RESUMO
In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.
Assuntos
Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Microscopia de FluorescênciaRESUMO
Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.
Assuntos
Inteligência Artificial , Microscopia , Humanos , Microscopia/métodos , Imageamento Tridimensional/métodos , SinapsesRESUMO
Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.
Assuntos
Neuroimagem/métodos , Óptica e Fotônica/métodos , Animais , Proteínas de Bactérias , Embrião não Mamífero , Feminino , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Masculino , Camundongos , Peixe-ZebraRESUMO
Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 µm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.
Assuntos
Membrana Celular/ultraestrutura , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Mitocôndrias/ultraestrutura , Animais , Células COS , Chlorocebus aethiops , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Células LLC-PK1 , Suínos , Peixe-Zebra/embriologiaRESUMO
Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.
Assuntos
Microscopia Confocal/instrumentação , Microscopia Confocal/estatística & dados numéricos , Óptica e Fotônica/instrumentação , Animais , Linhagem Celular , Óptica e Fotônica/normas , Fatores de Tempo , Peixe-ZebraRESUMO
We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.
Assuntos
Encéfalo/metabolismo , Luz , Neuroimagem/métodos , Óptica e Fotônica , Animais , Caenorhabditis elegans , Corantes Fluorescentes/química , Análise de Fourier , Histonas/química , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Processamento de Proteína Pós-Traducional , Proteínas/química , Pupila/fisiologia , Córtex Visual/fisiologia , Peixe-ZebraRESUMO
A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 µm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to â¼0.3 µm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Cromossomos/ultraestrutura , Células HeLa , Humanos , Células LLC-PK1 , Proteínas Luminescentes , Microscopia/instrumentação , Microscopia Confocal/métodos , Mitose , Fenômenos Ópticos , SuínosRESUMO
In traditional zonal wavefront sensing for adaptive optics, after local wavefront gradients are obtained, the entire wavefront can be calculated by assuming that the wavefront is a continuous surface. Such an approach will lead to sub-optimal performance in reconstructing wavefronts which are either discontinuous or undersampled by the zonal wavefront sensor. Here, we report a new method to reconstruct the wavefront by directly measuring local wavefront phases in parallel using multidither coherent optical adaptive technique. This method determines the relative phases of each pupil segment independently, and thus produces an accurate wavefront for even discontinuous wavefronts. We implemented this method in an adaptive optical two-photon fluorescence microscopy and demonstrated its superior performance in correcting large or discontinuous aberrations.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Microscopia de Contraste de Fase/métodosRESUMO
Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes have limited imaging depth, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed "photochemical sectioning," a spatially precise, light-based sample sectioning process. By combining photochemical sectioning with volumetric lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelination sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory-bulb-wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative mouse, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach.
RESUMO
Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction-limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 microm.
Assuntos
Aumento da Imagem/instrumentação , Iluminação/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Artefatos , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Lentes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Light sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.
RESUMO
Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet microscopy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of "harmonic balanced" lattice light sheet that improves performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.
RESUMO
The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
Assuntos
Enzima de Conversão de Angiotensina 2 , Rodopsina , Camundongos , Animais , Potenciais de Ação/fisiologia , Rodopsina/genética , Neurônios/fisiologia , Mutação/genéticaRESUMO
Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.
Assuntos
Conectoma , Microscopia , Animais , Conectoma/métodos , Sinapses/fisiologia , Drosophila , Expansão de TecidoRESUMO
Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.
Assuntos
Microscopia/métodos , Pupila/fisiologia , Adaptação Ocular , Algoritmos , Humanos , Luz , Microscopia/estatística & dados numéricos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Microscopia de Fluorescência por Excitação Multifotônica/estatística & dados numéricos , Fenômenos Ópticos , Espalhamento de RadiaçãoRESUMO
Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
Assuntos
Células/ultraestrutura , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Células COS , Adesão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Congelamento , Células HeLa , Humanos , CamundongosRESUMO
Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.
Assuntos
Encéfalo/diagnóstico por imagem , Nanotecnologia , Neuroimagem/métodos , Imagem Óptica/métodos , Animais , Axônios , Espinhas Dendríticas , Drosophila , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Rim/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Imagens de Fantasmas , Córtex Somatossensorial/diagnóstico por imagem , SinapsesRESUMO
True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.
Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Movimento Celular , Endocitose , Olho/ultraestrutura , Humanos , Mitose , Organelas , Análise de Célula Única , Peixe-ZebraRESUMO
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
Assuntos
Citoesqueleto/ultraestrutura , Endocitose , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Organelas/ultraestrutura , Actinina/análise , Actinas/análise , Animais , Linhagem Celular , Clatrina/análise , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/ultraestrutura , Citoesqueleto/química , Citoesqueleto/metabolismo , Endossomos/química , Endossomos/ultraestrutura , Complexo de Golgi/ultraestrutura , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Organelas/química , Organelas/metabolismo , Proteínas rab5 de Ligação ao GTP/análiseRESUMO
Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.