Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biomed Opt Express ; 15(6): 3715-3726, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38867795

RESUMO

In standard SMLM methods, the photoswitching of single fluorescent molecules and the data acquisition processes are independent, which leads to the detection of single molecule blinking events on several consecutive frames. This mismatch results in several data points with reduced localization precision, and it also increases the possibilities of overlapping. Here we discuss how the synchronization of the fluorophores' ON state to the camera exposure time increases the average intensity of the captured point spread functions and hence improves the localization precision. Simulations and theoretical results show that such synchronization leads to fewer localizations with 15% higher sum signal on average, while reducing the probability of overlaps by 10%.

2.
J Phys Chem B ; 127(3): 732-741, 2023 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-36638265

RESUMO

Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. Here, we study the versatile role of thiols in cyanine photoswitching at varying intensities generated in a single acquisition by a microelectromechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analyzed as a function of the thiolate concentration are photon budget, on-state and off-state lifetimes and the corresponding impact on image resolution. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum of ∼1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1-16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes.


Assuntos
Benchmarking , Quinolinas , Corantes Fluorescentes , Carbocianinas , Imagem Individual de Molécula/métodos
3.
Sci Rep ; 12(1): 20535, 2022 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-36446811

RESUMO

The regulated translocation of the glucose transporter, GLUT4, to the surface of adipocytes and muscle is a key action of insulin. This is underpinned by the delivery and fusion of GLUT4-containing vesicles with the plasma membrane. Recent studies have revealed that a further action of insulin is to mediate the dispersal of GLUT4 molecules away from the site of GLUT4 vesicle fusion with the plasma membrane. Although shown in adipocytes, whether insulin-stimulated dispersal occurs in other cells and/or is exhibited by other proteins remains a matter of debate. Here we show that insulin stimulates GLUT4 dispersal in the plasma membrane of adipocytes, induced pluripotent stem cell-derived cardiomyocytes and HeLa cells, suggesting that this phenomenon is specific to GLUT4 expressed in all cell types. By contrast, insulin-stimulated dispersal of TfR was not observed in HeLa cells, suggesting that the mechanism may be unique to GLUT4. Consistent with dispersal being an important physiological mechanism, we observed that insulin-stimulated GLUT4 dispersal is reduced under conditions of insulin resistance. Adipocytes of different sizes have been shown to exhibit distinct metabolic properties: larger adipocytes exhibit reduced insulin-stimulated glucose transport compared to smaller cells. Here we show that both GLUT4 delivery to the plasma membrane and GLUT4 dispersal are reduced in larger adipocytes, supporting the hypothesis that larger adipocytes are refractory to insulin challenge compared to their smaller counterparts, even within a supposedly homogeneous population of cells.


Assuntos
Adipócitos , Insulina , Humanos , Células HeLa , Tamanho Celular , Insulina/farmacologia , Translocação Genética , Miócitos Cardíacos
4.
Commun Biol ; 5(1): 218, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35264712

RESUMO

Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.


Assuntos
Nanotecnologia , Receptores de Superfície Celular , Animais , Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo
5.
ACS Photonics ; 8(9): 2728-2736, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34553004

RESUMO

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tunable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.

6.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33734291

RESUMO

Understanding biological function requires the identification and characterization of complex patterns of molecules. Single-molecule localization microscopy (SMLM) can quantitatively measure molecular components and interactions at resolutions far beyond the diffraction limit, but this information is only useful if these patterns can be quantified and interpreted. We provide a new approach for the analysis of SMLM data that develops the concept of structures and super-structures formed by interconnected elements, such as smaller protein clusters. Using a formal framework and a parameter-free algorithm, (super-)structures formed from smaller components are found to be abundant in classes of nuclear proteins, such as heterogeneous nuclear ribonucleoprotein particles (hnRNPs), but are absent from ceramides located in the plasma membrane. We suggest that mesoscopic structures formed by interconnected protein clusters are common within the nucleus and have an important role in the organization and function of the genome. Our algorithm, SuperStructure, can be used to analyze and explore complex SMLM data and extract functionally relevant information.


Assuntos
Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Corantes Fluorescentes/administração & dosagem
8.
Sci Rep ; 8(1): 605, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330515

RESUMO

The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.


Assuntos
Fusarium/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Esporos Fúngicos/crescimento & desenvolvimento , Microscopia , Software
9.
Nat Methods ; 14(1): 41-44, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869814

RESUMO

We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.


Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Imagem Molecular/métodos , Fotometria/métodos , Soroalbumina Bovina/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Algoritmos , Animais , Bovinos , Simulação por Computador , Humanos , Fótons , Análise de Célula Única/métodos
10.
Methods Appl Fluoresc ; 4(2): 022004, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28809165

RESUMO

For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research.


Assuntos
Neurônios , Corantes Fluorescentes , Imageamento Tridimensional , Substâncias Macromoleculares , Microscopia , Microscopia de Fluorescência , Imagem Molecular , Nanotecnologia , Imagem Óptica , Análise Espectral
11.
Sci Rep ; 5: 15348, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26481189

RESUMO

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.


Assuntos
Luz/efeitos adversos , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Corantes Fluorescentes , Humanos , Iluminação , Microtúbulos/metabolismo , Fatores de Risco
12.
ACS Nano ; 9(8): 8122-30, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26173009

RESUMO

We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.


Assuntos
Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Oligonucleotídeos/química , Coloração e Rotulagem/métodos , Carbocianinas/química , Cisteamina/química , Eletrodos , Corantes Fluorescentes/química , Glicerol/química , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Álcool de Polivinil/química , Eletricidade Estática , Processos Estocásticos
13.
Nat Commun ; 6: 5980, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25609143

RESUMO

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.


Assuntos
Microscopia/métodos , Imagem Óptica/métodos , Simplexvirus/ultraestrutura , Animais , Anticorpos Monoclonais/química , Linhagem Celular , Microscopia Crioeletrônica , Feminino , Herpesvirus Humano 1 , Humanos , Processamento de Imagem Assistida por Computador , Queratinócitos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Proteínas Recombinantes/química , Proteínas do Envelope Viral/química , Proteínas Virais/química , Proteínas Estruturais Virais/química , Vírion/metabolismo
14.
Nano Lett ; 15(2): 1374-81, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25533766

RESUMO

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.


Assuntos
Corantes Fluorescentes/química , Osteossarcoma/química , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Nanotecnologia
15.
J Cell Sci ; 127(Pt 20): 4351-5, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25146397

RESUMO

Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.


Assuntos
Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Proteínas de Xenopus/ultraestrutura , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Oócitos/ultraestrutura , Multimerização Proteica , Proteínas de Xenopus/química , Xenopus laevis
16.
Nat Commun ; 5: 4650, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25130366

RESUMO

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Drosophila/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Animais , Fenômenos Eletrofisiológicos/fisiologia , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Relação Estrutura-Atividade , Sinapses/fisiologia , Transmissão Sináptica/fisiologia
17.
Opt Express ; 22(9): 10304-16, 2014 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-24921733

RESUMO

In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

18.
Cell Microbiol ; 16(8): 1224-43, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24528559

RESUMO

Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER-vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER-vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER-SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER-SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.


Assuntos
Chlamydiales/patogenicidade , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Vacúolos/microbiologia , Antibacterianos/farmacologia , Infecções por Chlamydiaceae/patologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Mitocôndrias/metabolismo , Tapsigargina/farmacologia , Tunicamicina/farmacologia
19.
Chem Soc Rev ; 43(4): 1076-87, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23942584

RESUMO

Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching, bio-labeling compatibility, and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore, we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.


Assuntos
Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Processos Fotoquímicos , Imagem Óptica/métodos
20.
Chemphyschem ; 15(4): 651-4, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24227751

RESUMO

Crystal clear: The authors introduce a miniaturized localization microscopy setup based on cost-effective components. They demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.


Assuntos
Processamento de Imagem Assistida por Computador/economia , Microscopia de Fluorescência/economia , Linhagem Celular Tumoral , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Microscopia de Fluorescência/instrumentação , Software
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...