality of the images may vary on multiple scales,and these differences can lead to misconceptions.Current mapping methods fail to finely estimate the local quality,challenging to associate the SR scale content.Here,we develop a rolling Fourier ring correlation(rFRC)method to evaluate the reconstruction uncertainties down to SR scale.To visually pinpoint regions with low reliability,a filtered rFRC is combined with a modified resolution-scaled error map(RSM),offering a comprehensive and concise map for further examination.We demonstrate their performances on various SR imaging modalities,and the resulting quantitative maps enable better SR images integrated from different reconstructions.Overall,we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.
基金supported by the National Natural Science Foundation of China(81925022,61827825,32227802,92054301)the Fundamental Research Center Project of the National Natural Science Foundation of China(T2288102)+4 种基金the National Science and Technology Major Project Program(2022YFC3400600)Beijing Natural Science Foundation Key Research Topics(Z20J00059)UMHS-PUHSC Joint Institute for Translational and Clinical Research(BMU2019JI009)Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases(BZ0317)China Postdoctoral Science Foundation(2021M690465)。
文摘Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been associated with hypomyelination.However,how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood.We used superresolution Hessian structured illumination microscopy(Hessian-SIM)and spinning discconfocal structured illumination microscopy(SD-SIM)to find that the wild-type TMEM106B protein is targeted to the plasma membrane,filopodia,and lysosomes in human oligodendrocytes.The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress.Most importantly,we detected reductions in the length and number of filopodia in cells expressing the D252N mutant.PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B,and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells.Therefore,interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes,which may be sustained by the delivery of these proteins from lysosomes via exocytosis.
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.