RESUMO
Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.
Assuntos
Imagem Molecular/métodos , Biologia Celular , Microscopia de Fluorescência/métodos , Imagem Molecular/instrumentação , Imagem Molecular/tendênciasRESUMO
MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on-off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets. Our method is exemplified with three-color recordings of mitochondria in human cells.
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DNA , Corantes Fluorescentes , Humanos , Microscopia de Fluorescência/métodos , Mitocôndrias , FótonsRESUMO
Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2-3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.
Assuntos
Lentes , Imagem Individual de Molécula , Animais , Artefatos , Mamíferos , Microscopia , Imagem ÓpticaRESUMO
With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with "clickable" side chains into proteins of interest. Well-defined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl-tRNA synthetase (chPylRS2020) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label ß-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecular-scale optical three-dimensional resolution.
Assuntos
Aminoacil-tRNA Sintetases , Código Genético , Imagem Óptica , RNA de Transferência , Aminoácidos/química , Aminoácidos/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Linhagem Celular Tumoral , Estudos Transversais , Fluorescência , Humanos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , RNA de Transferência/química , RNA de Transferência/genéticaRESUMO
Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam-scanning STED nanoscopy, require the co-alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single-mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off-switching beam into a focal-plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots. Here we describe novel designs of such so-called 'easySTED phase plates' and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths.
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Luz , Microscopia de Fluorescência/métodosRESUMO
Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.
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Bioluminescence imaging of single cells is often complicated by the requirement of exogenous luciferins that can be poorly cell-permeable or produce high background signal. Bacterial bioluminescence is unique in that it uses reduced flavin mononucleotide as a luciferin, which is abundant in all cells, making this system purely genetically encodable by the lux operon. Unfortunately, the use of bacterial bioluminescence has been limited by its low brightness compared with other luciferases. Here, we report the generation of an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in Escherichia coli; ilux can be used to image single E. coli cells with enhanced spatiotemporal resolution over several days. In addition, since only metabolically active cells produce bioluminescent signal, we show that ilux can be used to observe the effect of different antibiotics on cell viability on the single-cell level.
Assuntos
Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Medições Luminescentes , Mutagênese Sítio-Dirigida , Óperon , Photorhabdus/enzimologia , Photorhabdus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Célula ÚnicaRESUMO
Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to â¼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.
Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Proteínas Luminescentes/metabolismo , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Sinapses/metabolismo , Córtex Visual , Animais , Proteína 4 Homóloga a Disks-Large/genética , Proteínas Luminescentes/genética , Camundongos , Sinapses/genética , Córtex Visual/citologia , Córtex Visual/metabolismo , Proteína Vermelha FluorescenteRESUMO
The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to â¼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.
RESUMO
Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.
Assuntos
Algoritmos , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Fluorescência , Lasers de Corante , Compostos Orgânicos/química , Fotodegradação , Xenopus laevisRESUMO
We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.
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Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several µm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.
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Electro-optical scanning (>1,000 frames/s) with pixel dwell times on the order of the lifetime of the fluorescent molecular state renders stimulated emission depletion (STED) nanoscopy temporally stochastic. Photon detection from a molecule occurs stochastically in one of several scanning frames, and the spatial origin of the photon is known with subdiffraction precision. Images are built up by binning consecutive frames, making the time resolution freely adjustable. We demonstrated nanoscopy of vesicle motions in living Drosophila larvae and the cellular uptake of viral particles with 5- to 10-ms temporal resolution.
Assuntos
Aumento da Imagem/instrumentação , Sistemas Microeletromecânicos/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Nanotecnologia/instrumentação , Fotometria/instrumentação , Interpretação Estatística de Dados , Desenho de Equipamento , Análise de Falha de Equipamento , Processos EstocásticosRESUMO
Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Algoritmos , Animais , Rastreamento de Células/métodos , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Cinética , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Receptores Patched , Receptor Patched-1 , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Receptor SmoothenedRESUMO
Nanowires hold great promise as tools for probing and interacting with various molecular and biological systems. Their unique geometrical properties (typically <100 nm in diameter and a few micrometers in length) enable minimally invasive interactions with living cells, so that electrical signals or forces can be monitored. All such experiments require in situ high-resolution imaging to provide context. While there is a clear need to extend visualization capabilities to the nanoscale, no suitable super-resolution far-field photoluminescence microscopy of extended semiconductor emitters has been described. Here, we report that ground state depletion (GSD) nanoscopy resolves heterostructured semiconductor nanowires formed by alternating GaP/GaInP segments ("barcodes") at a 5-fold resolution enhancement over confocal imaging. We quantify the resolution and contrast dependence on the dimensions of GaInP photoluminescence segments and illustrate the effects by imaging different nanowire barcode geometries. The far-red excitation wavelength (â¼700 nm) and low excitation power (â¼3 mW) make GSD nanoscopy attractive for imaging semiconductor structures in biological applications.
RESUMO
The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or 'nanoscopy' offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent 'on' and 'off' states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.
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Lentes , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Animais , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Processos EstocásticosRESUMO
We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 µm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.
Assuntos
Membrana Celular/química , Dispositivos Lab-On-A-Chip , Lipídeos de Membrana/química , Microscopia/métodos , Pontos Quânticos/química , Células HeLa , HumanosRESUMO
Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 "intensity minima"). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.
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Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.