Live-cell Super-resolution Microscopy Research Articles

Synapse-Specific Trapping of SNARE Machinery Proteins in the Anesthetized Drosophila Brain.

General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super-resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.

Macroscale structural changes of thylakoid architecture during high light acclimation in Chlamydomonas reinhardtii.

Photoprotection mechanisms are ubiquitous among photosynthetic organisms. The photoprotection capacity of the green alga Chlamydomonas reinhardtii is correlated with protein levels of stress-related light-harvesting complex (LHCSR) proteins, which are strongly induced by high light (HL). However, the dynamic response of overall thylakoid structure during acclimation to growth in HL has not been fully understood. Here, we combined live-cell super-resolution microscopy and analytical membrane subfractionation to investigate macroscale structural changes of thylakoid membranes during HL acclimation in Chlamydomonas. Subdiffraction-resolution live-cell imaging revealed that the overall thylakoid structures became thinned and shrunken during HL acclimation. The stromal space around the pyrenoid also became enlarged. Analytical density-dependent membrane fractionation indicated that the structural changes were partly a consequence of membrane unstacking. The analysis of both an LHCSR loss-of-function mutant, npq4 lhcsr1, and a regulatory mutant that over-expresses LHCSR, spa1-1, showed that structural changes occurred independently of LHCSR protein levels, demonstrating that LHCSR was neither necessary nor sufficient to induce the thylakoid structural changes associated with HL acclimation. In contrast, stt7-9, a mutant lacking a kinase of major light-harvesting antenna proteins, had a slower thylakoid structural response to HL relative to all other lines tested but still showed membrane unstacking. These results indicate that neither LHCSR- nor antenna-phosphorylation-dependent HL acclimation are required for the observed macroscale structural changes of thylakoid membranes in HL conditions.

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a 'before-and-after' experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons.

It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD. In our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Vesicle‐Associated Actin Assembly by Formins Promotes TGFβ‐Induced ANGPTL4 Trafficking, Secretion and Cell Invasion (Adv. Sci. 9/2023)

Actin Dynamics In article number 2204896, a novel role of the formin FMNL2 is uncovered by Dennis Frank, Christel Jessica Moussi, Carsten Schwan, Robert Grosse, and co-workers. TGFβ-induced processes, such as cell invasion and the secretion and trafficking of the pro-metastatic factor ANGPTL4 require phospho-activated FMNL2. Live cell super-resolution microscopy allows the observation of FMNL2 directly polymerizing actin at motile ANGPTL4-containing vesicles. A time course of FMNL2 (green) and actin polymerization (cyan) at an ANGPTL4 vesicle (magenta) is shown, starting from the association of FMNL2 (top left) and ending with the dissociation of the formin (bottom right).

A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

Facilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years. However, distinguishing between plasma membrane-bound or internalised ligands remains a major challenge for the studies of cargo sorting to endosomal compartments, especially in small and round cells such as lymphocytes. The specific hybridization internalisation probe (SHIP) assay, developed for flow cytometry studies, employs a ssDNA fluorescence internalisation probe and a complementary ssDNA quenching probe to unambiguously detect the internalized receptors/cargo. Here, we adopted the SHIP assay to study the trafficking of receptor/ligand complexes using B lymphocytes and B cell receptor-mediated antigen internalization as a model system. Our study demonstrates the potential of the SHIP assay for improving the imaging of internalized receptor/ligand complexes and establishes the compatibility of this assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.

Development of fluorescence labeling technique for multi-modal live-cell super-resolution microscopy

Live-cell super-resolution microscopy is a powerful tool that reveals spatiotemporal dynamics of biomolecules with nano-scale resolution in live specimens. However, the photobleaching of fluorescent molecules labeled to the target molecule is a significant obstacle to continuously obtaining super-resolution images for a long period. In this study, we developed a fluorescent labeling technique called de-quenching of organic dye emission (DeQODE) that enables long-term observation with a super-resolution microscope without being affected by photobleaching. In the DeQODE, a non-fluorescent probe is designed to become fluorescent by sequential binding to tag protein, an antibody against the probe, enabling maintaining the fluorescence signal for a long period. After optimizing the binding-dissociating property between tag protein and the probe, several intracellular organelles and the distribution of proteins labeled with DeQODE were imaged with a nano-scale resolution for tens of minutes by multiple super-resolution microscopies including stimulated emission depletion microscopy (STED), single-molecule localization microscopy (SMLM), and structured illumination microscopy (SIM). Our results show that DeQODE can contribute to research clarifying the nano-scale dynamics of functional molecules by live-cell super-resolution imaging.

Super-Resolution Radial Fluctuations (SRRF) Microscopy.

Super-resolution Radial Fluctuations (SRRF) imaging is a computational approach to fixed and live-cell super-resolution microscopy that is highly accessible to life science researchers since it uses common microscopes and open-source software plugins for ImageJ. This allows users to generate super-resolution images using the same equipment, fluorophores, fluorescent proteins and methods they routinely employ for their studies without specialized sample preparations or reagents. Here, we discuss a step-by-step workflow for acquiring and analyzing images using the NanoJ-SRRF software developed by the Ricardo Henriques group, with a focus on imaging chromatin. Increased accessibility of affordable super-resolution imaging techniques is an important step in extending the reach of this revolution in cellular imaging to a greater number of laboratories.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

Directionally Modified Fluorophores for Super-Resolution Imaging of Target Enzymes: A Case Study with Carboxylesterases.

In the need for improving the labeling quality of super-resolution imaging, multifarious fluorescent labeling strategies have sprang up. Among them, a small molecule inhibitor-probe (SMI-probe) shows its advancement in fine mapping due to its smaller size and its specific binding to a specific site. Herein, we report a novel protocol of mechanism-guided directional modification of fluorophores into fluorescent inhibitors for enzyme targeting, which could half the size of the SMI-probe. To confirm the feasibility of the strategy, carboxylesterase (hCE) inhibitors are designed and developed. Among the constructed molecule candidates, NIC-4 inhibited both isoforms of hCE1 and hCE2, with IC50 values of 4.56 and 4.11 μM. The CE-targeting specificity of NIC-4 was confirmed by colocalizing with an immunofluorescent probe in fixed-cell confocal imaging. Moreover, NIC-4 was used in live-cell super-resolution microscopy, which indicates dotlike structures instead of the larger staining with the immunofluorescent probe. Moreover, it enables the real-time tracking of dynamic flow of carboxylesterases in live cells.

Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.

Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

A dual role for H2A.Z.1 in modulating the dynamics of RNA polymerase II initiation and elongation.

RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.

Reversible Michael Addition of an Engineered Cysteine Residue to a Light Activated Ligand, a New Design Strategy for a Synthetic Photoswitchable Fluorescent Protein

Reversible photoswitchable fluorescent probes can transit between two different optical states when triggered by light. Because of the ability to switch, they become useful in live cell super-resolution microscopy (e.g. RESOLFT, psSIM, STORM, PALM), information storage, and optical control of protein activity. Currently, only a handful of fluorescent proteins and synthetic dyes exhibit reversible photoswitchability. Hence, there is a growing interest for new systems with enhanced functionality. We are currently focused on utilizing a synthetic ligand capable of reversible Michael addition of an active cysteine residue, within the binding pocket of human cellular retinoid binding protein (hCRBPII).

Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics

Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesised RNA, RNA processing proteins, and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs) 1–3. Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localisation microscopy and correlative electron microscopy, and find that MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates 4. Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

Live-SIMBA: an ImageJ plug-in for the universal and accelerated single molecule-guided Bayesian localization super resolution microscopy (SIMBA) method.

Live-cell super-resolution fluorescence microscopy techniques allow biologists to observe subcellular structures, interactions and dynamics at the nanoscale level. Among of them, single molecule-guided Bayesian localization super resolution microscopy (SIMBA) and its derivatives produce an appropriate 50 nm spatial resolution and a 0.1-2s temporal resolution in living cells with simple off-the-shelf total internal reflection fluorescence (TIRF) equipment. However, SIMBA and its derivatives are limited by the requirement for dual-channel dataset or single-channel dataset with special design, the time-consuming calculation for extended field of view and the lack of real-time visualization tool. Here, we propose a universal and accelerated SIMBA ImageJ plug-in, Live-SIMBA, for time-series analysis in living cells. Live-SIMBA circumvents the requirement of dual-channel dataset using intensity-based sampling algorithm and improves the computing speed using multi-core parallel computing technique. Live-SIMBA also better resolves the weak signals inside the specimens with adjustable background estimation and distance-threshold filter. With improved fidelity on reconstructed structures, greatly accelerated computation, and real-time visualization, Live-SIMBA demonstrates its extended capabilities in live-cell super-resolution imaging.

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Graphene-Enabled, Spatially Controlled Electroporation of Adherent Cells for Live-Cell Super-resolution Microscopy.

The incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, it remains a challenge to deliver tailored, often cell-impermeable, fluorescent probes into live cells for target labeling. Here, utilizing the outstanding mechanical, electrical, and optical properties of graphene, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent mammalian cells and in situ super-resolution microscopy on the same device. Approximately 90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate good spatiotemporal controls, which, in combination with the ready patternability of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate.

Competitive Substrate Binding Coordinates the Two Antagonistic Motors of the Bacterial Type IV Pilus

The bacterial type IV pilus is an intricate nano-machine that coordinates two antagonistic molecular motors to extend and retract a semi-flexible polymer to the environment. Filament retraction is driven by the strongest molecular motor known and used for a variety of tasks such as uptake of DNA, surface sensing, and motility. However, how both motors are coordinated to dynamically extend and retract pili is unknown. To fill this void, we genetically engineered a fluorescent label for the pilus fiber. This enables us to characterize the dynamic cycles of extension and retraction of the pilus in an unprecedented way. Careful characterization of pilus dynamics reveals that pili are short and individual cells make a new pilus every 10 seconds. This is in stark contrast to the current dogma of the field that was shaped by static cyro electron images. To explain this discrepancy, we introduce a simplistic three-state model that describes the mutually exclusive binding/unbinding of the motor proteins to the pilus machine by a Poisson process. We combined live cell super-resolution microscopy and Monte-Carlo simulations to determine the rate constants of the model. We explain how these rates determine biological function such as pilus length and rate of pilus assembly. Intriguingly, we find that the major throttle of pilus production is the unbinding step of the retraction motor. Furthermore, we measured pilus activity with and without surface contact by combining fluorescence microscopy and line-scanning optical tweezers. Cells show similar levels of pilus activity, overturning the current model that surface contact drives pilus retraction. Together, our results shape a comprehensive picture of the physical processes that determine pilus activity and suggest that the pilus was optimized for multitasking of different tasks such as DNA uptake and motility.

Live-Cell Superresolution Microscopy of Faa4 Re-Distribution on Lipid Droplets during Metabolic Transitions in Yeast

Lipid droplets (LDs) are energy storage organelles of a cell with a neutral lipid core surrounded by a phospholipid monolayer. Cells achieve their lipid and energy homeostasis by actively regulating the synthesis and the breakdown of neutral lipids stored in LDs during metabolic transitions. In order to accommodate the change in size, number and neutral lipid content of LDs, cells regulate the abundance and the density of specific proteins involved in the synthesis and the breakdown of neutral lipids. In yeast, Faa4 encodes for one of the fatty acyl-CoA synthetases required for the activation of fatty acids essential for making neutral lipids. In this study, we employ correlative Photo-Activated Localization Microscopy (PALM) and conventional fluorescence microscopy to resolve and quantify the density of Faa4 on LDs below the optical diffraction limit, on the ER, and on the vacuole as yeast cells transition between exponential, stationary and lag phases based on the nutrient availability. We find that the Faa4 density on LDs increases as LDs grow in size and re-localize to the vacuolar membrane from ER when the cell progresses to the stationary phase from the exponential growth phase. However, Faa4 is predominately localized to vacuolar lumen within ∼2 hrs of dilution in fresh media suggesting rapid consumption of LDs upon growth resumption. The increase in Faa4 density on LDs that are about to enter the vacuole for breakdown may therefore be the major mechanism how Faa4 shuttles into the vacuole to activate fatty acids. Our approach to resolve and quantify the density of regulatory proteins on LDs in living cells may help in future studies to better understand how cells differentially regulate their density to maintain lipid and energy homeostasis based on the metabolic needs.