Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Lipid rafts are micro domains within the plasma membrane that serve as organizing centers for cellular processes such as signaling and trafficking. Here, lipid raft partitioning at the plasma membrane of living cells is quantified using fluorescence correlations spectroscopy to examine lipid raft partitioning of the ganglioside GM one. Mammalian cells are probed with Alexa 4 88 labeled B subunits of chole toxin.

The fluorescent proteins bind to ganglioside GM one, which is preferentially partitioned into lipid rafts using confocal microscopy. Minute fluorescence fluctuations at the plasma membrane are measured. Auto correlation curves are generated and fitted with appropriate mathematical diffusion models to determine diffusion times of Fluor.

Since probes diffuse much faster outside than inside dense lipid rafts, the results obtained indicate the level of partitioning. The main advantage of this technique of our existing methods, such as detergent resistant membrane isolation, or co patching with antibodies is that FCS enables the determination of the absolute and not the related lipid graft pattern of the target. Moreover, it can be used for both lipids and proteins as long as they're fluorescently tagged.

This method can help answering key question in the Alzheimer's disease field, such as what is the importance of lipid on amyloid beta production? This will lead to important discoveries such as new inhibitors binding specifically to lipid graft components. Begin this procedure by calibrating the fluorescence correlation spectroscopy or FCS system.

This FCS setup is composed of a confocal microscope connected to an external detection unit with a detection filter and an avalanche photo diode, and a time correlated single photon counting system. Start the confocal microscope lasers, confocal imaging computer and FCS acquisition computer. Then turn on the incubator and set it to 37 degrees Celsius and 5%carbon dioxide.

Make sure the single photon avalanche diode or spad is switched on and the fluorescence filter inside the spad is well suited to the sample. Check that the spad is synchronized in time. Once the spad settings are ready for acquisition, start the FCS PICO quant software.

Prepare a fresh solution of cholera toxin Alexa 4 88 diluted in PBS at a final concentration of one microgram per milliliter. Pipette the solution onto a cover slip. Next, adjust the Z settings on the microscope to find the plane in which the fluorescence is the most intense.

To switch to point scanning mode, click on the bleach point button, then click on the image screen to choose a point region of interest in the solution sample. Using the software, set the exposure time to greater than five minutes. This ensures that the laser illumination is long enough to perform the FCS acquisitions.

Then using the FCS software, switch to external detection by the spad. Ensure that the fluorescent signal is in the range of 10, 000 to 50, 000 counts per second. The goal is to obtain a good signal but no saturation.

Acquire 10 sets of 32nd measurements on the same field. The acquisition time should be optimized for the sample so that high quality sampling is achieved without photo bleaching. Once the optimization sample data has been acquired, it is used to generate auto correlation curves from which the diffusion times of fluorescent species will be determined.First.

Using the FCS software, set the correlation interval and generate an auto correlation curve corresponding to the fluorescent species diffusing in solution. For each 32nd measurement, the software will analyze each 32nd sample independently and generate auto correlation curves from the fluorescence intensities using a classical mathematical function that reports the similarity between observations as a function of the time separation between them. The correlation interval will typically range from the shortest resolvable LAC time up to the longest time possible.

As can be seen here, the measurement becomes less accurate as the maximum correlation time approaches The total acquisition time Here, the maximum lag time is limited to 80%of the acquisition time. Next, for each sample, export the files corresponding to fluorescence fluctuations and auto correlation as a function of time. Then import the files into a data analysis and fitting software such as Origin Pro to check that photo bleaching did not occur during acquisition.

Examine the fluorescence fluctuations file. The fluorescence should be stable during the 30 seconds and should be represented as a flat line. When fluorescence measures display photo bleaching.

Do not use the corresponding FCS curves for further analysis as this may cause artificial longer diffusion times with the remaining FCS curves. Determine the mean FCS curve by averaging auto correlation values for each time point. Finally, to fit the curve with the appropriate mathematical model, use the fitting module of Origin Pro with the appropriate equation.

This fit will give the diffusion time of molecules and solution. In this case, there is only one population diffusing freely in diffusion. If diffusion time obtained is correct, approximately 0.2 milliseconds.

The setup is well calibrated and can be used for further experiments. Now that the setup has been checked for calibration, the system can be used to measure the lipid RAFs partitioning of a flora. Four of interest living cells remove HEC 2 93 cells from the incubator.

The cells should have been plated on eight wheel lab Tech coated slides with poly L lysine the day before so that they have time to adhere medium without phenol. Red should be used to ensure there is no perturbation of the fluorescence signal. Use a pipette to gently remove most of the medium from the well, ensuring that the cells are not allowed to dry out.

Then gently add HBSS to the, well. Repeat this wash once to each. Well add 500 microliters of diluted cholera toxin, Alexa 4 88 to the cells for 30 minutes.

At 37 degrees Celsius, cholera toxin will bind to ganglioside GM one known to be preferentially partitioned in lipid rafts following the incubation. Wash the cells twice with HBSS as before the cells are now ready for FCS data acquisition. Place the stained cells on the microscope stage and check the temperature and carbon dioxide settings.

Optimize confocal imaging of a cell of interest using internal detection of the microscope. Find a cell with a well stained membrane. Switch to point scanning mode as before, and choose a point in the plasma membrane of the cell of interest.

Take one to two snapshots of the cell to make sure the cell is not moving during acquisition. Switch to external detection by the spad and to the FCS software as before to ensure the proper signal 10, 000 to 50, 000. Modify the Z position gain and or laser power.

Acquire 10 samples of 32nd measurements as most cells are autofluorescent. There may be a decrease in fluorescence during the first acquisitions due to autofluorescence fading. Analyze the fluorescence fluctuations as before to determine the mean FCS curve.

Then to fit the multiphasic mean FCS curve with the appropriate mathematical model. Use the fitting module of Origin Pro with the appropriate equation. This fit will give the diffusion times and the proportion of molecules diffusing with these diffusion times.

This figure shows an example of an FCS calibration with a cholera toin Alexa 4 88 solution. After checking the individual measures of fluorescence as a function of time did not show any photo bleaching individual and mean FCS curves were calculated. Mean FCS curves were fitted with equations corresponding to various diffusion models.

The parameter classically considered to determine the quality of a fit is the coefficient of determination R squared. The closer R squared is to one, the better the fit in this case, the most accurate model to fit. The mean FCS curve is the one describing a population of fluorescent molecules diffusing freely in three dimensions.

The diffusion time derived from the fit is 0.32 milliseconds. Residuals from curve fitting and R squared factor give an estimate of the quality of the fit here. The r squared value is 0.99906 indicating a good fit.

An example of FCS analysis for cholera toxin Alexa 4 88 stained HC 2 93 cells is shown here. In this example, the toxin is beginning to be endocytose as demonstrated by the presence of small intracellular fluorescent dots to assess diffusion. Time of cholera toin Alexa 4 88 at the plasma membrane.

Data was collected and analyzed as described in this video. The multiphasic mean FCS curve shape reveals the existence of populations of fluorescent molecules with different diffusion times. The best fit for this curve corresponds to a model with three populations of fluorescent probes, two with hindered diffusion.

Diffusion in two dimensions as in the membrane plane, and one freely diffusing in three dimensions. This latter population corresponds to fluorescent molecules moving outside of the membrane plane. In other words, the fluorescent molecules are either binding or unbinding to membrane targets reaching the membrane through the secretion or recycling pathway, or leaving the membrane by endocytosis.

Two diffusion times were seen at the membrane. 25%of molecules had diffusion rates of two milliseconds corresponding to diffusion outside of lipid rafts. 50%of molecules had diffusion times of 75 milliseconds corresponding to diffusion in lipid rafts.

Following this procedure by coupling alternative optical configuration and then single molecule detection can be performed below the defraction limit, which then permit to access a larger range of diffusion times After its development. This technique paved the way for researchers in the field of lipid drafts to study disease related dynamics of lipid draft partitioning after drug edition membrane lipid composition change in cell lines and primary cultures.