Summary

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

Published: May 04, 2012
doi:

Summary

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.

Abstract

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 ≤5 s up to several hours, depending on the nature of the microinjected sample.

Protocol

1. Млекопитающие культуре клеток 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…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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

Materials

Name of the Reagent Company Catalogue Number Comments
Photoetched Coverslip and Live-cell Plate MatTek P35G-2-14-CGRD  
Texas Red Invitrogen D3329  
Cascade Blue Invitrogen D7132  
Rhodamine Labeling Kit Thermo Scientific 53002  
0.22 μm centrifugal filtration unit Millipore UFC30GV00  
Borosilicate Glass Pipettes Sutter Instruments BF100-50-10  
Micropipette Puller Sutter Instruments Model P-97 Use program #4 after performing ramp test
FemtoJet Microinjection System Eppendorf Injectman NI2  
Coverslip Removal Fluid MatTek PDCF OS 30  
Technai Transmission Electron Microscope FEI Technai G2 BioTWIN  
Ultramicrotombe Leica EM UC6  

Referências

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Reddick, L. E., Alto, N. M. Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets. J. Vis. Exp. (63), e3650, doi:10.3791/3650 (2012).

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