RESUMO
As a basic functional unit in neural circuits, each neuron integrates input signals from hundreds to thousands of synapses. Knowledge of the synaptic input fields of individual neurons, including the identity, strength, and location of each synapse, is essential for understanding how neurons compute. Here, we developed a volumetric super-resolution reconstruction platform for large-volume imaging and automated segmentation of neurons and synapses with molecular identity information. We used this platform to map inhibitory synaptic input fields of On-Off direction-selective ganglion cells (On-Off DSGCs), which are important for computing visual motion direction in the mouse retina. The reconstructions of On-Off DSGCs showed a GABAergic, receptor subtype-specific input field for generating direction selective responses without significant glycinergic inputs for mediating monosynaptic crossover inhibition. These results demonstrate unique capabilities of this super-resolution platform for interrogating neural circuitry.
Assuntos
Neurônios/citologia , Imagem Óptica/métodos , Sinapses/metabolismo , Animais , Encéfalo/citologia , Proteínas de Transporte , Imuno-Histoquímica , Proteínas de Membrana , Camundongos , Rede Nervosa , Vias Neurais , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Células Ganglionares da Retina/metabolismo , Neurônios Retinianos/metabolismoRESUMO
Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.
Assuntos
Sistemas CRISPR-Cas/genética , Processamento de Imagem Assistida por Computador/métodos , Hibridização in Situ Fluorescente/métodos , RNA Longo não Codificante , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Edição de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Sondas Moleculares/química , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Análise de Célula Única/métodos , Algoritmos , Divisão Celular , Linhagem Celular Tumoral , Replicação do DNA , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , RNA/metabolismo , Reprodutibilidade dos TestesRESUMO
By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.
Assuntos
Actinas/química , Citoesqueleto/química , Linhagem CelularRESUMO
Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Nanoestruturas/ultraestrutura , Organelas/ultraestrutura , Compostos de Boro/química , Carbocianinas/química , Membrana Celular/ultraestrutura , Dendritos/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Hipocampo/citologia , Bicamadas Lipídicas , Lisossomos/ultraestrutura , Microscopia de Fluorescência/instrumentação , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Pseudópodes/ultraestrutura , Processos EstocásticosRESUMO
In super-resolution imaging techniques based on single-molecule switching and localization, the time to acquire a super-resolution image is limited by the maximum density of fluorescent emitters that can be accurately localized per imaging frame. In order to increase the imaging rate, several methods have been recently developed to analyze images with higher emitter densities. One powerful approach uses methods based on compressed sensing to increase the analyzable emitter density per imaging frame by several-fold compared to other reported approaches. However, the computational cost of this approach, which uses interior point methods, is high, and analysis of a typical 40 µm x 40 µm field-of-view super-resolution movie requires thousands of hours on a high-end desktop personal computer. Here, we demonstrate an alternative compressed-sensing algorithm, L1-Homotopy (L1H), which can generate super-resolution image reconstructions that are essentially identical to those derived using interior point methods in one to two orders of magnitude less time depending on the emitter density. Moreover, for an experimental data set with varying emitter density, L1H analysis is ~300-fold faster than interior point methods. This drastic reduction in computational time should allow the compressed sensing approach to be routinely applied to super-resolution image analysis.
Assuntos
Algoritmos , Diagnóstico por Imagem/métodos , Aumento da Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , HumanosRESUMO
Optimal analysis of single molecule localization microscopy (SMLM) data acquired with a scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera relies on statistical compensation for its pixel-dependent gain, offset and readout noise. In this work we show that it is also necessary to compensate for differences in the relative quantum efficiency (RQE) of each pixel. We found differences in RQE on the order of 4% in our tested sCMOS sensors. These differences were large enough to have a noticeable effect on analysis algorithm results, as seen both in simulations and biological imaging data. We discuss how the RQE differences manifest themselves in the analysis results and present the modifications to the Poisson maximum likelihood estimation (MLE) sCMOS analysis algorithm that are needed to correct for the RQE differences.
Assuntos
Artefatos , Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/instrumentação , Algoritmos , Animais , Calibragem , Desenho de Equipamento , Camundongos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/normas , Distribuição de Poisson , Pontos Quânticos/normas , Semicondutores/normas , Imagem Individual de Molécula/normas , Tálamo/diagnóstico por imagemRESUMO
Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we report a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot, properties that are important for MERFISH imaging. Using this amplification approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.
Assuntos
Perfilação da Expressão Gênica/métodos , Hibridização in Situ Fluorescente/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/análise , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Processamento de Imagem Assistida por Computador , Osteossarcoma/patologia , RNA Neoplásico/análise , Análise de Célula ÚnicaRESUMO
This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally resolved SMLM. We also provide open-source software for simultaneous control of multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.
RESUMO
The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia , Modelos Teóricos , Imagem Molecular , Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Imagem Molecular/métodosRESUMO
Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.
Assuntos
Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Humanos , Microscopia Eletrônica/instrumentação , Microscopia de Fluorescência/instrumentação , Microtomia , Microtúbulos/ultraestrutura , Orthomyxoviridae/ultraestrutura , Processos Estocásticos , Inclusão do Tecido , Liberação de Vírus/fisiologiaRESUMO
We examine the dynamics of DNA molecules in mixed flows where the ratio of vorticity to strain rate may be slightly above or below unity via Brownian dynamics simulation. We find that the chain dynamics in these flows are dramatically different than those found for simple shear flow. When the strain rate exceeds vorticity, the dynamics are found to be driven by the extra amount of straining. For vorticity-dominated flows, a periodicity in chain extension is observed with considerable chain deformation.
Assuntos
DNA/química , Fenômenos Químicos , Físico-Química , Modelos Moleculares , Conformação de Ácido Nucleico , Reologia , TermodinâmicaRESUMO
Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of ßII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of ßII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of ßII spectrin in neurites.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Actinas/química , Anquirinas/metabolismo , Espectrina/químicaRESUMO
The question of how genetic materials are trafficked in and out of the cell nucleus is a problem of great importance not only for understanding viral infections but also for advancing gene-delivery technology. Here we demonstrate a physical technique that allows gene trafficking to be studied at the single-gene level by combining sensitive fluorescence microscopy with microinjection. As a model system, we investigate the nuclear import of influenza genes, in the form of ribonucleoproteins (vRNPs), by imaging single vRNPs in living cells in real time. Our single-particle trajectories show that vRNPs are transported to the nuclear envelope by diffusion. We have observed heterogeneous interactions between the vRNPs and nuclear pore complexes with dissociation rate constants spanning two orders of magnitude. Our single-particle tracking experiments also provided new insights into the regulation mechanisms for the nuclear import of vRNPs: the influenza M1 protein, a regulatory protein for the import process, downregulates the nuclear import of vRNPs by inhibiting the interactions between vRNPs and nuclear pore complexes but has no significant effect on the transport properties of vRNPs. We expect this single-particle tracking approach to find broad application in investigations of genetic trafficking.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Análise de Injeção de Fluxo/métodos , Genes Virais/fisiologia , Microscopia de Fluorescência/métodos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Citometria de Fluxo/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de VídeoRESUMO
Highly extensible Escherichia coli DNA molecules in planar extensional flow were visualized in dilute solution by fluorescence microscopy. For a narrow range of flow strengths, the molecules were found in either a coiled or highly extended conformation, depending on the deformation history of the polymer. This conformation hysteresis persists for many polymer relaxation times and is due to conformation-dependent hydrodynamic forces. Polymer conformational free-energy landscapes were calculated from computer simulations and show two free-energy minima for flow strengths near the coil-stretch transition. Hysteresis cycles may directly influence bulk-solution stresses and the development of stress-strain relations for dilute polymer flows.
Assuntos
Biopolímeros/química , DNA Bacteriano/química , Escherichia coli , Conformação de Ácido Nucleico , Fenômenos Químicos , Físico-Química , Simulação por Computador , Microscopia de Fluorescência , TermodinâmicaRESUMO
Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cell's constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.
Assuntos
Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Orthomyxoviridae/patogenicidade , Actinas/química , Animais , Transporte Biológico , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Endocitose , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Modelos Biológicos , Fatores de TempoRESUMO
Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli , Hidrólise , Espectrometria de FluorescênciaRESUMO
Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.
Assuntos
RNA Catalítico/química , RNA/química , Tetrahymena/química , Animais , Relação Dose-Resposta a Droga , Cinética , Magnésio/farmacologia , Modelos Biológicos , Conformação de Ácido Nucleico , Dobramento de Proteína , Espalhamento de Radiação , Sódio/farmacologia , Ésteres do Ácido Sulfúrico/farmacologia , Termodinâmica , Fatores de Tempo , Raios XRESUMO
We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.
Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Carbocianinas/metabolismo , Catálise , Enzimas Imobilizadas , Fluorescência , Ligação de Hidrogênio , Cinética , Nepovirus/genética , Conformação de Ácido Nucleico , RNA Satélite , Espectrometria de Fluorescência , TermodinâmicaRESUMO
How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.