[Credits: Manoj V Mathew, Centre for Cellular and Molecular Platforms (CCAMP), Bangalore, India.
Experiments were conducted at the Central Imaging and Flow Cytometry Facility (CIFF), National Center for Biological Sciences (NCBS), Bangalore, India.]
FRAP stands for Fluorescence Recovery After Photobleaching. This is a very simple technique to study dynamics like diffusion rates in biological samples at the microscopic level. A better way to study dynamics would be at the single-molecule level. For such studies, FCS (Fluorescence Correlation Spectroscopy) might be a better tool. However, FCS is not easy, requires specialized hardware and some complex analysis modules. FRAP, on the other hand, can be easily performed on most confocal fluorescence microscopes. For a better understanding of the differences between FRAP and FCS kindly see (Link).
FRAP involves photobleaching a Region of Interest (ROI) in the biological sample and studying the recovery of fluorescence in that region over time. Since photobleached molecules cannot recover the ability to fluoresce, the only way recovery can happen is by the diffusion of non-bleached molecules from outside ROI. The average intensity of the ROI can be measured as a function of time and plotted as a graph. This can then be fitted onto standard diffusion models to compute parameters like diffusion rates.
In this write-up, I will describe how to use the Olympus FV3000 confocal microscope for performing FRAP experiments.
The main interface of the FV3000 software is shown in Figure 1708/1.
Step 1: Image the sample
Use the software interface to generate an image of the sample. Adjust the laser power and PMT gain such that the image is not saturated but covers most of the dynamic range of the system. This can be ensured by using the HiLo mode in the image visualization window. I used a fixed thin section of convallaria as the sample. While this is definitely not a good sample to demonstrate FRAP, it is certainly enough for us to learn the software features. Convallaria is autofluorescent for excitations with most of the available visible laser excitation sources. I used 488nm excitation source and spectral detector settings commonly used for Alexa 488 dye for detection.
In general, it is always recommended to use the 10% AOTF dampening (Laser ND filter set to 10%) to protect the detectors against excessive light exposure. This is especially important when using GaAsP detectors. However, when performing FRAP we need laser powers higher than 10%. Hence imaging should also be performed with the 10% AOTF dampening factor removed (Laser ND filter set to 'None'). In this case be extremely careful with the AOTF laser power setting and PMT gain setting to make sure you donot saturate the detectors.
Step 2: Enable the 'LSM Stimulation' and 'Synchronization' tabs
For performing FRAP experiments you need to enable the 'LSM Stimulation' and 'Synchronization' tabs in the software. Ths can be done by going to 'Tools Window' in the main menu of the software. This is shown in Figure 1708/2.
Once enabled they will appear within one of the tab containers in the interface as shown in Figure 1708/3.
Step 3: Set the 'LSM Stimulation' tab
Go to the 'LSM Stimulation' tab and set the photobleaching parameters. The 'LSM Stiumation' settings are shown in Figure 1708/4.
The 'LSM Stimulation' tab provides the following:
- ROI tools: A number of tools to select one or more ROIs on the acquired image or the Live view
- Bleaching Dwell Time: The bleaching dwell time can be set different from the imaging dwell time. The image dwell time is entered in the 'LSM Imaging' tab. It might be useful to set a higher bleaching dwell time for effective bleaching.
- Bleaching Duration: You can set the bleaching duration to 'continuous'. In this case, the beaching continues till the user stops it. You can also enter a total bleach duration. For FRAP experiments it might be better not to use the continuous mode.
- Bleaching Laser selection and powers: Bleaching is usually most effective with the same laser wavelength as the excitation wavelength of the fluorophore. You would certainly need much more power for bleaching than what you use for imaging. But increasing the laser power much beyond the value where the fluorophore saturates is counterproductive. This is because at saturation, fluorophores achieve the maximum possible excitation-emission cycle rate. For photobleaching, most people simply blast the sample with maximum available laser power. This is not ideal. It would be great to perform a fluorophore saturation experiment first. Then set the bleaching laser power to about 10% more than what would saturate the fluorophore. After that play with the bleaching dwell time and total bleach duration for optimized bleaching. (Perform a few pilot bleach tests to optimize the parameters before commencing the actual experiment).
Step 4: Select one or more ROIs for bleaching and analysis
Once the "LSM Stimulation' parameters are set, select one or more ROIs on the image using the ROI tools in the 'LSM Stimulation' tab. Please note that selecting ROIs using the ROI tools on the visualization tab will not help.
I used the circle ROI tool to select a circular ROI. This is shown in Figure 1708/5.
Step 5: Set the Time Series
Set the Time Series from the 'Series' tab as shown in Figure 1708/6.
The number of cycles and interval depend on how fast the fluorescence recovery is. If the recovery is fast, set the system to image as fast as possible ('Freerun'). It would also help to explore other ways of improving imaging speed like:
- Zooming into as small a regions as possible for imaging
- Decreasing imaging dwell time
- Decreasing the number of pixels (sometimes at the cost of resolution)
Please note that the total number of cycles set in the 'Time Series' also includes the pre-bleach cycles.
Step 6: Set the 'Synchronization' tab
Set the pre-bleach and post-bleach imaging cycles in the 'Synchronization' tab. This is shown in Figure 1708/7.
Do the following:
- Base Method: Select 'LSM Imaging'
- Rest in stimulation: Select 'OFF'
- Stimulation Wait: Enter the number of required pre-bleach cycles.
No: Post-Bleach cycles= Time Series Cycles - No: Pre-Bleach cycles
In my case: Time Series Cycles=30, Pre-Bleach Cycles=5, so Post-Bleach cycles=30-5=25.
Step 7: Run the photo-bleaching protocol
If only photo-bleaching is required and FRAP analysis is not required then go to the 'Normal' tab in the 'Aquire'' container and press 'Stimulation Start' as shown in Figure 1708/8.
Photobleaching will be performed on the selected ROIs as per the ROIs selected and settings in the 'LSM Stimulation' tab. A time series will not be run.
If FRAP analysis is required then, go to the 'Sync' tab in the 'Acquire' container and press 'Synchronization Start' as shown in Figure 1708/9.
The system will now run the time series: acquire the pre-bleach images, bleach the ROI/s and then acquire the post-bleach images as per the settings in the 'LSM Stimulation', 'Time Series' and 'Synchronization' tabs. The whole set will be saved as a time series.
Note: Don't forget the mention the target folder for saving the 'Synchronization' files. This location is not mapped to be same as LSM acquisition ('Normal') folder location.
The resultant image after bleaching and recovery is shown in Figure 1708/10.
Note: Since I used a fixed sample there is hardly any recovery (about 25 seconds post bleaching).
Step 8: Load the time lapse data into CellSens software for analysis
Open the acquired time series (with pre-beach and post-bleach) data in the CellSens software. This can either be done by opening the CellSens software and loading the file from the target folder or by right-clicking on the time lapse data on the acquisition software and clicking 'Export to CellSens'.
Once the time series data is opened in CellSens, we need to define two more ROIs in addition to the bleach ROI which is already defined and indicated in the time series data. The two additional ROIs are:
- Background: ROI in a region where there is no signal. This ROI would be used for background correction.
- Photobleaching Correction: ROI in a region which has signal but has not been bleached. This ROI will be used for correcting any bleaching that occurs during the image acquisition process after bleaching.
Note that the bleach ROI is already embedded in the time series data. But if you want to modify that or create a fresh one you can.
Make sure you select the appropriate ROI tools. This is shown in Figure 1708/11.
Once the three ROIs (Bleach, Background, and Photo Bleaching Correction) are defined the time series data will look as shown in Figure 1708/12.
Step 8: Plot the Intensity Profile
Click on the measurement tab on the top panel of CellSens. At the bottom of the tab, you will find tools for FRET and FRAP analysis. This is shown in Figure 1708/13.
Click on the 'Intensity Profile' option. It presents a GUI shown in Figure 1708/14. Select the Time Series file location, Bleach ROI, and Background ROI and then press 'Execute'.
This will display a background corrected intensity profile over time (both pre-bleach and post-bleach). This intensity profile as obatianed in my experiment shown in Figure 1708/15.
Note that you can choose to display the average, integral, maximum or minimum intensity on the intensity profile GUI. For the fixed sample I performed FRAP on, after bleaching while the fluorescence intensity drops very close to zero there is understandably no recovery of fluorescence over time.
Step 9: Perform FRAP analysis
You can now open the FRAP analysis GUI by clicking on the button on the top right corner of the intensity profile (Figure 1708/15) or use the option in the measurement tab (Figure 1708/13).
The FRAP analysis GUI is shown in Figure 1708/16.
As shown in Figure 1708/16 enter the following in the FRAP analysis GUI.
- Time Series file location
- Bleach ROI
- Single or double exponential fit. If the diffusion has only one component, use the 'Single Exponential Fit' and if the diffusion has two components select the 'DoubleExponential Fit'. For more complex diffusion scenario consider using some other fitting algorithm.
- Time series cycles over which the recovery data is fitted.
- Background ROI
- Photobleaching Correction ROI
The data fitting results are shown in the same GUI window. It also shows the raw and fitted data graphs. By pressing 'Execute' you can export the fitted data and results.