This video article describes how to build a customized epi fluorescence fret imaging system from commercially available components and shows how to use it for real-time monitoring of intracellular cyclic a MP in intact cells. First, a step-by-step procedure for assembling the fret imaging is detailed. Next, the process for installing and configuring the software is shown once the system is completely set up.
Its use in measuring intracellular cyclic. A MP levels in transfected cells is demonstrated data obtained using this system exhibit its usefulness in monitoring intracellular cyclic A MP dynamics by measuring fret ratios. The main advantage of this technique is that you can visualize SEC messengers biochemical events or protein protein interactions with high spatial and temporal resolution in intact cells and tissues.
And this is not possible with existing biochemical methods like ELIZA or Radio immunoassays. We use this method to gain insight into cyclic nucleotide dynamics, but this can be easily applied onto other systems using fret biosensors. These biosensors can be either expressed in cell lines or cells and tissues of transgenic mice to study intracellular mechanisms in a more physiological context.
And now we will show you how to set up a fret system and how to use it for simple imaging experiments. In principle, any inverted fluorescence microscope that 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.
The hardware devices, including the light source, the shutter, and the camera are integrated into and controlled by the imaging software, which allows image acquisition and analysis. Begin the setup by connecting the light source to the microscope. For example, use a single wavelength light emitting diode, which selectively excites enhanced cyan fluorescent protein, which is used as a donor in most fret biosensors.
Place an appropriate filter cube into the microscope for routine fret measurements with CFP and enhanced YFP or any of their variants as a fret pair. Use a simple filter cube containing an ET 4 36 30 M excitation filter, and A-D-C-L-P 4 5 5 dichroic mirror. Next, connect the beam splitter via a CM mount to one of the microscope's emission ports.
For example, use the DV two dual view from photometrics, which splits the emission light into donor and acceptor channels that can be simultaneously monitored on a single CCD camera chip. Connect the CCD camera to the beam splitter. Use a fire wire cable to connect the camera to the IEEE 1394 computer interface without switching on the camera.
Install the camera drivers from the software CD to establish the communication between the computer and the light source. Use A BNC cable to connect the Arduino input output board to the LED. Finally, connect the assembled board to the computer via USB.
Now that the hardware has been set up, imaging software should be installed and configured here. The open source micromanager freeware version 1.4 0.5 is used. This software offers a high degree of flexibility for low cost imaging and can be downloaded from the site shown here.
Next, download the software to control the Arduino board from the web. Follow the instructions found on this website and run this software just one time prior to starting micromanager. Now download a film ware source code for use of the board with micromanager software from the website shown on the screen.
Then upload it onto the board once the software has been installed and initialized switch on the LED and the camera. Start the software and configure micromanager communication with the camera and LED by selecting tools. Then hardware Configuration wizard.
In the software, add the required components including the camera, Arduino board, Arduino Switch, Arduino hub, and Arduino shutter devices. During the next steps, use the default setting suggested by the wizard, save the new system configuration. When prompted by the wizard, use the main menu to open tools.
Then device property browser. Scroll down to Arduino. Switch State and select one.
Close the dialogue window. Make sure that the auto shutter box is ticked in the main software dialogue. Press file.
Then save system state to save the software configuration, which can be opened any time after starting the software. This will establish the communication between the board and the software needed to perform image acquisition. Next, press the live button to monitor the signal coming from the camera.
Make sure that the fluorescent light goes on any time the live or snap function is selected. Start the imaging software. Then to load the previously defined system configuration, select file, then select load system state and choose the name of the previously saved configuration file.
Next, using forceps, carefully mount a cover slide with a adherent non COFLUENT cyclic A MP sensor TRANSFECTED 2 9 3 A cells into the imaging chamber. Using a pipette rinse the cells once with 400 microliters of fret buffer. Then add 400 microliters of fret buffer.
Put a drop of immersion oil onto the objective and transfer the imaging chamber onto the microscope. Focus on the cell layer using trans illumination light Switch on the fluorescent light by pressing the live button and select a cell. For the experiment.
Choose a cell with an optimal sensor expression, meaning that it should be not too bright and not too dim. After finding an appropriate cell switch off the fluorescent light immediately to avoid photo bleaching of the fret sensor. Under camera settings, adjust the exposure time to 10 to 50 milliseconds and press the snap button to take an image, check the signal to noise ratio and adjust the exposure time as needed to obtain an image with a good signal to noise ratio.
Two long excitation times might result in photobleaching while two short times result in low image quality. Press the multi D acquisition button and set the number of time points and the time interval for image acquisition. For this cyclic, A MP fret sensor one image will be acquired every five seconds.
Start the measurements by pressing the acquire button during the measurement to monitor fret ratio changes. Run the fret online plugin by selecting plugins. Then fret online in the Image J software menu.
Then using the freehand selections tool, select a region of interest in the fret ratio image and add the region into the ROI manager. In the time series analyzer window press the get average button. The fret ratio trace will be displayed to update the trace during the measurement.
Run the fret ratio online to plugin and press the get average button as soon as the fret ratio has reached a stable baseline pipette isoproterenol solution into the chamber and wait for a signal to come. Then just after a new stable baseline is reached, add propanolol solution. After finishing the experiment, save the time-lapse stack of images.
Remove the measurement chamber from the microscope and clean the objective using objective tissue when all of the data has been collected. Follow the instructions in the accompanying document for analysis. A fret system assembled as described in this video, was used to monitor cyclic A MP levels in response to beta adrenergic agonists and antagonists.
In cyclic A MP sensor TRANSFECTED 2 9 3 A cells as can be seen here, the fret ratio showed a stable baseline until the beta adrenergic agonist isoproterenol was added. At frame number 30 after the application, there was a slow decrease in the monitored fret ratio indicating an increase in intracellular cyclic A MP.When the beta blocker propanolol was added at frame number 60, the isoproterenol signal was reversed and cyclic A MP levels decreased to basal levels to obtain a corrected fret ratio trace the data were analyzed offline and bleed through of CFP into the YFP channel was corrected. As can be seen here.
The corrected fret ratio trace shows a bigger amplitude of the fret signal, which reflects the actual fret response. Following this procedure, other methods like Western blot analysis or functional assays can be performed in order to answer additional questions on the activity of downstream signaling pathways and cellular response to CAMP stimulating agents. The development of this technique paved the way for researchers in the field of cellular biology to explore real time dynamics of messengers such as C-A-M-P-C-G-M-P, or calcium in intact cells or tissues.
After watching this video, you should have a good idea how to build a simple fat imaging system and how to control it by the software to perform your experiments.
