From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

The overall goal of this procedure is to measure the diffusion law of lipids and proteins in live cell membranes to do this. First, the point spread function of the microscope is calibrated by imaging nanoscopic fluorescent beads. Next, liposome based vectors, including a fluorescently tagged molecule of interest, are prepared and introduced into live cells either by transfection or direct incorporation.

Fast imaging of the live cell plasma membrane is performed by total internal reflection fluorescence microscopy. Finally, the correlation function is calculated and the molecular diffusion law is extracted by Gaussian fitting. The resulting data.

Identify the mode of motion of the lipid or protein under investigation. The main advantage of this technique over conventional fluorescence recovery after photo bleaching approach or classical single point fluorescence correlation spectroscopy is that it allows measuring molecular diffusion law. We know a priority knowledge of the system.

Also contrary to classical single particle tracking methodologies. Here, mular displacement are measured with no need to extract particle trajectories. As a consequence, this metal can be easily used in combination with relatively deemed and dense molecule as, for instance, genetically encoded fluorescent proteins.

Overall, this method can help answer key question in the cellular molecular biology field, such as how does protein or lipid motion regulate plasma membrane structure and functional organization answer to these and related question will be made available to everyone and by using just a commercial microscope, demonstration of the overall procedure will be performed by zo, a PhD student from the lab. Begin this protocol by calibrating the point spread function of the imaging system. Begin by diluting 10 microliters of a fluorescent bead solution in 90 microliters of distilled water previously added to a 500 microliters centrifuge tube.

Sonicate the solution for 20 minutes in a sauna cater bath using a scalpel, cut a one centimeter square piece of 3%AGA rose gel and place it in a Petri dish. Deposit 10 microliters of the diluted fluorescent bead solution on the top of the gel. Then using forceps, invert the piece of gel in a two centimeter glass Petri dish.

Again, using forceps. Push down on the piece of gel to disperse the drop. Next turn on the acquisition setup, which consists of a commercial total internal reflection fluorescence microscope.

Equipped with an incubator and a fast E-M-C-C-D camera connected to a computer running li a application suite, advanced fluorescent software and and or Solis image acquisition software. Place the sample in the holder on the microscope stage using the I piece and transmitted light. Focus on the border of the gel.

Then center the gel over the objective and adjust the focus to start the laser alignment procedure in the Leica application suite, advanced fluorescent software select auto align and follow the auto alignment procedure. After the laser has been aligned in the and or Solis acquisition software, click on the settings button. A window will open.

Set the camera exposure to 100 milliseconds, the EM gained to 1000, the repetition to 100, and check the frame transfer checkbox. Then click on the auto save window and insert the name of the experiment. Find a field of view with isolated single spots.

Focus on a bright spot, which usually represents a bead aggregate. Then acquire 100 frames. Repeat this step five to six times to acquire images of several single spots.

Import the acquired series to a data processing program such as MATLAB and average the stack in time. Then display the measured average intensity image. Zoom on a bright, small fluorescent spot and roughly identify the center as the brightest pixel of the spot.

Finally, select the size of the region of interest You want to draw around the particle, typically one micron next to fit the selected intensity distribution with a Gaussian function. Select the command gause fit. Inspect the obtained residuals to verify the goodness of the fit.

Finally, calibrate the camera as described in the accompanying document. Once the system is calibrated, it is time to label the cells for image acquisition. Begin preparing the liposomes required for lipid incorporation by dissolving one milligram of DOPE, one milligram of D-O-T-A-P and one milligram of PPE ADO 4 88 each in a separate micro centrifuge tube containing one milliliter of chloroform.

Next, combine 0.5 milliliters each of the DOPE and D-O-T-A-P solutions with 25 microliters of PPE ATO 4 88 solution. Put the tube in a centrifugal evaporator and initiate the spin at 280 gs. Start the vacuum pump and allow it to spin for 24 hours.

The next day, add 0.5 milliliters of 20 millimolar heis buffer to the tube and vortex it for 15 minutes. Then place the tube in a sonic cater bath and sonicate for 15 minutes at 40 degrees Celsius. To prepare the cells wash a P 100 dish of confluent Chinese hamster ovary or CHOK one cells three times with 10 milliliters of PBS.

Then add one milliliter of trypsin and incubate at 37 degrees Celsius and 5%carbon dioxide for five minutes. After the incubation, add nine milliliters of D-M-E-M-F 12 supplemented with 10%of FBS to suspend the cells seed 150 microliters of this cell containing solution. In a 22 millimeter glass bottom Petri dish containing 800 microliters of the same medium.

Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. For lipid incorporation, replace the cell medium with 500 microliters of serum free medium, and incubate for 30 minutes. After the incubation, add two microliters of liposome solution and incubate for another 15 minutes.

Then wash the cells with PBS and add new DM EMF 12. Before imaging at least 12 hours before the experiment, turn on the microscopes incubator to bring the microscope stage and optical components up to experimental temperature. The most difficult step of this procedure is that acquisition.

In order to ensure success, be sure to optimize every acquisition parameter for the system. Under study. On the day of the experiment, turn on the microscope, wait for the cameras to reach the working temperature.

To perform the experiment, we will use two cameras. Camera one will be used for imaging and camera two will be used to select the cell. This configuration will allow for the fastest achievable acquisition time in the software.

To set the parameters for transmitted light imaging. Enter 20 milliseconds For the exposure time, enter one for the EM gain for both cameras. Then open the contrast mode window in the software and left.

Click on BF to select the bright field option. Put the samples in the holder while viewing the sample through the eye pieces. Bring the sample into focus.

Then manually send the light to camera one and gently push the slits so that only the ROI to be imaged is illuminated. Next, move a cell into the selected region and using the software, send the light to camera two. Then in the software, draw an ROI to generate a reference.

First, to align the laser by software select auto align. Then to start the auto alignment procedure, select enable collimator. Then open the incubator on the top and collimate the laser beam.

When the laser is aligned in the bottom left corner of the software window left, click the 70 button to set the penetration depth to 70 nanometers. This will correspond to an incidence angle of the laser on the bottom glass of about 70 degrees in the Leica application suite. Advanced fluorescent software exposure time text box.

Enter 70 milliseconds in the EM gain text box. Enter 1000 to do the same on camera two. Open the settings window and enter the same numbers in the corresponding text boxes.

Next click on the live button. The fluorescence will be sent to camera one, and an image of the sample will appear on the screen. Move the sample until a fluorescent cell reaches the reference region of interest.

Then push the button on the front of the microscope body to send the light to camera two and move the Z position wheel accurately to focus on the cell membrane in the and or sole software. Open the settings window and set the minimum exposure time achievable 1000 EM gain cropped sensor mode 10 to the fifth repetitions, and set the auto save option to start acquiring the image series. Click on the camera icon of the software and wait for the acquisition to finish.

Then open the settings window, disable the EM gain and remove the check from the cropped mode option. During the acquisition, the temperature of the camera usually increases about two to three degrees. Thus, before acquiring images from a new cell, wait until the optimal working temperature is reached.

Again, once the imaging is completed, follow the instructions in the accompanying document to calculate the mean square displacement from imaging to obtain a fluorescently labeled variant of the molecule of interest. CHOK one cells were either labeled with ADO 4 88 PPE or transfected with a transfer in receptor or T-F-R-G-F-P and imaged by TIRF as described in this video. As can be seen here, the measured diffusion law for ADO 4 88 PPE is flat, indicating a mostly free diffusion as previously shown by stimulated emission depletion fluorescence, correlation spectroscopy or stead FCS measurements following transfection with the T-F-R-G-F-P, the measured diffusion law for T-F-R-G-F-P is initially flat remaining below 100 nanometers of average molecular displacement with an average apparent diffusion coefficient of about 0.7 micron squared per second.

This is followed by a rapid decrease in apparent diffusivity down to 0.2 microns squared per second. The value typically measured by diffraction limited FCS. It is interesting to note that as shown by this last example, the present approach can easily measure average molecular displacement with a resolution of a few tens of nanometers even by using relatively dim and dense labels such as GFP.

Moreover, the spatial scale at which the apparent diffusion coefficient starts to decrease sets the characteristic size of protein partial confinement by the membrane skeleton at around 120 nanometers in keeping with previous estimates. Once mastered the procedure of data analysis and that acquisition can be accomplished in few minutes. We believe that this technique will pave the way for researcher the field of cellular and molecular biology to easily and quantitatively explore the spatiotemporal regulation of protein and lipid diffusion in life samples.