Existing fluorescence recovery after photobleaching, FRAP protocols, are either incomplete or overly complex. The protocol presented in this video is significant in that it is comprehensive, practical, and straightforward. The main advantage of this FRAP technique is that it allows for the quantification of protein mobility within and between subcellular compartments in live cells.
FRAP is being studied for both therapeutic and diagnostic purposes. One such use is to quantify diffusion within drug delivery systems in different tissues in vivo. This information can by used to optimize a drug structure and composition.
The FRAP microscopy technique has provided insights in a broad range of fields in which understanding diffusion phenomenon is of interest. These include cell and molecular biology, developmental biology, neuroscience, material science, advanced biomaterials, and drug discovery. Although the protocol described is for use with mammalian cells, the concepts can be applied to other systems, such as plant cells, yeast cells, and bacteria.
An individual who has never performed this technique may struggle in determining the optimal image acquisition parameters, creating an acquisition region of interest that is suitable for their experiment, and calculating the mobile fraction and halftime of recovery. When attempting this procedure for the first time, be sure to perform several pilot experiments to optimize the protocol in the context of the aims of your experiment. Demonstrating the procedure will be Maleen Cabe, a technician from my lab, and David Rademacher, the core imaging facility manager.
To begin, prepare a plate of YFP-p62 transfected RAW264.7 macrophages, and treat them with Lipopolysaccharide as described in the accompanying text protocol. Next, rinse the treated cells with ice cold Tyrode's buffer one time. Then, add two milliliters of Nocodazole-supplemented Tyrode's buffer to the plate, and allow the plates to incubate at four degrees Celsius for 15 to 20 minutes prior to FRAP imaging and analysis.
Allow the plates to incubate at four degrees Celsius for 15 to 20 minutes prior to FRAP imaging and analysis. Over at the imaging setup, select the 514 nanometer line of the Argon/2 laser, as the fluorescence marker has a peak excitation at 512 nanometers, and a peak emission at 527 nanometers. Next, in the Laser Control window, click Argon/2, 458, 477, 488, 514, and then click the Standby button.
After waiting about three minutes for the laser to warm up, click the On button. Then set the laser power of the laser to 100%and press Enter. Next, set the transmission of the laser line to 5%On the microscope, turn to the Plan-Apochromat 63x magnification 1.40 numerical aperture oil objective.
View the specimen through the microscope eyepiece and place an aggresome-like induced structure in the center of the field of view. Then, in the acquisition software, set the frame size to 512 by 512 pixels, the scan speed to eight, and the data depth to 12 Bit. Also set the scan average to one, the optical zoom to three, and press Enter.
Next, se the pinhole to 1.95 error units and detector gain to 582, which is just below saturation. Create a square-shaped region of interest for the image acquisition, that has the dimensions 150 by 150 pixels. Set up the time series such that the region of interest is scanned 35 times, once every 30 seconds.
Then, set the bleach control such that the photobleaching will occur after scan number five, to collect five prebleach images of the region of interest. Press the Enter key to confirm. Next, go to the Bleach Regions window and create a circular-shaped bleach region of interest within the aggresome-like induced structure area, that has a diameter of 10 pixels.
Now, set the number of iterations to 300, and again press the Enter key. Then, set the laser line to 100%power, and enter 100 in the transmission percent field for the laser, and press Enter. Finally, run the image acquisition program to collect FRAP data from 10 aggresome-like induced structures in 10 cells with less than about three microns of drift.
To begin data analysis, first transfer the AIM lsm files to a personal computer for data analysis. Correct for image drift by aligning or matching the stack of time series images of the acquisition region of interest by opening each AIM lsm file with an image processing program. Then, select Plugins, Registration, StackReg, and Translation, followed by selecting Plugins, Registration, StackReg, and then Rigid Body.
Next, set up the region of interest manager to measure the signal intensity in the bleach region of interest. For this, select the oval tool, then draw a circle in the bleach region of interest with a diameter of 10 pixels, and then click the Add button. Once added, set up the program to measure signal intensity in the control region of interest.
Select the rectangle tool, then draw a square of 20 by 20 pixels in the control region, and click the Add button. Finally, set up the region of interest manager to measure signal intensity in the background region of interest. Select the rectangle tool, draw a square of 20 by 20 pixels in the background region, and then click the Add button.
After adding the regions to be analyzed in ROI Manager, rename them Bleach ROI, Control ROI, and Background ROI, accordingly. Measure signal intensity in each region. To accomplish this, select Bleach ROI, Control ROI, and Background ROI, and then go to More, and select Multi Measure.
Paste the results to a spreadsheet into columns labeled Bleach ROI, Control ROI, and Background ROI, respectively. Start by background correcting the signal intensity in the Bleach ROI and the Control ROI by subtracting the values in the column labeled Background ROI from the values in the column labeled Bleach ROI, and the column labeled Control ROI. Label these new columns Corrected Bleach ROI and Corrected Control ROI.
Next, normalize the signal in the Bleach ROI to the background-corrected signal in the Control ROI. Divide the values in the column labeled Corrected Bleach ROI by the values in the column labeled Corrected Control ROI. Label this new column, Normalized Corrected Bleach ROI.
Then, normalize the signal in the Normalized Corrected Bleach ROI column to the average of the five prebleach values in the Bleach ROI. Divide the values in the column labeled Normalized Corrected Bleach ROI by the average of the five prebleach values in the Bleach ROI. Label this new column, Normalized Corrected Prebleach Average Bleach ROI.
Finally, open a program for image processing. Curve fit the normalized and corrected bleach ROI data by copying the postbleach normalized corrected bleach ROI values and the corresponding time values. Paste them into the Curve Fitter window, select exponential recovery from the Curve Fitter dropdown menu, and select Fit.
Shown here is a typical video of fluorescence recovery after photobleaching for RAW264.7 cells treated with LPS. The fluorescence recovery within the aggresome-like induced structure region of interest, is slow. This experiment uses photobleaching to examine the degree of mobility of p62.
p62 fluorescence in the aggresome-like induced structures, prebleach and postbleach, is shown here. As described in this video, the fluorescence intensity can be tracked over time. Following background correction and normalization, the p62 fluorescence can be calculated to describe the mobile fraction as 22%the immobile fraction as 78%and describes the halftime of recovery within the aggresome-like induced structures, as 2.14 minutes.
Remember, it is especially important to properly define the acquisition region of interest so that it includes an ALIS of interest, a control region of interest, and a background region of interest. Following this procedure, other quantitative fluorescent microscopy techniques may be applied, such as fluorescence loss in photobleaching, FLIP, and selective photobleaching. Whereas FLIP measures the degree of continuity and communication between subcellular compartments, selective photobleaching provides kinetic information about active and passive transport of proteins into organelles, or between protein-rich areas.
FRAP was initially developed to quantify the lateral movement of proteins in cell membranes. Subsequently, it has been used to quantify protein mobility within and between a variety of subcellular compartments, which paved the way for researchers to answer questions in a variety of fields, including cell and molecular biology, developmental biology, neuroscience, material science, advanced biomaterials, and drug discovery.
