RESUMO
Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20-30 nm lateral and 50-70 nm axial resolution within an area of 500 µm × 500 µm and a depth of 0.6 µm to several micrometers. We employ single-molecule localization microscopy (SMLM) on immunolabeled fixed brain tissue slices. tomoSTORM utilizes array tomography to achieve SMLM in 40 nm thick resin-embedded sections. dSTORM of cryo-sectioned slices uses optical sectioning in 0.1-4 µm thick hydrated sections. Both approaches deliver 3D nanolocalization of two or more labeled proteins within a defined tissue volume. We review sample preparation, data acquisition, analysis, and interpretation.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Imagem Individual de Molécula/métodos , Tomografia/métodos , Biomarcadores , Processamento de Imagem Assistida por ComputadorRESUMO
The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1's function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31-Cdc31 interactions between Sfi1-Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution dSTORM microscope (direct stochastic optical reconstruction microscopy). The coordinate transfer was based on fixed cells as reference points without the use of additional fiducial markers. This procedure is suitable to combine any kind of fluorescence microscopy technique, in order to gain further insights on the observed specimen at multiple temporal or special scales.
Assuntos
Microscopia de Fluorescência/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Fluorescência/instrumentação , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
Photophysical properties of any fluorophore are governed by the chemical nanoenvironment. In the context of imaging biological samples, this translates to different photophysical properties for different labels and probes. Here, we demonstrate that the nanoenvironment of fluorophores within a probe can be advantageously used to induce particular properties such as light-induced photoswitching. We demonstrate efficient photoswitching and single-molecule super-resolution imaging for various fluorophore-phalloidin conjugates in aqueous buffer without the addition of further chemicals. We further demonstrate the utility of two-color imaging of fluorophore-phalloidin and a photoactivatable fluorescent protein in presynaptic nerve terminals.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Faloidina , Triptofano/metabolismo , Animais , Células Cultivadas , Células HeLa , Hipocampo/citologia , Humanos , Ratos , Espectrometria de Fluorescência/métodosRESUMO
High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for scalable experiments. The workflow consists of a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently re-localizing those cells with micrometer precision for confocal and super-resolution imaging. As a case study, we visualized and quantified cis- and trans-Golgi markers at increasing resolution.
Assuntos
Complexo de Golgi/ultraestrutura , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Processamento de Imagem Assistida por Computador/métodos , Rim/citologia , RatosRESUMO
Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods-collectively referred to as "single-molecule localization microscopy" (SMLM)-are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.Key to this strategy is the use of fluorophores that can be switched "on" and "off" in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.
Assuntos
Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência/métodos , Fenômenos Ópticos , Cor , Corantes Fluorescentes/química , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Imagem Molecular , Coloração e RotulagemRESUMO
We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging.
Assuntos
Corantes Fluorescentes/química , Ácidos Nucleicos/química , Sondas de Oligonucleotídeos/química , Estrutura Molecular , Hibridização de Ácido NucleicoRESUMO
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
Assuntos
Anatomia Transversal/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Tomografia/métodos , Animais , Encéfalo/citologia , Encéfalo/ultraestrutura , Mitocôndrias/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestruturaRESUMO
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.