Live-cell Super-resolution Imaging Research Articles

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).

First person – Takuro Tojima

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Takuro Tojima is first author on ‘Spatiotemporal dissection of the trans-Golgi network in budding yeast’, published in JCS. Takuro is a Research Scientist in the lab of Akihiko Nakano at Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan, investigating molecular mechanisms of membrane traffic.

Multicolor re-scan super-resolution imaging of live cells.

Multicolor fluorescence microscopy has proved essential in biological studies. However, the application of conventional multicolor microscopy to imaging subcellular organelles is restricted by its diffraction-limited spatial resolution. Re-scan confocal microscopy (RCM), a novel super-resolution imaging technique, can effectively address this problem. However, previous multicolor RCM imaging methods usually led to spatial mismatch in images due to the sequential scanning of the sample with multiple excitation lasers. We present a new RCM system to achieve multicolor super-resolution imaging. A spectrograph was used as the multicolor detection system, and a linear spectral unmixing algorithm was applied to separate different fluorophores in the spectral image. Moreover, since the image reconstruction process induced an artificial resolution improvement, a gamma correction was introduced to restore the multicolor super-resolution image. By imaging phalloidin-labeled F-actin in breast cancer cells, we found that the lateral resolution of our system is approximately 171 nm, which is a 1.8-fold improvement over that of wide-field imaging. The successful identification of three types of fluorescent beads indicated that our multicolor RCM can resolve different fluorophores whose spectra largely overlap with each other. Finally, we demonstrated that our method is suitable for imaging multicolor-labeled organelles of live cells. Our novel RCM system can acquire multicolor super-resolution images of live cells without spatial mismatch, obvious photobleaching or photodamage. This system may provide a new imaging tool for monitoring dynamic events involving interactions between multiple molecules and organelles in cells.

A ratiometric near-infrared fluorescence strategy based on spiropyran in situ switching for tracking dynamic changes of live-cell lysosomal pH

Abnormal lysosomal pH values have been associated with cellular dysfunctions and many diseases. However, tools to monitor dynamic changes of pH values in live-cell lysosomes are limited. Herein, biocompatible near-infrared (NIR) fluorescent dyes HAN1-4 for lysosome-targeting staining were rationally designed and synthesized with the aid of a spiropyran in situ switching (SIS) strategy. The novel SIS-based dyes share the following special characteristics: 1) pH-dependent ratiometric fluorescent patterns matched nicely with pH windows of lysosomes, 2) light-independent real-time spiropyran in situ switching behavior in lysosomes and 3) pH-sensitive fluorescence in the NIR region, thereby ensuring their applications in obtaining highly specific images of live-cell lysosomes with a weak background signal. Among them, the HAN2 probe was found to induce the best ratiometric response toward pH in the range corresponding to lysosomal pH window, thus being further applied for the real-time tracking of lysosomal subtle pH changes in stressed cells undergoing drug and heat stimulations. Live-cell super-resolution and confocal fluorescent imaging indicate that our SIS-based NIR probes are robust and can be applied for the accurate detection of lysosomal pH in complex biosystems.

Spiropyran in Situ Switching: A Real-Time Fluorescence Strategy for Tracking DNA G-Quadruplexes in Live Cells.

DNA G-quadruplexes (G4s) in vivo have been linked to cancer and other diseases such as neurological disorders. Nondestructive fast detection of endogenous DNA G4s can provide specific real-time information, which is of particular interest for clinic accurate diagnosis. However, tools to probe live-cell endogenous DNA G4s in real time are very limited. Herein, we report the design and development of a fluorescent molecule QIN for the real-time detection of endogenous DNA G4s in live cells with the aid of a new spiropyran in situ switching (SIS) strategy. The lipophilic spiropyran-linked QIN differs from the other probes in that it can enter live cells readily within 15 s and can be in situ induced by DNA G4s to adopt its charged open form, causing a large red shift in the fluorescent emission wavelength. Live-cell super-resolution fluorescent imaging suggests that the SIS-based probe has high photostability and can be applied for the accurate detection of DNA G4s in complex biosystems with very high sensitivity and selectivity.

Nanoscale monitoring of mitochondria and lysosome interactions for drug screening and discovery

Technology advances in genomics, proteomics, and metabolomics largely expanded the pool of potential therapeutic targets. Compared with the in vitro setting, cell-based screening assays have been playing a key role in the processes of drug discovery and development. Besides the commonly used strategies based on colorimetric and cell viability, we reason that methods that capture the dynamic cellular events will facilitate optimal hit identification with high sensitivity and specificity. Herein, we propose a live-cell screening strategy using structured illumination microscopy (SIM) combined with an automated cell colocalization analysis software, Cellprofiler™, to screen and discover drugs for mitochondria and lysosomes interaction at a nanoscale resolution in living cells. This strategy quantitatively benchmarks the mitochondria-lysosome interactions such as mitochondria and lysosomes contact (MLC) and mitophagy. The automatic quantitative analysis also resolves fine changes of the mitochondria-lysosome interaction in response to genetic and pharmacological interventions. Super-resolution live-cell imaging on the basis of quantitative analysis opens up new avenues for drug screening and development by targeting dynamic organelle interactions at the nanoscale resolution, which could facilitate optimal hit identification and potentially shorten the cycle of drug discovery.

E-syt1 Re-arranges STIM1 Clusters to Stabilize Ring-shaped ER-PM Contact Sites and Accelerate Ca2+ Store Replenishment

In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.

Live Cell Super-Resolution Imaging with Red-Shifted States of Conventional Bodipy Fluorophores

Live Cell Super-Resolution Imaging with Red-Shifted States of Conventional Bodipy Fluorophores

Live cell super resolution imaging by radial fluctuations using fluorogen binding tags.

Fluorescence-Activating and absorption-Shifting Tag (FAST) is a novel genetically encoded optical highlighter probe. Since the fluorescence of FAST originates from the stochastic and reversible diffusive association of a fluorogenic ligand, we investigate the application of FAST using Super-Resolution Radial Fluctuations (SRRF) to achieve routine imaging below the diffraction limit in a widefield epifluorescence microscope. We show that intensity fluctuation analysis like SRRF allows the imaging of FAST-tagged proteins with sub - 100 nm resolution in live cells. FAST co-labeled with conventional fluorophores enables real time multicolour 2D and 3D super-resolution imaging, indicating that FAST can be used for the observation of sub-diffraction limited structures in both living and fixed samples.

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy

Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.

Live-cell imaging of human spermatozoa using structured illumination microscopy.

Structural details of spermatozoa are interesting from the perspectives of fundamental biology and growing reproductive health problems. Studies of nanostructural details of these extremely motile cells have been limited to fixed cells, largely using electron microscopy. Here we provide the protocols for and demonstrate live-cell multi-color super-resolution imaging of human spermatozoa using structured illumination microscopy (SIM). By using patches of agarose for immobilization, we achieved four-channel 3D SIM imaging of the plasma membrane, nucleus, mitochondria and microtubulin in the same living sperm cells. We expect that high-resolution imaging of living spermatozoa will be implemented for research on fundamental cellular mechanisms together with morphological aberrations involved in male infertility for a future improved cell selection process in in vitro fertilization treatments.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells.

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.

Artifact-free high-density localization microscopy analysis

High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

Spontaneously Blinking Fluorescent Protein for Simple Single Laser Super-Resolution Live Cell Imaging.

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

Malic Acid Carbon Dots: From Super-resolution Live-Cell Imaging to Highly Efficient Separation.

As-synthesized malic acid carbon dots are found to possess photoblinking properties that are outstanding and superior compared to those of conventional dyes. Considering their excellent biocompatibility, malic acid carbon dots are suitable for super-resolution fluorescence localization microscopy under a variety of conditions, as we demonstrate in fixed and live trout gill epithelial cells. In addition, during imaging experiments, the so-called "excitation wavelength-dependent" emission was not observed for individual as-made malic acid carbon dots, which motivated us to develop a time-saving and high-throughput separation technique to isolate malic acid carbon dots into fractions of different particle size distributions using C18 reversed-phase silica gel column chromatography. This post-treatment allowed us to determine how particle size distribution influences the optical properties of malic acid carbon dot fractions, that is, optical band gap energies and photoluminescence behaviors.

Deep learning massively accelerates super-resolution localization microscopy.

The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution. Super-resolution reconstructions are even possible from widefield images alone, though adding localization data improves image quality. We demonstrate super-resolution imaging of >1,000 fields of view containing >1,000 cells in ∼3 h, yielding an image spanning spatial scales from ∼20 nm to ∼2 mm. The drastic reduction in acquisition time and sample irradiation afforded by ANNA-PALM enables faster and gentler high-throughput and live-cell super-resolution imaging.