Correlative और एक उपकरण के रूप में इलेक्ट्रॉन माइक्रोस्कोपी (भूखों मरना) लाइट कल्पना Microinjected अणु और उनके यूकेरियोटिक उप सेलुलर लक्ष्य
Summary
भूखों मरना तकनीक झिल्ली organelles, और subcellular संरचनाओं microinjected अणुओं से प्रभावित ultrastructural आकारिकी का विश्लेषण करने के लिए अनुकूलित किया गया है. इस विधि / micromanipulation microinjection के शक्तिशाली तकनीक, confocal माइक्रोस्कोपी फ्लोरोसेंट, और इलेक्ट्रॉन माइक्रोस्कोपी जोड़ती बहु nanometer संकल्प मिलीमीटर की अनुमति. इस तकनीक के लिए आवेदनों की एक विस्तृत विविधता के लिए उत्तरदायी है.
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
Discussion
यहाँ प्रस्तुत पद्धति यूकेरियोटिक cytoplasm शुट्ठ प्रोटीन, न्यूक्लिक एसिड, या छोटे अणुओं के प्रत्यक्ष वितरण सक्षम बनाता है है और फ्लोरोसेंट और इलेक्ट्रॉन माइक्रोस्कोपी के संबंध के माध्यम से अल्ट्रा उच्च स…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम उपयोगी विचार – विमर्श के लिए ऑल्टो प्रयोगशाला के सदस्यों को धन्यवाद. हम भी UT दक्षिण मेडिकल सेंटर, विशेष रूप से डॉ. क्रिस Gilpin, टॉम Januszewski, और लॉरी म्यूएलर में तकनीकी विशेषज्ञता और सलाह के लिए सेलुलर और आण्विक इमेजिंग सुविधा धन्यवाद. पांडुलिपि के महत्वपूर्ण पढ़ने के लिए डॉ. Xionan दांग धन्यवाद. इस काम NIH अनुदान AI083359 द्वारा NMA समर्थित है
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 |
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