Förster 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.
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.
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 controlled by the imaging software which allows image acquisition and analysis. Below we describe a procedure to assemble a simple FRET system from commercially available components.
2. Setting up the Imaging Software
To control and synchronize the light source with image capturing by the camera, imaging software should be installed on the computer. There are several commercially available software packages including MetaFluor (Molecular Devices), Slidebook (Intelligent Imaging Innovations), VisiView (Visitron Systems). Here we demonstrate the use of the open-source Micro-Manager freeware which offers a high degree of flexibility for low-cost imaging.
3. Cell Culture and Transfections
4. FRET Measurements in Live Cells
5. Offline Data Analysis
FRET imaging data can be analyzed offline at any time after the experiment using ImageJ software. As a supplement to this protocol, we provide the “FREToffline” plug-in used in our laboratory to split the acquired images into donor and acceptor channels and to measure fluorescent intensities in multiple regions of interest. These intensities can further be copy-pasted into an Excel or Origin spreadsheet to calculate the corrected FRET ratio. To visualize the FRET changes of unimolecular biosensors, simple ratiometry is often used. In this case, only the donor fluorophore (CFP) is excited, and two images are taken at CFP and YFP emission peaks. The calculated YFP/CFP ratio (sometimes also referred to as FRET/CFP ratio) represents the degree of FRET between the two fluorophores. In unimolecular biosensors, the numbers of CFP and YFP moieties are equal, so that the simple ratiometry is sufficient to represent the FRET efficiency12.
6. Representative Results
Figure 1 shows an example of a fully assembled FRET imaging setup consisting of a Nikon inverted microscope, CoolLED, DV2 DualView and the Hamamatsu ORCA-03G CCD camera. To establish the communication between the hardware components and the computer, the I/O Arduino board is connected to the computer and to the CoolLED as shown in Figure 2. To control the light source and image capturing by the camera in a synchronized fashion, Micro-Manager software has to be installed and properly configured (see Figure 3). This freeware can be easily adapted to individual experimental needs by adding necessary plug-ins. Figure 4A shows a representative raw FRET ratio trace from a measurement using the cAMP sensor Epac1-camps13 expressed in 293A cells to monitor the effects of β-adrenergic agonist isoproterenol applied at time point 150 sec and the β-blocker propranolol added at time point 300 sec (performed as described in 4.3-4.10). These data can be analyzed offline and corrected for the bleedthrough of CFP into the YFP channel as described in 5.1-5.7 to obtain the corrected FRET ratio trace shown in Figure 4B. This representative experiment shows a slow decrease in the monitored FRET ratio upon isoproterenol treatment which indicates an increase of intracellular cAMP. Propranolol as a β-blocker reverses the isoproterenol signal, leading to a decrease of cAMP to basal levels. These changes in FRET signal can be monitored online during any experiments (as described in 4.9). Such experiments can be performed with a variety of commonly used biosensors designed to monitor different second messengers or biochemical processes.
Figure 1. Layout of the FRET imaging setup comprised of a CoolLED, inverted Nikon microscope, DV2 DualView, and ORCA-03G CCD camera.
Figure 2. Arduino I/O board and its connections. The board is positioned in a self-mounted plastic box. A standard BNC cable connects the LED to the pins 8 and GND (0) of the board.
Figure 3. Screenshots demonstrating integration of the system components using Hardware Configuration Wizard. A) Start the Hardware Configuration Wizard. B) Add the required devices as mentioned in 2.3. Click here to view larger figure.
Figure 4. A representative FRET experiment which measures cAMP levels in 293A cells transfected with Epac1-camps. First, cells were stimulated with the β-adrenergic agonist isoproterenol (100 nM, at frame 30 or at 150 sec) to increase cAMP (observed as a decrease in YFP/CFP FRET ratio). Cells were subsequently treated with the β-blocker propranolol (10 μM, at frame 60 or at 300 sec) which leads to an increase of FRET ratio, reflecting a decrease in cAMP. A) Raw online FRET ratio trace from one region of interest corresponding to a single cell monitored during the experiment. B) Corrected ratio trace after offline data analysis performed as described in 5.1-5.7.
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. In the case of LED, another single LED line, for example 490 nm to excite green fluorescent protein can be used. Single-wavelength LEDs can be easily (within seconds) dismounted and exchanged. Alternatively, there are LED arrays available which contain several lines to excite various fluorescent proteins (e.g. pE-2 CoolLED). To enable measurements with alternative FRET pairs, other fluorescent filter cubes can be placed into the microscope, and eventually the emission beam-splitter filters should be exchanged. Photometrics offers additional emission filter sliders for DV2 DualView. Some recently developed applications use two or more biosensors simultaneously to monitor multiple processes at the same time, for example cAMP and cGMP together in one cell15. In this case, a QuadView (Photometrics) containing four emission channels can be used, the ImageJ plug-in for image splitting and analysis can be then adapted to be used with four channels and to calculate two FRET ratios. ImageJ software is very flexible in terms of image analysis and online representation of the results. Simple text-edited plug-ins allow adaptation of the software algorithm to the needs of any individual imaging system and experiment. This is sometimes very helpful and offers rapid solutions to the technical problems, which can require long times when they have to be implemented into any commercial software package.
When doing FRET experiments, it is crucial to avoid photobleaching which appears when the excitation times are too long or when the images are taken too frequently. In this case, a photon-induced chemical damage or covalent modifications of fluorophores may occur and decrease FRET efficiency. To avoid photobleaching, one can reduce the exposure time and the frequency of image acquisition. There are also established protocols12,16 to correct for this phenomenon. During data analysis, it is possible to correct for the cross-talk between donor and acceptor channels (bleedthrough). When using unimolecular FRET biosensors (in which case donor and acceptor fluorophores are always expressed at the same level) for simple ratiometric measurements, it can be enough to correct just for the bleedthrough of the donor into the acceptor channel or even omit this correction altogether because it does not qualitatively affect the shape of the FRET ratio curve. The bleedthrough of the acceptor into the donor channel is usually negligible. When bimolecular biosensors comprised of two different proteins are used, these may be expressed at various levels. In this case, the bleedthrough correction and an additional correction for the direct YFP excitation by the 440 nm light are also advisable. Please, refer to the published protocol12 where all correction procedures are described in more detail. Additional comprehensive information about the development of biosensors, FRET microscopy, possible pitfalls of the technique and about the data analysis are available in previously published protocols17-18. In conclusion, the simple and powerful imaging system described here provides a flexible platform to monitor various biochemical events and signaling molecules with high temporal and spatial resolution in live cells.
The authors have nothing to disclose.
The authors would like to thank Anke Rüttgeroth 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 Göttingen Medical Center (“pro futura” grant to V.O.N.).
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 |