Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms.

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400-650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

An open-source microscopy framework for simultaneous control of image acquisition, reconstruction, and analysis.

We present a computational framework to simultaneously perform image acquisition, reconstruction, and analysis in the context of open-source microscopy automation. The setup features multiple computer units intersecting software with hardware devices and achieves automation using python scripts. In practice, script files are executed in the acquisition computer and can perform any experiment by modifying the state of the hardware devices and accessing experimental data. The presented framework achieves concurrency by using multiple instances of ImSwitch and napari working simultaneously. ImSwitch is a flexible and modular open-source software package for microscope control, and napari is a multidimensional image viewer for scientific image analysis. The presented framework implements a system based on file watching, where multiple units monitor a filesystem that acts as the synchronization primitive. The proposed solution is valid for any microscope setup, supporting various biological applications. The only necessary element is a shared filesystem, common in any standard laboratory, even in resource-constrained settings. The file watcher functionality in Python can be easily integrated into other python-based software. We demonstrate the proposed solution by performing tiling experiments using the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope, a high-throughput super-resolution microscope based on reversible saturable optical fluorescence transitions (RESOLFT).

Multi-foci parallelised RESOLFT nanoscopy in an extended field-of-view.

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastic-targeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers 45 - 65 nm $45 - 65\;{\rm{nm}}$ resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of ( 100 - 130 µ m ) 2 ${( {100 - 130\;{\rm{\mu m}}} )^2}$ . We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post-synaptic density protein Homer1c in living hippocampal neurons.

Super-Resolution Microscopy Opens New Doors to Life at the Nanoscale.

Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 µm. It is at this scale where most of the unknown and exciting action is and where cell biologists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its performance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy techniques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Quantitative Super-Resolution Microscopy to Assess Adhesion of Neuronal Cells on Single-Layer Graphene Substrates.

Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG.

STED and parallelized RESOLFT optical nanoscopy of the tubular endoplasmic reticulum and its mitochondrial contacts in neuronal cells.

The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites. Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at ~50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites.

Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy.

Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells. We achieved rapid (1-2 Hz) acquisition of large fields of view (~40 × 40 µm2) by highly parallelized image acquisition with an interference pattern that creates an array of 3D-confined and equally spaced intensity minima. This allowed us to reversibly turn switchable fluorescent proteins to dark states, leading to a targeted 3D confinement of fluorescence. We visualized the 3D organization and dynamics of organelles in living cells and volumetric structural alterations of synapses during plasticity in cultured hippocampal neurons.

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales.

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

Hypericin-Apomyoglobin: An Enhanced Photosensitizer Complex for the Treatment of Tumor Cells.

Bioavailability of photosensitizers for cancer photodynamic therapy is often hampered by their low solubility in water. Here, we overcome this issue by using the water-soluble protein apomyoglobin (apoMb) as a carrier for the photosensitizer hypericin (Hyp). The Hyp-apoMb complex is quickly uptaken by HeLa and PC3 cells at submicromolar concentrations. Fluorescence emission of Hyp-apoMb is exploited to localize the cellular distribution of the photosensitizer. The plasma membrane is rapidly and efficiently loaded, and fluorescence is observed in the cytoplasm only at later times and to a lesser extent. Comparison with cells loaded with Hyp alone demonstrates that the uptake of the photosensitizer without the protein carrier is a slower, less efficient process, that involves the whole cell structure without preferential accumulation at the plasma membrane. Cell viability assays demonstrate that the Hyp-apoMb exhibits superior performance over Hyp. Similar results were obtained using tumor spheroids as three-dimensional cell culture models.

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems.

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW cm-2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40-50 frames) and large (50 × 50 µm2) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy.

Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.

Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy. Numerous dark states of Dendra2 are observed both in inactive (green fluorescent) and active (orange fluorescent) forms; the interconversion rates are both light- and pH-dependent, as observed for other PAFPs. The dark states limit the detected count rate per molecule, which is a crucial parameter for localization microscopy. We then developed, to our knowledge, a new mathematical estimate for the resolution in localization microscopy as a function of the measured photophysical parameters of the probe such as photobleaching quantum yield, count rate per molecule, and intensity of saturation. The model was used to predict the dependence of resolution on acquisition parameters such as illumination intensity and time per frame, demonstrating an optimal set of acquisition parameters for a given probe for a variety of measures of resolution. The best possible resolution was then compared for Dendra2 and other widely used probes, including Alexa dyes and quantum dots. This work establishes a framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore, and suggests that development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.

Nanoscale Molecular Reorganization of the Inhibitory Postsynaptic Density Is a Determinant of GABAergic Synaptic Potentiation.

Gephyrin is a key scaffold protein mediating the anchoring of GABAA receptors at inhibitory synapses. Here, we exploited superresolution techniques combined with proximity-based clustering analysis and model simulations to investigate the single-molecule gephyrin reorganization during plasticity of inhibitory synapses in mouse hippocampal cultured neurons. This approach revealed that, during the expression of inhibitory LTP, the increase of gephyrin density at postsynaptic sites is associated with the promoted formation of gephyrin nanodomains. We demonstrate that the gephyrin rearrangement in nanodomains stabilizes the amplitude of postsynaptic currents, indicating that, in addition to the number of synaptic GABAA receptors, the nanoscale distribution of GABAA receptors in the postsynaptic area is a crucial determinant for the expression of inhibitory synaptic plasticity. In addition, the methodology implemented here clears the way to the application of the graph-based theory to single-molecule data for the description and quantification of the spatial organization of the synapse at the single-molecule level.SIGNIFICANCE STATEMENT The mechanisms of inhibitory synaptic plasticity are poorly understood, mainly because the size of the synapse is below the diffraction limit, thus reducing the effectiveness of conventional optical and imaging techniques. Here, we exploited superresolution approaches combined with clustering analysis to study at unprecedented resolution the distribution of the inhibitory scaffold protein gephyrin in response to protocols inducing LTP of inhibitory synaptic responses (iLTP). We found that, during the expression of iLTP, the increase of synaptic gephyrin is associated with the fragmentation of gephyrin in subsynaptic nanodomains. We demonstrate that such synaptic gephyrin nanodomains stabilize the amplitude of inhibitory postsynaptic responses, thus identifying the nanoscale gephyrin rearrangement as a key determinant for inhibitory synaptic plasticity.

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

Subdiffraction localization of a nanostructured photosensitizer in bacterial cells.

Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.

Engineered chimeras reveal the structural basis of hexacoordination in globins: a case study of neuroglobin and myoglobin.

BACKGROUND: Myoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure-function relationships within the globin superfamily. METHODS: In this work we generated chimeric proteins by swapping a previously identified regulatory segment - the CD region - and evaluated comparatively the structural and functional properties of the resulting proteins by molecular dynamics simulations, and spectroscopic and kinetic investigations. RESULTS: Our results show that chimeric proteins display heme coordination properties displaced towards those expected for the corresponding CD region. In particular, in the absence of exogenous ligands, chimeric Mb is found as a partially hexacoordinated bis-histidyl species, whereas chimeric Ngb shows a lower equilibrium constant for forming a hexacoordinated bis-histidyl species. CONCLUSIONS: While these results confirm the regulatory role of the CD region for bis-histidyl hexacoordination, they also suggest that additional sources contribute to fine tune the equilibrium. General significance Globins constitute a ubiquitous group of heme proteins widely found in all kingdoms of life. These findings raise challenging questions regarding the structure-function relationships in these proteins, as bis-histidyl hexacoordination emerges as a novel regulatory mechanism of the physiological function of globins.

Photochromic conversion in a red/green cyanobacteriochrome from Synechocystis PCC6803: quantum yields in solution and photoswitching dynamics in living E. coli cells.

The protein encoded by the gene slr1393 from the cyanobacterium Synechocystis sp. PCC6803 (Slr1393) is composed of three GAF domains, a PAS domain, and a histidine kinase motif. The third GAF domain (referred to as GAF3) was previously characterized as the sole domain in this protein, being able to carry phycocyanobilin (PCB) as the chromophore and to accomplish photochemistry. GAF3 shows photochromicity, and is able to switch between a red-absorbing parental state (GAF3R, λmax = 649 nm) and a green-absorbing photoproduct state (GAF3G, λmax = 536 nm) upon appropriate irradiation. In this study we have determined the photochemical quantum yields for the interconversion between both forms using two methods: an "absolute" method and a reference-based control. The latter is a comparative procedure which exploits a well-characterized blue-light photoreceptor, YtvA from Bacillus subtilis, and the cyanobacterial phytochrome Cph1 as actinometers. The former is an ad hoc developed, four laser-based setup where two cw lasers provide the pump beams to induce photoswitching (red to green and green to red, respectively) and two cw lasers simultaneously monitor the appearance and disappearance of the two species. Interestingly, fit analysis of the recorded transient absorbance changes provided a quantum yield for the green → red conversion (≈0.3) at least three times larger than for the red → green conversion (≈0.08). These data are in agreement with the results from the comparative method documenting the usefulness of the 'direct' method developed here for quantum yields' determination. The light-induced switching capability of this photochromic protein allowed measuring the kinetics of GAF3 immobilized on a glass plate, and within living, overexpressing Escherichia coli cells.

The dark recovery rate in the photocycle of the bacterial photoreceptor YtvA is affected by the cellular environment and by hydration.

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration.