Name: correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets
Uploaded: 2012-05-04T12:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

Мы благодарны сотрудникам лаборатории Alto за полезные обсуждения. Мы также благодарим Молекулярная и клеточная изображений объекта в UT Юго-западного медицинского центра, в частности, д-р Крис Гилпин, Том Янушевский, и Лори Мюллер на техническую экспертизу и консультации. Мы также благодарим доктора Xionan Dong за критическое прочтение рукописи. Эта работа выполнена при поддержке гранта NIH AI083359 в NMA

1. Млекопитающие культуре клеток <ol><li> Нормальные почки крысы (NRK), увековеченный рака шейки матки (HeLa), или других соответствующих линий клеток млекопитающих культивируют при 37 ° C, 5% CO 2, на фототравленые стекла покровные сетке прикреплены к живой клетке блюд (см. Таблицу <s...

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Метод, представленный здесь, дает прямую поставку очищенных белков, нуклеиновых кислот, или небольших молекул в цитоплазме эукариотической и обеспечивает сверхвысокое разрешение анализу на основе соотношения люминесцентных и электронной микроскопии. Использование этого метода явл?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Фототравленые Покровные и Live-клеточной пластинки </td><td> Маттек </td><td> P35G-2-14-CGRD </td><td></td></tr><tr><td> Texas Red </td><td> Invitrogen </td><td> D3329 </td><td></td></tr><tr><td> Каскад синий </td><td> Invitrogen </td><td> D7132 </td><td></td></tr><tr><td> Родамина маркировки Kit </td><td> Thermo Scientific </td><td> 53002 </td><td></td></tr><tr><td> 0,22 мкм центробежные блок фильтрации </td><td> Millipore </td><td> UFC30GV00 </td><td></td></tr><tr><td> Боросиликатного стекла Пипетки </td><td> Sutter Instruments </td><td> BF100-50-10 </td><td></td></tr><tr><td> Микропипетки съемник </td><td> Sutter Instruments </td><td> Модель P-97 </td><td> Используйте программу № 4 после выполнения теста рампы </td></tr><tr><td> FemtoJet Микроинъекция системы </td><td> Eppendorf </td><td> InjectMan Ni2 </td><td></td></tr><tr><td> Покровные удаления жидкости </td><td> Маттек </td><td> PDCF OS 30 </td><td></td></tr><tr><td> Technai просвечивающего электронного микроскопа </td><td> FEI </td><td> Technai G2 BioTWIN </td><td></td></tr><tr><td> Ultramicrotombe </td><td> Leica </td><td> EM UC6 </td><td></td></tr></tbody></table>

correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Техника CLEM была адаптирована для анализа ультраструктурным морфологии мембран, органеллы, и субклеточных структур, пострадавших от микроинъекции молекул. Этот метод сочетает в себе мощные методы микроманипуляция / микроинъекции, конфокальной флуоресцентной микроскопии и электронной микроскопии, чтобы миллиметра до нескольких нанометровым разрешением. Этот метод поддается широкий спектр приложений.

Корреляционный световой и электронной микроскопии (CLEM) как инструмент для визуализации микроинъекции молекул и их Эукариотические Sub-клеточных мишеней


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Research

JoVE Journal

Biology

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Electroantennographic биопроб в качестве скринингового инструмента для летучих растения-хозяина

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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В естественных условиях клоновых Tracking гемопоэтических стволовых и клеток-предшественников оцениваться пятью флуоресцентных белков с помощью конфокальной и многофотонной микроскопии

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Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Сканирующая электронная микроскопия из размоченной ткани для визуализации внеклеточный матрикс

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

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Подготовка прокариот и эукариот организмов, с использованием химических сушки для морфологического анализа в растровая электронная микроскопия (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

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Novel Protocol for Simultaneous Fluorescence and Ultrastructure Preservation

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Author Spotlight: Advancements in Correlative Light and Electron Microscopy with Fluorescent Protein Preservation

Epon Post Встраивание коррелятивной световой и электронной микроскопии

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence ...