RESUMO
We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reconhecimento Automatizado de Padrão/métodos , Animais , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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 <20â 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 laevisRESUMO
One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41 ± 7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Carbocianinas , Feminino , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Modelos Moleculares , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Oócitos/metabolismo , Oócitos/ultraestrutura , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas de Xenopus/ultraestrutura , Xenopus laevisRESUMO
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.
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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 , SoftwareRESUMO
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
Assuntos
DNA/química , Proteína HMGA1a/química , Motivos AT-Hook , Sequência de Aminoácidos , Aminoácidos Básicos/química , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , DNA/ultraestrutura , Proteína HMGA1a/análise , Proteína HMGA1a/genética , Dados de Sequência Molecular , MutaçãoRESUMO
A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.
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Encéfalo/anatomia & histologia , Imageamento Tridimensional , Pesquisa , Animais , Masculino , Camundongos , Microscopia Confocal , Neurônios/citologia , Ratos , Análise de Célula Única , Sinapses/fisiologiaRESUMO
In experimental animal research body temperature (BT) is measured for the objective determination of an animals' physiological condition. Invasive, probe-based measurements are stressful and can influence experimental outcome. Alternatively BT can be determined touch-free from the emitted heat of the organism at a single spot using infrared thermometers [1]. To get visual confirmation and find more appropriate surfaces for measurement a hand-held thermal imager was equipped with a self-made, cheap, 3D-printable close-up lens system that reproducibly creates eight-time magnified thermal images and improves sensitivity. This setup was used to establish ocular surface temperature (OST), representing the temperature of the brain-heart axis, as a touch-free alternative for measurement of BT in mice, rats, rabbits and humans.OST measurement after isoflurane exposure and myocardial infarction (MI) experiments in mice revealed high physiological relevance and sensitivity, the possibility to discriminate between MI and sham operations in one hour and even long-term outcome-predictive capabilities of OST after MI. Summarized here we present: â¢Self-made close-up lens for thermal imaging cameras for eight-time magnificationâ¢Establishment of OST for touch-free determination of BT in rodents and humansâ¢Short- and long-term predictive capabilities of OST in experimental MI in mice.
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Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.
Assuntos
Estruturas Celulares/ultraestrutura , Química Click/métodos , Proteínas de Fluorescência Verde/química , Imagem Molecular/métodos , Animais , Linhagem Celular , DNA/química , DNA/metabolismo , Humanos , Camundongos , Microscopia Confocal , Oxigênio/química , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.
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Estruturas Celulares/ultraestrutura , Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , HumanosRESUMO
Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of â¼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.