Light-induced cell damage in live-cell super-resolution microscopy

Monitoring the Organization, Dynamics, and Interactions of Early B Cell Receptor Signaling Proteins using 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 microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching.

This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.

Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.

Live Cell Super-Resolution Microscopy Measures Membrane-Driven Sorting of B Cell Receptor Signaling Partners

We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with 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.

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

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.

Quantitative single-cell optical technologies.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Subcellular organization is important for bacterial cell physiology. Especially, bacterial secretion systems are tightly regulated in a temporal and spatial manner to efficiently fulfill their function. Among them, the contact-dependent Type VI secretion (T6SS) has an important role in inter-bacterial competitions and pathogenicity of Gram-negative bacteria. T6SS translocates effector proteins into target cells using the contraction of a long cytosolic sheath, which pushes an inner tube together with a sharp tip and associated effectors across target cell membranes. This mode of action allows bacteria to use T6SS against a broad range of prokaryotic and eukaryotic organisms. However, the contact-dependency limits the target range and the efficiency of effector translocation is low because only a small number of effectors can be delivered per one round of T6SS assembly. Recent advances in live-cell fluorescence and super resolution microscopy led to the revelation that T6SS activation patterns and dynamics are surprisingly diverse in different bacteria. These differences in T6SS assembly dynamics likely reflect different strategies to overcome the disadvantages of T6SS mode of action. However, the spatio-temporal regulation behind these different T6SS firing patterns are not well understood. My PhD thesis provides new insights into how different subcellular localizations of T6SS assembly are achieved. The Threonine phosphorylation pathway (TPP) is a unique posttranslational regulation mechanism, which allows Pseudomonas aeruginosa to activate its T6SS apparatus in response to membrane damage inflicted by an attack from neighboring bacteria and to localize it to the site of attack. While the involved components are identified, it is not clear how the periplasmic sensor module integrates spatial and temporal information for precise and fast T6SS assembly initiation. To test if relocation of TPP components from outer to inner membrane (IM) is important for T6SS activation, I changed their subcellular localization by mutating their N-terminal signal sequences. Relocation of one TPP component to IM indeed hyper-activated T6SS assembly, however, the exact mechanism of T6SS localization remains to be elucidated. In collaboration with Prof. Kevin Foster, University of Oxford, we tested the benefit and cost of TPP-dependent localization of T6SS during bacterial competitions. Our results from in silico and imaging experiments suggested that P. aeruginosa uses TPP to kill competing bacteria by localized and repeated T6SS assemblies and thus inflicting more damage than it encounters from attacking competitors. In collaboration with Prof. Petr Broz, University of Lausanne, we characterized the unique Francisella pathogenicity island (FPI), which encodes a non-canonical T6SS essential for phagosomal escape. The FPI lacks a specialized unfoldase required for recycling of contracted sheaths and for dynamics of canonical T6SS. Furthermore, the FPI encodes genes with unknown function. By live-cell fluorescence microscopy, we showed that F. novicida T6SS dynamics is comparable to canonical T6SS dynamics. Moreover, we found that general-purpose unfoldase ClpB recycles contracted sheaths and is essential for phagosomal escape in vivo. By analyzing T6SS dynamics and virulence of single deletion mutants in vitro and in vivo, we could group FPI components with unknown function into structural components, which are required for T6SS function, and putative effectors, which are critical for virulence but not for T6SS assembly. Moreover, we showed that F. novicida T6SS assembles exclusively at bacterial poles. This unique polar localization raised the question of how Francisella T6SS is localized to the poles and whether it is important for T6SS function. I analyzed the dynamics of membrane complex formation, which is the first step of T6SS assembly, by live-cell fluorescence microscopy and structured illumination microscopy. I showed that the membrane complex is stably formed on the poles even in the absence of other FPI components. In addition, the membrane complex formation was insufficient to initiate sheath assembly indicating that additional signals are required to activate T6SS in F. novicida. To investigate the contribution of FPI components and localization of T6SS to Francisella virulence in more detail, I established Galleria mellonella larvae as infection model. Besides, I constructed two expression plasmids for F. novicida, which are mobilized by conjugation and have tetracyline inducible promoters for tunable gene expression. These new tools will be invaluable in the future research of mechanism required for F. novicida pathogenesis.

Every collected photon is precious in live-cell super-resolution (SR) microscopy. Here, we describe a data-efficient, deep learning-based denoising solution to improve diverse SR imaging modalities. The method, SN2N, is a Self-inspired Noise2Noise module with self-supervised data generation and self-constrained learning process. SN2N is fully competitive with supervised learning methods and circumvents the need for large training set and clean ground truth, requiring only a single noisy frame for training. We show that SN2N improves photon efficiency by one-to-two orders of magnitude and is compatible with multiple imaging modalities for volumetric, multicolor, time-lapse SR microscopy. We further integrated SN2N into different SR reconstruction algorithms to effectively mitigate image artifacts. We anticipate SN2N will enable improved live-SR imaging and inspire further advances.

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.