Super-resolution Microscopy Imaging Research Articles

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

KCBC – a correlation-based method for co-localization analysis of super-resolution microscopy images using bivariate Ripley's K functions

Motivated by the high demand for co-localization analysis methods for super-resolution microscopy images which are featured with nanoscale precise locational information of molecules, this paper establishes a novel correlation-based method, KCBC, named after the Coordinated-Based Colocalization (CBC) method proposed by Malkusch et al. in 2012, by using bivariate Ripley's K functions. The local KCBC values are to quantify the local spatial co-localization of molecules between two species by measuring the correlation of bivariate Ripley's K functions over equal-area concentric rings around the base species within a near distance. The mean of local KCBC values is proposed to quantify the co-localization degree of cross-channel to base-channel molecules for the whole image. It could effectively correct the false positives with reduced variance and increased power within the user-defined proximity size. We provide extensive simulation studies under different scenarios to demonstrate the unbiasedness of the KCBC method, and its ability to filter noise signals and random over-counting. Our real data application for super-resolution mitochondria image data illustrates the applicability of our methods with increased effectiveness and power.

Machine learning-based 3D segmentation of mitochondria in polarized epithelial cells

Mitochondria are dynamic organelles that alter their morphological characteristics in response to functional needs. Therefore, mitochondrial morphology is an important indicator of mitochondrial function and cellular health. Reliable segmentation of mitochondrial networks in microscopy images is a crucial initial step for further quantitative evaluation of their morphology. However, 3D mitochondrial segmentation, especially in cells with complex network morphology, such as in highly polarized cells, remains challenging. To improve the quality of 3D segmentation of mitochondria in super-resolution microscopy images, we took a machine learning approach, using 3D Trainable Weka, an ImageJ plugin. We demonstrated that, compared with other commonly used methods, our approach segmented mitochondrial networks effectively, with improved accuracy in different polarized epithelial cell models, including differentiated human retinal pigment epithelial (RPE) cells. Furthermore, using several tools for quantitative analysis following segmentation, we revealed mitochondrial fragmentation in bafilomycin-treated RPE cells.

Fokker–Planck Analysis of Superresolution Microscopy Images

A method for the analysis of super-resolution microscopy images is presented. This method is based on the analysis of stochastic trajectories of particles moving on the membrane of a cell with the assumption that this motion is determined by the properties of this membrane. Thus, the purpose of this method is to recover the structural properties of the membrane by solving an inverse problem governed by the Fokker–Planck equation related to the stochastic trajectories. Results of numerical experiments demonstrate the ability of the proposed method to reconstruct the potential of a cell membrane by using synthetic data similar those captured by super-resolution microscopy of luminescent activated proteins.

Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations.

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins.

Deep learning enables stochastic optical reconstruction microscopy-like superresolution image reconstruction from conventional microscopy

Despite its remarkable potential for transforming low-resolution images, deep learning faces significant challenges in achieving high-quality superresolution microscopy imaging from wide-field (conventional) microscopy. Here, we present X-Microscopy, a computational tool comprising two deep learning subnets, UR-Net-8 and X-Net, which enables STORM-like superresolution microscopy image reconstruction from wide-field images with input-size flexibility. X-Microscopy was trained using samples of various subcellular structures, including cytoskeletal filaments, dot-like, beehive-like, and nanocluster-like structures, to generate prediction models capable of producing images of comparable quality to STORM-like images. In addition to enabling multicolour superresolution image reconstructions, X-Microscopy also facilitates superresolution image reconstruction from different conventional microscopic systems. The capabilities of X-Microscopy offer promising prospects for making superresolution microscopy accessible to a broader range of users, going beyond the confines of well-equipped laboratories.

Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and invivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Nanoscale reorganisation of synaptic proteins in Alzheimer's disease.

Synaptic strength depends strongly on the subsynaptic organisation of presynaptic transmitter release and postsynaptic receptor densities, and their alterations are expected to underlie pathologies. Although synaptic dysfunctions are common pathogenic traits of Alzheimer's disease (AD), it remains unknown whether synaptic protein nano-organisation is altered in AD. Here, we systematically characterised the alterations in the subsynaptic organisation in cellular and mouse models of AD. We used immunostaining and super-resolution stochastic optical reconstruction microscopy imaging to quantitatively examine the synaptic protein nano-organisation in both Aβ1-42-treated neuronal cultures and cortical sections from a mouse model of AD, APP23 mice. We found that Aβ1-42-treatment of cultured hippocampal neurons decreased the synaptic retention of postsynaptic scaffolds and receptors and disrupted their nanoscale alignment to presynaptic transmitter release sites. In cortical sections, we found that while GluA1 receptors in wild-type mice were organised in subsynaptic nanoclusters with high local densities, receptors in APP23 mice distributed more homogeneously within synapses. This reorganisation, together with the reduced overall receptor density, led to reduced glutamatergic synaptic transmission. Meanwhile, the transsynaptic alignment between presynaptic release-guiding RIM1/2 and postsynaptic scaffolding protein PSD-95 was reduced in APP23 mice. Importantly, these reorganisations were progressive with age and were more pronounced in synapses in close vicinity of Aβ plaques with dense cores. Our study revealed a spatiotemporal-specific reorganisation of synaptic nanostructures in AD and identifies dense-core amyloid plaques as the major local inductor in APP23 mice.

DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.

Keyframe-aided resolution enhancement network for dynamic super-resolution structured illumination microscopy.

Deep learning has been used to reconstruct super-resolution structured illumination microscopy (SR-SIM) images with wide-field or fewer raw images, effectively reducing photobleaching and phototoxicity. However, the dependability of new structures or sample observation is still questioned using these methods. Here, we propose a dynamic SIM imaging strategy: the full raw images are recorded at the beginning to reconstruct the SR image as a keyframe, then only wide-field images are recorded. A deep-learning-based reconstruction algorithm, named KFA-RET, is developed to reconstruct the rest of the SR images for the whole dynamic process. With the structure at the keyframe as a reference and the temporal continuity of biological structures, KFA-RET greatly enhances the quality of reconstructed SR images while reducing photobleaching and phototoxicity. Moreover, KFA-RET has a strong transfer capability for observing new structures that were not included during network training.

Scattering properties of Bessel beams on metal particles

Based on Generalized Lorenz-Mie Theory (GLMT), the scattering phenomenon caused by Bessel beam irradiation on metal particles is studied. The order and polarization mode of Bessel beam, as well as the influence of the size of metal particles on the total intensity distribution of the light field around metal particles are investigated by using the software simulation calculation. The results show that for spherical metal particles of the same size, the higher order Bessel beam is more likely to bypass the metal particles in the forward propagation, while the lower order Bessel beam bypassed the particles in the propagation is related to the beam polarization mode. The results have potential applications in microscopy super-resolution imaging and detection of tiny particles.

Apoptotic mitochondrial poration by a growing list of pore-forming BCL-2 family proteins.

The pore-forming BCL-2 family proteins are effectors of mitochondrial poration in apoptosis initiation. Two atypical effectors-BOK and truncated BID (tBID)-join the canonical effectors BAK and BAX. Gene knockout revealed developmental phenotypes in the absence the effectors, supporting their roles in vivo. During apoptosis effectors are activated and change shape from dormant monomers to dynamic oligomers that associate with and permeabilize mitochondria. BID is activated by proteolysis, BOK accumulates on inhibition of its degradation by the E3 ligase gp78, while BAK and BAX undergo direct activation by BH3-only initiators, autoactivation, and crossactivation. Except tBID, effector oligomers on the mitochondria appear as arcs and rings in super-resolution microscopy images. The BH3-in-groove dimers of BAK and BAX, the tBID monomers, and uncharacterized BOK species are the putative building blocks of apoptotic pores. Effectors interact with lipids and bilayers but the mechanism of membrane poration remains elusive. I discuss effector-mediated mitochondrial poration.

Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

A fundamental understanding of gene regulation requires a quantitative characterization of the spatial organization and dynamics of chromatin. The advent of fluorescence super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) presented a breakthrough to visualize structural features with a resolution of ~20 nm in fixed cells. However, until recently the long acquisition time of super-resolution images prevented high-resolution measurements in living cells due to spreading of localizations caused by chromatin motion. Here, we present a step-by step protocol for our recently developed approach for correlatively imaging telomeres with conventional fluorescence and PALM, in order to obtain time-averaged super-resolution images and dynamic parameters in living cells. First, individual single molecule localizations are assigned to a locus as it moves, allowing to discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. By subtracting the telomere trajectory from the localization of bound molecules, the motion blurring is then corrected, and high-resolution structural characterizations can be made. These structural parameters can also be related to local chromatin motion or larger scale domain movement. This protocol therefore improves the ability to analyze the mobility and time-averaged nanoscopic structure of locus-specific chromatin with single-molecule sensitivity.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Nonblinking Core–Multishell InP/ZnSe/ZnS Quantum Dot Bioconjugates for Super-resolution Imaging

Colloidal quantum dots (QDs) are vital fluorescent probes with several optical features superior to those found in organic dyes for microscopy imaging. However, the emission blinking behavior of a single QD is an intrinsic drawback for stimulated emission depletion microscopy (STED) or structured illumination microscopy techniques. Herein, we present a rational strategy for the synthesis of a type of deterministic, environmentally friendly, small nonblinking core–multishell InP/ZnSe/ZnS QD using a hydrofluoric acid (HF) etching passivation. The strategy includes four steps: synthesis of the InP core, synthesis of oil-phase core–multishell InP/ZnSe/ZnS QDs using hydrogen fluoride and trioctylphosphine etching passivation, promotion of the water solubility of QDs modified by 3-mercaptopropionic acid (MPA), and enhanced biotargeting of core–multishell InP/ZnSe/ZnS QDs by surface coupling technology. The suppressed blinking mechanism was primarily attributed to surface wrapping shells and HF etching passivation of surface dangling bonds, which reduced surface nonradiative recombination. Nonblinking QDs exhibit high fluorescence efficiency (∼90%), good photostability (>20 min), a large Stokes shift (>135 nm), biocompatibility of the cell viability (>90%), and high targeting. Furthermore, super-resolution multifocal structured illumination microscopy imaging of subcellular structures labeled using these QDs confirmed a resolution improvement of greater than 2.6-fold compared to wide-field microscopy. The results indicate that the nonblinking QDs can function as excellent probes for use in the long-term super-resolution visualization of subcellular dynamics.

Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.

Quantifying the Impact of Charge Rate and Number of Cycles on Structural Degeneration of Li-Ion Battery Electrodes

A quantitative link between crack evolution in lithium-ion positive electrodes and the degrading performance on cells is not yet well established nor is any single technique capable of doing so widely available. Here, we demonstrate a widely accessible high-throughput approach to quantifying crack evolution within electrodes. The approach applies super-resolution scanning electron microscopy (SEM) imaging of cross-sectioned NMC532 electrodes, followed by segmentation and quantification of crack features. Crack properties such as crack intensity, crack width and length are quantified as a function of charge rate (1C, 6C, and 9C) and cycle number (25, 225, and 600 cycles). Hundreds of particles are characterized for statistical confidence in the quantitative crack measurements. The data on crack evolution is compared to electrochemical data from full cells and half cells with the NMC532 positive electrodes. We show that while crack evolution strongly correlates with capacity fade in the first 25 cycles, it does not correlate well for the following hundreds of cycles indicating that cracking may not be the dominant cause of capacity fade throughout the cycle-life of cells.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells

BackgroundIncreasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified.ResultsA fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all.ConclusionsThe fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.