A. Cell Preparation Protocol
1. Generate cell lines stably expressing membrane receptors of interest and cells expressing monomeric or dimeric forms of the fluorescent marker protein; see previously described protocol12,13
2. Maintain a healthy cell culture on T-25 flask. If cells are being revived from deep freeze, allow for at least two cell passages. Subculture cells if they are more than 85% confluent on flask. Subculturing of cells should be performed in a sterile environment using standard cell culture procedures
3. Lift cells from T-25 flask and plate on poly-d-lysine coated chambered cover glass. Seed between 500-1000∙ cells/mm2 of coverslip growth area onto poly-d-lysine coated chambered #1.5 cover glass. The amount of cells seeded will ultimately depend on the doubling time of the cell line being studied. Allow the cells to grow for 36-48 hours in humidified incubator in order to spread out on the glass surface.
4. Prepare cells for imaging
i. Aspirate cell growth medium
ii. Wash cells twice with PBS
iii. If exposing cells to ligand, add ligand solution to cells for desired time. If no ligand is being used, simply add PBS to cells for same amount time.
iv. Remove ligand solution or PBS solution and add 1.5 mL of 4% formaldehyde fixing solution. Leave cells in fixing solution for 20 minutes. Leave room lights off for duration of time
v. Remove fixing solution
vi. Resuspend cells in PBS; cells now ready for imaging.
B. Image Acquisition Protocol
1. Set the pixel dwell time for the measuring system to be at least a factor of five times shorter than the two-dimensional characteristic diffusion time, τD, of the receptor under study. The characteristic diffusion time is the average time a receptor molecule will reside within the measurement volume before diffusing out and is related to the laser beam waist, ω, and the diffusion coefficient, D, through the following relation τD=ω2/4D. The laser beam waist is defined as the distance from the center of the focused beam to where the intensity of the beam drops to e-2 its maximum value.
2. Obtain fluorescence images from several hundred cells for each type of experimental condition.
i. Search for cell/cells using scanning parameters with a reduced power or much shorter exposure time (preview scan) compared to the settings used for the actual data collection (fluorescence scan) so as not to expose cells to too much laser light.
ii. Focus on the basolateral membrane of a cell or group of cells using the preview scan.
iii. Take fluorescence scan of the focused cell/cells.
iv. Repeat steps i-iii until fluorescence images have been acquired from several hundred cells
3. Obtain fluorescence images from cells separately expressing monomeric and tandem dimer forms of the fluorescent marker which has been fused to receptor of interest. Apply the exact same imaging conditions which were used to collect images of cells expressing fluorescently labeled receptor of interest.
4. Obtain fluorescence images of sub-diffraction sized fluorescent microspheres for determining the laser beam waist size.
5. Obtain measurements from a light source with constant intensity (i.e. no temporal fluctuations). Signal from a constant light source can be obtained from a number of different methods:
i. Laser spot scanning the surface of a mirror slide in the plane of focus of the microscope. Remove filters used to eliminate laser light from striking the detector from the detection pathway. Acquire scans for a range of laser powers.
ii. Transmitted light illuminator turned on during acquisition. The same integration time settings can be used as was applied to the fluorescence scans. Acquire scans with a range of transmitted light intensity levels
C. Analysis Protocol
1. Determine average background intensity of fluorescence images. All fluorescence images must be corrected for background prior to computing brightness values. Contributions to intensity levels due to background include the electronic offset (i.e. bias level) added to the output signal of the detector as well as dark noise. Determine this contribution by measuring the mean intensity in multiple ~10,000 pixel subregions of the acquisitions where there are no cells/fluorophores present and averaging the results from these multiple subregions.
2. Determine slope and intercept for a plot of variance vs intensity for constant light source measurements.
i. Fluorescence intensity traces must be corrected for fluctuations in intensity arising due to the detector5,7.
ii. Measure both the variance as well as mean intensity from small subregions of the scans taken of the constant light source.
iii. Construct a scatter plot of the variance vs average intensity from all of the subregions measured. The relationship between the detector variance and intensity is linear and therefore the scatter plot can be fit with a straight line of slope S, and intercept, σ2.
3. Use FIF Spectrometry Suite to demarcate regions of interest (ROI) on fluorescence images of cells separately expressing monomeric and tandem dimer forms of the fluorescent marker
i. Load 2D fluorescence images for a given sample
ii. Select ROIs using a polygon tool. Draw the polygon such that it encompasses the majority of the region inside the cell boundary. Multiple ROIs can be selected for each loaded image.
iii. Segment each ROI into smaller segments. The segmentation procedure is fully automated, the only user input is the maximum size of each segment. We have used a maximum segment size of 500 pixels.
4. Use FIF Spectrometry Suite to calculate the effective brightness for each segment generated in the images of cells expressing monomeric and dimeric fluorescent protein constructs. Make sure to input the values for average background intensity, S, and σ2. determined in steps C1 and C2 above.
i. Determine the monomeric effective brightness value, εproto, from the distribution of monomer and dimer brightness values calculated in C4
ii. Simultaneously fit the two spectrograms with Gaussian functions
iii. Set the Gaussian peak position simulating the monomer distribution to be εproto and the Gaussian peak position simulating the dimer peak position to be 2εproto . Use the same standard deviation for both Gaussian functions, but allow it to vary as a fitting parameter. Use the best fit value of εproto as the monomeric effective brightness value.
5. Repeat step C3 for images of cells expressing the receptor of interest under appropriate experimental conditions.
i. Use FIF Spectrometry Suite to extract brightness and concentration from each segment generated in C6
ii. Calculate the brightness and concentration of each segment using the mean and standard deviation obtained from the corresponding intensity histograms. The entire procedure of fitting starting at intensity histogram calculation and ending in the calculation of effective brightness and concentration of each of the segments is performed after a click of a single button. Be certain to input the values for εproto, average background intensity, S, and σ2 determined in steps C1, C2, and C5 above.
iii. Create a two-dimensional surface plot of the frequency of occurrence of each effective brightness-concentration pair for visual analysis of the effective brightness distribution
iv. Generate brightness spectrograms for various concentration ranges. The brightness spectrogram is a partitioning of the surface plot into one dimensional brightness spectrograms for a chosen concentration range.
6. Use FIF Spectrometry Suite to perform meta-analysis of brightness spectrograms for various concentration ranges and obtain oligomer species fraction plots as a function of protomer concentration
i. Fit the individual brightness spectrogram for a single concentration range with an array of Gaussian functions, S(εeff )=∑Anexp^[-(εeff-nεproto )2/(2σ2 )] The mean value of each Gaussian function, nεproto, corresponds to the peak brightness value from a particular oligomer size n, and are all linearly related to a multiple of the monomeric molecular brightness, εproto
ii. Calculate the area underneath each Gaussian (relative to the area underneath the entire histogram). This area reflects the relative abundance of the particular oligomer size corresponding to that Gaussian.
iii. Repeat fitting of spectrogram for all concentration ranges and obtain relative abundance values for each oligomer size