जीवित कोशिकाओं में घटनाओं संकेत Unimolecular Biosensors की वास्तविक समय की निगरानी के लिए माइक्रोस्कोपी झल्लाहट
Summary
Forster प्रतिध्वनि ऊर्जा हस्तांतरण (झल्लाहट) माइक्रोस्कोपी जीवित कोशिकाओं में घटनाओं संकेत संवाददाताओं से के रूप में विभिन्न biosensors का उपयोग करने की वास्तविक समय की निगरानी के लिए एक शक्तिशाली तकनीक है. यहाँ हम वर्णन कैसे एक स्वनिर्धारित epifluorescence बनाने के व्यावसायिक रूप से उपलब्ध उपकरणों और कैसे इसे इस्तेमाल के लिए प्रयोगों झल्लाहट झल्लाहट इमेजिंग प्रणाली.
Abstract
Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.
Protocol
Discussion
इस प्रोटोकॉल में, हम दिखाना है कि कैसे एक साधारण कम लागत लेकिन शक्तिशाली झल्लाहट उपलब्ध biosensors की एक किस्म के साथ नियमित अनुप्रयोगों के लिए इमेजिंग प्रणाली का निर्माण करने के लिए. यहाँ प्रस्तुत प्रणाली CFP…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए तकनीकी सहायता के लिए Anke Rüttgeroth और करीना Zimmermann धन्यवाद देना चाहूंगा. यह काम ड्यूश Forschungsgemeinschaft (अनुदान एनआई वॉन 1301/1-1) और गौटिंगेन मेडिकल सेंटर के विश्वविद्यालय ("समर्थक FUTURA" वॉन के लिए अनुदान) द्वारा समर्थित किया गया.
Materials
| Name of the reagent/equipment | Company | Catalogue number | Comments |
| BES Buffer Grade | AppliChem | A1062 | |
| CaCl2 dihydrate | Sigma-Aldrich | C5010 | |
| Glass coverslides | Thermo Scientific | 004710781 | Diameter 24 mm |
| Glass-bottomed cell-culture dishes | World Precision Instruments | FD3510-100 | |
| D-MEM medium | Biochrom AG | F0445 | |
| Fetal calf serum (FCS) | Thermo Scientific | SH30073.02 | |
| L-Glutamine | Biochrom AG | K0283 | |
| HEPES | Sigma | H4034 | |
| KCl | Sigma | P5405 | |
| MgCl2 hexahydrate | AppliChem | A4425 | |
| NaCl | AppliChem | A1149 | |
| Na2HPO4 | Sigma-Aldrich | S9707 | |
| Penicillin/Streptomycin | Biochrom AG | A2213 | |
| Inverted fluorescent microscope | e.g. Nikon | Request at Nikon | |
| CoolLED | CoolLED | pE-100 | 440 nm |
| DualView | Photometrics | DV2-SYS | |
| DualView filter slider | Photometrics | 05-EM | |
| CFP/YFP filter set | Chroma Technology | 49052 | without the emission filter |
| ORCA-03G camera | Hamamatsu Photonics | C8484-03G02 | |
| Arduino I/O board | Sparkfun Electronics | DEV-00666 | |
| Attofluor cell chamber | Invitrogen | A-7816 | |
| Personal computer with WindowsXP or Windows7 system | Any supplier | Include hard-drive with high capacity |
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