High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

The overall goal of the following experiment is to visualize single receptors on the surface of living cells and analyze their location, motion, and dynamic association into supramolecular complexes. This is achieved by expressing snap tagged receptors at very low levels on the surface of living cells and labeling them with bright organic fluorophores to permit single molecule detection. As a second step, cells are visualized by total internal reflection fluorescence microscopy, which allows acquiring a fast image sequence of individual receptor particles moving on the cell surface.

Next, automated detection and tracking algorithms are applied to the image sequence in order to obtain the position and intensity of each receptor particle over time. Results are obtained that show a precise analysis of the size of receptor complexes in this example, beta two adrenergic receptors based on a mixed Gaussian fitting of the intensity distribution of the particles at the beginning of the image sequence. This method can help answer key questions in the cell biology field, such as how our receptors organize in od domains of the surface of living cells.

How do they interact with each other to form dimers and oligomers, and how dynamic events at the basis of receptor signaling are actually taking place. The main advantage of this technique over standard biochemical and microscopy methods is that it's investigate the behavior of single receptors in the natural environment, thus allowing us to study important aspects which are typically hidden in ensemble measurements. The cover slips for the samples must be extensively cleaned to minimize background fluorescence During imaging, use clean tweezers to place 24 millimeter diameter.

Glass cover slips into a cover slip holder that separates individual cover slips. Put the holder with cover slips into a beaker and add chloroform until the cover slips are cover. Cover the beaker with aluminum foil to reduce evaporation and sonicate in a bath.

Sonicate for one hour at room temperature. After one hour, take the cover slip holder out of the beaker and let the cover slips dry. Next, place the holder with the cover slips into another beaker and add five molar sodium hydroxide solution until the cover slips are covered.

As before, cover the beaker with foil and sonicate for one hour at room temperature, transfer the cover slip holder into a new beaker and wash three times with distilled water. Lastly, put cleaned cover slips in a glass cell culture dish filled with 100%ethanol. Shown here are cover slips before and after cleaning, imaged by total internal reflection fluorescence or turf microscopy.

Cal calibration samples are used to estimate the intensity of single fluorescent molecules during turf microscopy. To prepare the samples, dissolve the fluorescent dye in the appropriate solvent. Prepare a one to 10 serial dilution of the fluorescent dye ranging from one picaMolar to one ano molar.

In filter sterilized water. Wash the previously cleaned cover slips by transferring them to a cell culture dish filled with filter sterilized water. After that place each cover slip in a well of a six well cell culture plate and wait until they dry spot 20 microliters of each fluorescent dye dilution on a separate cleaned cover slip.

Let the cover slips dry under a sterile hood. Protect the cover slips from light and dust until use prior to transfection. Prepare a six well cell culture plate Wash the cleaned cover slips with sterile PBS and place one cover slip into each well of the six well cell culture plate.

The CHO cells to be transfected for this study are cultured at 37 degrees Celsius in 5%CO2 in one, one-to-one ECCOs modified eagle medium nutrient mixture F 12 supplemented with 10%fetal bovine serum penicillin and streptomycin after trypsin and counting the cells following standard methods seed at a density of three times 10 to the fifth cells per well. In the sixth well cell culture plate containing the cover slips, let the cells grow in an incubator for 24 hours in order to achieve approximately 80%co fluency, which is the optimal cell density. For transfection.

The most difficult aspect of this protocol is to achieve extremely low expression level of membrane receptors on the cell surface to ensure success. The transfection condition. For example, the amount of plus media NA and time after transfection must be optimized On the day of transfection.

Prepare the transfection reagents for each will dilute two micrograms of the desired plaid DNA in 500 microliters of optimum medium in another tube. For each well dilute six microliters of Lipectomy 2000 in 500 microliters of Optum medium. Incubate both tubes at room temperature for five minutes.

After five minutes, combine both solutions into one tube and mix to obtain a transfection mixture. Incubate the transfection mixture at room temperature for 20 minutes. In the meantime, prepare the CHO cells.

Wash the cells twice with pre-warned PBS After the second wash, replace PBS with one milliliter per well of phenol red free D-M-E-M-F 12 medium supplemented with 10%FBS, but without antibiotics. Add one milliliter of the transfection mixture, dropwise to each well and gently rock the plate back and forth to ensure complete mixing. Incubate at 37 degrees Celsius in 5%CO2 for two to four hours before proceeding immediately to the protein labeling.

To begin this procedure, dilute one microliter of the fluoro four conjugated benzo guine derivative or fluoro four BG stock solution In one milliliter of D-M-E-M-F 12 medium supplemented with 10%FBS to obtain a final concentration of one micromolar. Retrieve the transfected cells from the incubator and wash twice with prewarm PBS. Replace PBS with one milliliter of one micromolar fluoro four BG solution and incubate at 37 degrees Celsius in 5%CO2 for 20 minutes.

Next, wash the cells three times with D-M-E-M-F 12 medium supplemented with 10%FBS each time, followed by a five minute incubation at 37 degrees Celsius. Used tweezers to transfer cover slips with labeled cells to an imaging chamber. Wash twice with 300 microliters of imaging buffer.

Add 300 microliters of fresh imaging buffer and proceed immediately to imaging images are acquired. Using a turf microscope equipped with an oil immersion, high numerical aperture objective, suitable lasers, an electron multiplying charge coupled device, camera, and incubator, and a temperature control. Set the desired microscope parameters that is the laser line turf angle exposure time frame rate, and number of images per movie.

Put a drop of immersion oil on the 100 x objective of the microscope. Place the imaging chamber with the labeled cells onto the specimen holder of the microscope and bring the cells into focus. Using brightfield illumination.

Switch to turf illumination. Keep laser power as low as possible to allow searching for the desired cell, but at the same time minimizing photo bleaching. Select the desired cell and fine.

Adjust the focus. Increase the laser power to a level that allows visualization of single flora fours. Acquire an image sequence and save the raw image sequence as a tiff file for calibration.

Assemble each calibration sample in the imaging chamber and place on the microscope. Choose a sample containing well separated diffraction. Limited spots that bleach in a single step.

Turf image sequences are subsequently acquired in the same way as demonstrated for the labeled cells. To prepare an image sequence, use an image processing software such as Image J to crop the images. Save individual frames as separate TIFF images in a new folder indicating the frame number on each image.

Particle detection and tracking is performed with a non-commercial software such as U Track in a MATLAB environment. From the MATLAB command prompt type movie selector, GUI. To open the movie selection interface, follow the instructions to create a new movie database starting from the previously saved separate images.

Provide the pixel size in nanometers time interval in seconds, numerical aperture camera bit depth and emission wavelength of the fluorophore required for particle detection and tracking. Save the movie database from the movie selection interface. Run the analysis choosing single particles as type of object.

Run the detection algorithm, then run the tracking algorithm. Use the movie viewer routine contained in the URAC package to visualize the tracks and check the quality of the detection and tracking. Open the dot MAT file to see the position and amplitude of the tracked particles at each frame.

The size of particles can also be calculated from the data acquired. Shown here is the first frame of a typical turf image sequence of a cell transfected with snap tagged beta two adrenergic receptors and labeled with a fluoro four conjugated benzyl guine derivative. The spots represent single receptors or receptor complexes.

A detection algorithm is applied to the same image sequence and each detected particle is indicated by a blue circle application of a tracking algorithm. To the same image sequence results in trajectories of the individual particles indicated in blue. Green segments represent merging events and red segments represent splitting events.

This method also captures dynamic events. In this example, two particles undergo a transient interaction, moved together for several frames and split again. The trajectories of individual particles are used to calculate their diffusion coefficients.

This representative result shows that the beta two adrenergic receptor has high mobility. While the gaba B receptor has limited mobility, the size of receptor complexes can be precisely estimated by fitting the distributions of particle intensities with a mixed Gaussian model. This analysis can also reveal the coexistence of monomers and dimers.

Shown here are examples of a monomeric receptor particle bleaching in one step and a diametric receptor particle bleaching in two steps. These procedures can be extended and adapted to work with other cell surface proteins, leveling methods and cell models. After watching this video, you should have a good understanding of how to produce clean cover sleep, how to obtain low expression levels and efficient labeling with bright organic fluoros, as well as how to acquire and analyze turf images, which represent all key steps for successful single molecule.T.