The authors would like to thank Anke R&uuml;ttgeroth and Karina Zimmermann for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (grant NI 1301/1-1 to V.O.N) and University of G&ouml;ttingen Medical Center ("pro futura" grant to V.O.N.).

1. Setting up a FRET Imaging Microscope
In principle, any inverted fluorescence microscope which is available in the lab and has a camera port can be adapted for FRET imaging. The final setup should include the following crucial components: a microscope, a light source with or without additional shutter, a beam-splitter for emission light and a CCD-camera (see Figure 1). The hardware devices, especially the light source, the shutter and the camera are integrated into and control...

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No conflicts of interest declared.

in this protocol, we demonstrate how to build a simple low-cost but powerful FRET imaging system for routine applications with a variety of available biosensors. The system presented here is designed for CFP and YFP, or similar types of fluorescent proteins, as the donor-acceptor pair. Meanwhile, other individual biosensors become available which use for example green and red fluorescent proteins14. To adapt the described system for other colors, appropriate light sources and/or filter sets should be selected....

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>BES Buffer Grade</td>
 <td>AppliChem</td>
 <td>A1062</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CaCl2 dihydrate</td>
 <td>Sigma-Aldrich</td>
 <td>C5010 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass coverslides</td>
 <td>Thermo Scientific</td>
 <td>004710781</td>
 <td>Diameter 24 mm</td>
 </tr>
 <tr>
 <td>Glass-bottomed cell-culture dishes</td>
 <td>World Precision Instruments</td>
 <td>FD3510-100 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-MEM medium</td>
 <td>Biochrom AG</td>
 <td>F0445</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal calf serum (FCS)</td>
 <td>Thermo Scientific</td>
 <td>SH30073.02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Biochrom AG</td>
 <td>K0283</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H4034</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Sigma</td>
 <td>P5405</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgCl2 hexahydrate</td>
 <td>AppliChem</td>
 <td>A4425</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>AppliChem</td>
 <td>A1149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Na2HPO4</td>
 <td>Sigma-Aldrich</td>
 <td>S9707</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Biochrom AG</td>
 <td>A2213</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted fluorescent microscope</td>
 <td>e.g. Nikon</td>
 <td>Request at Nikon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CoolLED</td>
 <td>CoolLED</td>
 <td>pE-100 </td>
 <td>440 nm</td>
 </tr>
 <tr>
 <td>DualView</td>
 <td>Photometrics</td>
 <td>DV2-SYS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DualView filter slider</td>
 <td>Photometrics</td>
 <td>05-EM</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CFP/YFP filter set</td>
 <td>Chroma Technology</td>
 <td>49052</td>
 <td>without the emission filter</td>
 </tr>
 <tr>
 <td>ORCA-03G camera</td>
 <td>Hamamatsu Photonics</td>
 <td>C8484-03G02 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Arduino I/O board </td>
 <td>Sparkfun Electronics</td>
 <td>DEV-00666</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Attofluor cell chamber </td>
 <td>Invitrogen</td>
 <td>A-7816</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Personal computer with WindowsXP or Windows7 system </td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>Include hard-drive with high capacity</td>
 </tr>
 </tbody>
</table>

fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors

F&ouml;rster 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 &beta;-adrenergic receptor agonist and blocker.

Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

fret-microscopy-for-real-time-monitoring-signaling-events-live-cells

Research

JoVE Journal

Biology

23.0K visualizações.  Georg August University Medical Center, Göttingen, Germany. Förster transferência de energia por ressonância (FRET) microscopia é uma técnica poderosa para monitoramento em tempo real de eventos de sinalização em células vivas usando biossensores diversos como repórteres. Aqui descrevemos como construir um epifluorescência personalizado FRET sistema de imagens a partir de componentes disponíveis comercialmente e como usá-lo para FRET experimentos.