The current view of the plasma membrane lateral organization remains incomplete. For this reason, it is important to implement some new innovative techniques such as spot variation Fluorescence Correlation Spectroscopy. This is a very powerful technique characterized by excellent time and spatial resolution.
Moreover, it is non-invasive and make it possible to study cell membranes under physiological conditions. Each day before running an experiment, open all of the iris diaphragms and use a power meter to measure the laser power with the first iris fully open. Turn the halfway plate to locate the maximum power.
If the laser power is lower than usual, use the irises to check the alignment and alternately move L1 and M1 as necessary. Then note the power value in a lab notebook. To check the detection path, place water, a cover slip and a droplet of two nanomolar rhodamine 6G solution onto the objective.
Then launch the correlator software and record the data. The auto correlation function should display a low amount of noise, a small waste size and a high count rate per molecule per second. To calibrate the spot size, place a cell culture coded cover slip onto the water immersion objective and add 200 microliters of two nanomolar rhodamine 6G solution to the cover slip.
Adjust the 488 nanometer laser beam power to 300 microwatts and in the software, select the svFCS illumination detection microscope port. Turn on the APD and open the iris until a signal drops to obtain the minimal waste size or close it for a bigger waste size. Record about 10, 20-second auto correlation functions to improve the statistical reproducibility and turn off the APD.
In an appropriate data analysis program, check and discard the runs with strong fluctuations due to molecular aggregates, then fit the average of the retained auto correlation function with a 3d diffusion model, extract the average diffusion time from the fitting parameters and save the data as a TXT file. For svFCS analysis, adjust the 488 nanometer beam powered between two to four microwatts and equilibrate the samples for 10 minutes at 37 degrees Celsius. Select the svFCS illumination detection microscope port, and on the APD.
Next, perform an XY scan and a Z scan of the selected cell. Select the plasma membrane at the top of the cell to locate the confocal spot at the maximal fluorescence intensity and start the data acquisition. In the correlator software, record one series of 20, five second runs.
After the last run, turn off the APD and discard any unexpected runs as demonstrated. Fit the average auto correlation function with a two species 2d diffusion model and save the fitting parameters into the previously saved TXT file. When all of the cells of interest have been recorded, analyze at least four waste sizes varying between 200 to 400 nanometers to establish a single diffusion law.
Then in MATLAB, select a folder containing all the TXT files corresponding to at least four waste size experiments and plot the diffusion time versus the square waste. In this figure, a diffusion law for Thy1-GFP expressed in Cos7 cells can be observed. The diffusion law has a positive time zero value, indicating that the Thy1-GFP is confined within nano domain structures of the plasma membrane.
In this analysis, the cholesterol oxidase treatment of the cells expressing Thy1-GFP resulted in the shift of the diffusion law time zero value to 7.36 plus or minus 1.34 milliseconds. Confirming that the nature of the Thy1-GFP confinement depends on the cholesterol content and is associated with lipid raft nano domains. A small but significant decrease in the total cholesterol content was also observed upon cholesterol oxidase treatment.
As this enzyme acts only on the cholesterol pool accessible at the outer leaflet of the plasma membrane, these data suggests that the observed decrease in cholesterol is associated only with the plasma membrane and causes destabilization of the lipid raft nano domains. The most important thing when attempting this procedure is the proper calibration of svFCS system and the preparation of cells for the analysis. svFCS is an excellent tool to study cell membrane organization.
However, it can also be used to analyze the diffusion of intracellular molecules. This technique can be successfully implemented in biomedicine in order to determine the influence of the potential drugs on living cells such as cancer cells.
