Live-cell Super-resolution Imaging Research Articles

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.

ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.

A Cyclometalated Iridium (III) Complex as a Microtubule Probe for Correlative Super-Resolution Fluorescence and Electron Microscopy.

The visualization of microtubules by combining optical and electron microscopy techniques provides valuable information to understand correlated intracellular activities. However, the lack of appropriate probes to bridge both microscopic resolutions restricts the areas and structures that can be comprehended within such highly assembled structures. Here, a versatile cyclometalated iridium (III) complex is designed that achieves synchronous fluorescence-electron microscopy correlation. The selective insertion of the probe into a microtubule triggers remarkable fluorescence enhancement and promising electron contrast. The long-life, highly photostable probe allows live-cell super-resolution imaging of tubulin localization and motion with a resolution of ≈30 nm. Furthermore, correlative light-electron microscopy and energy-filtered transmission electron microscopy reveal the well-associated optical and electron signal at a high specificity, with an interspace of ≈41 Å of microtubule monomer in cells.

Breaking and exiting

Cell Biology Some bacteria, including Salmonella and Shigella spp., can take up residence within vacuoles inside mammalian host cells. To gain access to the nutrient-rich host cytosol, the internalized bacteria must break out from their vacuole, but how? Ellison et al. used super-resolution live-cell imaging and correlative light and electron microscopy to characterize this process. They developed a reporter for the host lipid sphingomyelin, which is normally found on extracellular-facing membranes. The authors found that sphingomyelin became exposed to the cytosol on bacteria-containing vacuoles. This exposure occurred just before the vacuoles ruptured, releasing the bacteria into the cytosol. Sphingomyelin exposure can thus be used as an early indicator of bacterial invasion and potentially more widely as an indicator of membrane damage that threatens the integrity of the cytosol. Curr. Biol. 30 , 2974 (2020).

Dynamic regulation of promoter-proximal RNA polymerase II pausing

Transcriptional pausing is a widely used regulatory mechanism to control precise gene expression from bacteria to humans. In metazoans, transcription initiation requires the recruitment of general transcription factors and RNA polymerase II (Pol II) to gene promoters. After it initiates transcription and synthesizes a short RNA, Pol II pauses at the promoter-proximal region, standing by for further cues to enter the productive elongation stage. Promoter-proximal Pol II pausing is one of the rate-limiting steps during transcription, functioning as an early transcriptional elongation check point, to ensure the correct modification of transcription machinery and the addition of the 7-methylguanosine cap to nascent transcripts. Pol II pausing at promoter-proximal region is stabilized by the negative elongation factor (NELF) and the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). In response to developmental signals or environmental stress, the release of paused Pol II can be triggered by the positive transcription elongation factor b (P-TEFb), which is composed of the kinase CDK9 and its regulatory subunit CCNT1/2. The kinase activity of P-TEFb towards Pol II carboxy-terminal domain (CTD), NELF and DSIF is critical for P-TEFb to execute its function in pause release, and is thus tightly controlled. To date, it has been identified that P-TEFb mainly exists in three different complexes: the super elongation complex (SEC), BRD4-P-TEFb, and the inactive 7SK snRNP-P-TEFb complex. SEC, one of the most active P-TEFb containing complexes, is required for rapid gene expression in response to stress or developmental signals. The active and inactive forms of P-TEFb complexes co-exist in vivo , reaching equilibrium to meet the transcriptional needs of cells. The transition of Pol II from pause to productive elongation stage is achieved by dynamic exchange between the pausing factors and the positive elongation factors at the proximal promoter region. Liquid-liquid phase separation (LLPS) in living cells is a physicochemical process that confines biochemical reactions within membrane-less compartments. Low complexity intrinsically disordered regions (IDRs) within proteins can initiate and promote phase separation and formation of liquid droplets, which in turn facilitate the multivalent weak interactions among these IDR-containing proteins. Recent studies using super-resolution live cell imaging technology have revealed that RNA Pol II can form dynamic foci in cells through LLPS and transitions between different phases, which are likely mediated by phosphorylated CTD. Moreover, factors involved in various transcription steps can rapidly and dynamically concentrate and exchange at the hot regulatory genomic loci. For example, SEC components are able to compartmentalize and concentrate P-TEFb from its inactive partners. Upon serum stimulation, SEC assembles promptly into phase-separated droplets at the immediate early genes (IEG), triggering rapid transcription induction. Mutations in transcription regulators can lead to developmental defects and many human diseases, including cancers. For instance, abnormal stabilization of SEC components via MLL fusion plays a contributing role in leukemogenesis. Fusion of the SEC core component ENL with MLL can greatly enhance phase separation of SEC, through which the leukemic genes might be mis-activated. In addition, there is growing evidence that disease related mutations in transcriptional regulators can cause malfunction through aberrant condensation, providing potential targets for designing therapeutic strategies through phase separation interventions.

Homogeneous multifocal excitation for high-throughput super-resolution imaging.

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging.

Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.

Deep learning enables structured illumination microscopy with low light levels and enhanced speed

Structured illumination microscopy (SIM) surpasses the optical diffraction limit and offers a two-fold enhancement in resolution over diffraction limited microscopy. However, it requires both intense illumination and multiple acquisitions to produce a single high-resolution image. Using deep learning to augment SIM, we obtain a five-fold reduction in the number of raw images required for super-resolution SIM, and generate images under extreme low light conditions (at least 100× fewer photons). We validate the performance of deep neural networks on different cellular structures and achieve multi-color, live-cell super-resolution imaging with greatly reduced photobleaching.

A millisecond structured illumination microscope for super-resolution live cell imaging

In typical super-resolution microscopy, high temporal resolution is sacrificed to achieve a spatial resolution beyond the diffraction limit. In this study, we have developed a new structured illumination microscopy (SIM) system using two Pockels cells, achieving a temporal resolution of 1 ms. The introduction of the two Pockels cells enables control of the phases of the illumination pattern along the X- and Y-axes in microseconds. We obtained five images consecutively with different illumination patterns and reconstructed a super-resolution image. We observed fluorescent nanoparticles in living cells for 8 s with super resolution and a frame rate of 1,000 fps.

Magnaporthe oryzae fimbrin organizes actin networks in the hyphal tip during polar growth and pathogenesis.

Magnaporthe oryzae causes rice blast disease, but little is known about the dynamic restructuring of the actin cytoskeleton during its polarized tip growth and pathogenesis. Here, we used super-resolution live-cell imaging to investigate the dynamic organization of the actin cytoskeleton in M. oryzae during hyphal tip growth and pathogenesis. We observed a dense actin network at the apical region of the hyphae and actin filaments originating from the Spitzenkörper (Spk, the organizing center for hyphal growth and development) that formed branched actin bundles radiating to the cell membrane. The actin cross-linking protein Fimbrin (MoFim1) helps organize this actin distribution. MoFim1 localizes to the actin at the subapical collar, the actin bundles, and actin at the Spk. Knockout of MoFim1 resulted in impaired Spk maintenance and reduced actin bundle formation, preventing polar growth, vesicle transport, and the expansion of hyphae in plant cells. Finally, transgenic rice (Oryza sativa) expressing RNA hairpins targeting MoFim1 exhibited improved resistance to M. oryzae infection, indicating that MoFim1 represents an excellent candidate for M. oryzae control. These results reveal the dynamics of actin assembly in M. oryzae during hyphal tip development and pathogenesis, and they suggest a mechanism in which MoFim1 organizes such actin networks.

Superresolution Imaging of Live Cells with Genetically Encoded Silicon Rhodamine-Binding RNA Aptamers

The key role of fluorescence microscopy in the molecular life sciences rests to a large extent on the availability of genetically encoded fluorescence marker proteins, allowing facile labeling of specific proteins within living cells. Although there is an urgent need to establish a comparable, easy-to-use technology for specific, genetically encoded fluorescence labeling of ribonucleic acid (RNA) molecules, such strategies are less advanced. Here, we present studies exploring the suitability of SiRA, a novel, silicon rhodamine (SiR)-binding RNA aptamer for super-resolution microscopy in live cells. Selective binding of SiRA to the fluorescent, zwitterionic form of SiR dyes, rather than to the non-fluorescent, spiro-cyclized form, results in a significant fluorescence turn-on, generating an extremely photostable and, in fact, the brightest far-red light-up aptamer system reported to date. We have found that SiRA can be utilized as an excellent marker for stimulated emission depletion (STED) nanoscopy; further research toward applications in single-molecule based localization microscopy is presently ongoing.

Live-Cell Intracellular Storm in the Presence of Oxygen with Membrane-Impermeable Organic Fluorophores

Live-cell super-resolution imaging is a valuable tool for studying living systems. Labeling intracellular targets inside living cells easily, quickly, and inexpensively, particularly in the presence of atmospheric oxygen, has been a challenge. Here we deliver multicolor organic dyes, which have particularly good photostability, inside cells to conduct live-cell dSTORM in the presence of oxygen. The dyes pass through temporary pores in the membrane made by a bacterial toxin Streptolysin O, with no ill effects. Intracellular targets are labelled faster (< 30 min.) with smaller amount of dyes (< 200 nM) compared to other methods, with no special equipment. Virtually any combination of protein, tagging system and dye (e.g. Nanobodies, Halo-, SNAP- and eDHFR-tag) can be applied, although the choice of dyes is important. For instance, Alexa Fluor 488 (blue), CF 568 (yellow) and Atto 655 (red) are found particularly useful. Hence the technique can also be used to label endogenous proteins, such as endogenous actin labelled by Alexa Fluor 488-Phalloidin. With proper selection of organic dyes, 2-(and 3-)color live-cell intracellular dSTORM imaging is achieved without any oxygen scavenger system.

Far-red photoactivatable BODIPYs for the super-resolution imaging of live cells.

The identification of viable designs to construct switchable fluorescent probes and operate them in the interior of live cells is essential to allow the acquisition of SMLM images and permit the visualization of cellular components with sub-diffraction resolution. Our laboratories developed a mechanism to switch the fluorescence of BODIPY chromophores with the photoinduced cleavage of oxazine heterocycles under mild 405-nm illumination. With appropriate structural modifications, these switchable molecules can be engineered to immobilize covalently on large biomolecules within lysosomal compartments of live COS-7 cells and produce bright far-red fluorescence with optimal contrast upon activation. Such a combination of properties permits the acquisition of PALM images of the labeled organelles with localization precision of ca. 15nm. This article reports the experimental protocols for the synthesis of and live-cell labeling with these compounds as well as for the reconstruction of super-resolution images of the resulting biological preparations.

Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations.

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells.

Method for live-cell super-resolution imaging of mitochondrial cristae and quantification of submitochondrial membrane potentials.

The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.

Ultra-fast, universal super-resolution radial fluctuations (SRRF) algorithm for live-cell super-resolution microscopy.

For a long term, spatial resolution of fluorescence microscopy was strictly restricted by the diffraction limit. To solve this problem, various super-resolution technologies have been developed. Super-resolution radial fluctuations (SRRF), an emerging type of super-resolution microscopy, directly analyze raw images and generate super-resolution results without fluorophore localization, thereby showing more advantages in handling high-density data. Here, by speeding up the process of the algorithm with graphics processing unit (GPU) and programming with Python language, we expand the universality and improve the computing speed of the SRRF algorithm. We further apply our SRRF algorithm in different live-cell super-resolution microscopy methods with two types of fluorescence fluctuation sources: (i) direct stochastic optical reconstruction microscopy (dSTORM) in which fluorophores themselves blink under specific buffer and laser condition and (ii) structural illumination microscopy (SIM) and modulated Airyscan in which fluorescence fluctuations are artificially introduced with modulated laser illumination. With improved spatiotemporal resolution and image quality, our SRRF algorithm demonstrates its capability in live-cell super-resolution imaging, indicating its wide applications in life sciences.

Dynamic live-cell super-resolution imaging with parallelized fluorescence emission difference microscopy

The investigation of the cell biology with fluorescence microscopy remains challenging because of the requirement of both high temporal and spatial resolution. In this paper, parallelized fluorescence emission difference microscopy (pFED) is presented to provide 2-fold increasing imaging speed than conventional FED and achieve high spatial resolution (0.2 λ–0.3 λ) with a novel system design. The super-resolution images are obtained by aligning and subtracting two images, that are acquired simultaneously using parallelized scanning solid- and donut-shaped excitation beams with a lateral offset between two foci. Herein, we demonstrate the general applicability of pFED with time-lapse imaging of various subcellular dynamics in live cells over an extended period.

Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.

Simple and efficient delivery of cell-impermeable organic fluorescent probes into live cells for live-cell superresolution imaging

Numerous commercial organic fluorophores with excellent optical properties are precluded from live-cell superresolution imaging due to poor cell permeability. Here, we develop a simple but effective strategy that renders cells permeable to cell-impermeable, organic fluorescent probes by using a novel peptide vehicle, PV-1. By simple coincubation with PV-1, 22 different cell-impermeable, organic fluorescent probes were efficiently delivered into live cells and specifically labeled a variety of organelles. Moreover, PV-1 can simultaneously transfer up to three different probes into live cells. By using PV-1 and these cell-impermeable fluorescent probes, we obtained multicolor, long-term, live-cell superresolution images of various organelles, which allowed us to study the dynamic interactions between them. PV-1, together with these organic fluorescent probes, will greatly broaden the applications of superresolution imaging technology in diverse live-cell studies and opens up a new avenue in the design and application of peptide vehicles.

Making and breaking contacts

Cell Biology Within our cells, mitochondria frequently make contact and undergo fission and fusion events to regulate the mitochondrial network. Mitochondrial contact is important for normal cellular metabolism. Wong et al. used super-resolution live-cell imaging to examine intermitochondrial contacts sites in human cell lines. They found that intermitochondrial contact restricts the motility of individual mitochondria within the cell but can be modulated. Lysosome contact promotes mitochondrial untethering by RAB7 guanosine triphosphate (GTP) hydrolysis. By contrast, contact with endoplasmic reticulum (ER) does not prompt decoupling, although ER tubules marked intermitochondrial untethering events. Multiple lysosomal and mitochondrial GTPases appear to coordinate to regulate mitochondrial contact dynamics. If these dynamics are disrupted by mutation, for example, in cells from patients with the hereditary neuropathy Charcot-Marie-Tooth disease, mitochondrial contacts are slower to disassemble. This results in dysfunctional mitochondrial dynamics, which culminates in axonal degradation. Dev. Cell 50 , 339 (2019).