Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation

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.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation

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.

脑神经活动光学显微成像技术

大脑包含数亿至数千亿的神经元以及更为复杂的神经突触连接网络,是生物体中最复杂的器官.脑科学是21世纪以来最重要的前沿新兴学科之一,它的兴起标志着人类在认识自我、探索智慧和意识的本质中进入了一个新时代.在活体中对大脑神经活动进行长时间、大视野、高时空分辨率的观测,是解析大脑功能的关键.光学显微成像技术以其时空分辨率高,光学探针的特异性和多样性等优势,成为了脑神经活动研究的重要工具.针对大脑的高度散射、高速神经信号传递、超大神经元规模、精细突触连接结构等特性以及自由活动动物的脑神经活动观测需求,本文将从超深、超快、大视场、超分辨、微型化5个发展方向,概述包括多光子、红外二区、光声、光片、结构光以及自适应光学在内的多种光学显微成像技术在脑神经活动显微观测领域的发展进展及前沿动态,并展望脑神经活动光学显微成像技术的未来发展方向与前景.

Super-resolution fluorescence-assisted diffraction computational tomography reveals the threedimensional landscape of the cellular organelle interactome

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.

Miniature Fluorescence Microscopy for Imaging Brain Activity in Freely-Behaving Animals

An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.

Live-cell superresolution pathology reveals different molecular mechanisms of pelizaeus-merzbacher disease

Modern cellular pathology relies on conventional microscopes to study the etiology and pathogenesis of diseases,which suffer from limited spatial resolution,little dynamic information due to sample fixation,and the lack of molecular information.For example,Pelizaeus-Merzbacher disease(PMD)is a genetic disorder of the central nervous system caused by different duplications or mutations of the proteolipid protein 1 gene(PLP1)in oligodendrocytes,which leads to hypomyelination and leukodystrophy in patients classified into either classical,transitional,or connatal phenotype[1].However,the correlations between genotypes and phenotypes at cellular level remain elusive.

Ultrafast,accurate,and robust localization of anisotropic dipoles

The resolution of single molecule localization imaging techniques largely depends on the precision of localization algorithms.However,the commonly used Gaussian function is not appropriate for anisotropic dipoles because it is not the true point spread function.We derived the theoretical point spread function of tilted dipoles with restricted mobility and developed an algorithm based on an artifi cial neural network for estimating the localization,orientation and mobility of individual dipoles.Compared with fi tting-based methods,our algorithm demonstrated ultrafast speed and higher accuracy,reduced sensitivity to defocusing,strong robustness and adaptability,making it an optimal choice for both two-dimensional and threedimensional super-resolution imaging analysis.

3D Hessian deconvolution of thick light-sheet z-stacks for high-contrast and high-SNR volumetric imaging

Due to its ability of optical sectioning and low phototoxicity,z-stacking light-sheet microscopy has been the tool of choice for in vivo imaging of the zebrafish brain.To image the zebrafish brain with a large field of view,the thickness of the Gaussian beam inevitably becomes several times greater than the system depth of field(DOF),where the fluorescence distributions outside the DOF will also be collected,blurring the image.In this paper,we propose a 3D deblurring method,aiming to redistribute the measured intensity of each pixel in a light-sheet image to in situ voxels by 3D deconvolution.By introducing a Hessian regularization term to maintain the continuity of the neuron dis-tribution and using a modified stripe removal algorithm,the reconstructed z stack images exhibit high contrast and a high signal-to-noise ratio.These performance characteristics can facilitate subsequent processing,such as 3D neuron registration,segmentation,and recognition.

Alterations of the Ca2＋ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca2＋-dependent manner. In diabetic animal models, different aspects of the calcium signaling path- way in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca2＊ （[Ca2＊]~） via Ca2＋ influx, Ca2＊ mobilization from endoplasmic reticulum （ER） calcium stores, and the removal of Ca2＋ via multiple mechanisms in beta-cells from both diabetic db/db mice and non- diabetic C57BL/6J mice. We refined our previous quan- titative model to describe the slow [Ca2＋]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca2＋-ATPase （SERCA） pump, SERCA2 and SERCA3, were severely down-reg- ulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduc- tion in the Ca2＋ concentration in the ER, a compensatory up-regulation of the plasma membrane Na＋/Ca2＋ exchanger （NCX） and a reduction in depolarizationevoked Ca2＋ influx. As a result, the patterns of glucosestimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.

Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells

Although bulk endocytosis has been found in a number of neuronal and endocrine cells,the molecular mechanism and physiological function of bulk endocytosis remain elusive.In pancreatic beta cells,we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization.Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca^(2+) entry and suppressed by the inhibition of dynamin function.Moreover,defects in bulklike endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice,which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability.

Mitochondria determine the sequential propagation of the calcium macrodomains revealed by the super-resolution calcium lantern imaging

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.