In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The overall goal of the following experiment is to determine stoia metric and structural information about protein. All ligament complexes expressing living biological cells, yeast cells are genetically engineered to express the proteins of interest attached to either a donor or an acceptor fluorescent probe. Donor probes can be excited directly by a laser.

While acceptors can only be excited through a transfer of energy from a nearby excited donor, the cells are exposed to light from a pulsed near infrared laser. The fluorescent inseminating from the probes is separated into its spectral components by a transmission grating and projected onto the surface of an electron multiplying CCD array. Therefore, complete fluorescent spectra from individual, all ligaments are obtained for each position in the sample.

The spectral profile of the detected fluorescence depends on the specific interaction of the proteins of interest. The data are analyzed to determine for each image pixel of a scan cell, the separate emissions from the donor and acceptor probes. The efficiency of energy transfer is determined at each image pixel, and then pixels are bend according to their fret efficiency and histogram.

The histogram interpretation allows for the in vivo determination of protein, complex size and configuration. Hi, my name is Michael Stoneman from the Riku Research Group at the University of Wisconsin Milwaukee. My name is Singh.

I am from Rikus Lab in Wisconsin, Milwaukee. The main advantage of this technique is that the complete emission profiles from hundreds of all ligament complexes within a a cell are obtained with one complete scan of the cell. Typically, most fluorescence microscopy methods require the cell of interest to be scanned multiple times in order to accumulate enough spectral information in order to determine the fret efficiency at various regions of interest within the cell.

However, diffusion biochemical reactions may change the molecular makeup of these regions of interest and thereby limit the information obtained in the scan of the image. This method can help answer key questions in the study of protein, protein interaction in living cells, such as what is the size of oligomers harm? What stage of the protein lifecycle do the oligomers farm, and what is the role of oligomer of this protein in cellular function?

The spectrally resolved two photo microscope, accumulate spectral information about the olima complexes in the image cell by scanning the focus of the excitation beam across the sample of interest in a number of locations. A pair of orthogonal scanning mirrors is used to translate the focus of the laser beam across the sample in two different directions. The movement of the scanning mirrors is computer controlled and synchronized with the extraction of fluorescence intensity data from the CCD camera from the line scans fluorescence intensity spatial maps corresponding to a particular wavelength of light can be reconstructed.

In order to accurately reconstruct x-ray fluorescent intensity spatial maps and calculate the actual wavelength corresponding to each of these maps, the line scanning protocol must be calibrated using fluorescein solution. To begin the calibration procedure, send to the excitation spectrum at a wavelength of 800 nanometers, which is two times the maximum excitation wavelength of the donor tag GFP two two to send to the excitation spectrum, translate the prism located within the laser cavity to modify the group velocity dispersion using the computer program to which the camera is interfaced. Send a command to the CCD chip to lower the temperature of the CCD chip to its lowest attainable temperature.

In order to reduce dark noise. Pipette 10 microliters of fluorescein solution onto a microscope slide cover with a cover slip so that a thin layer of the sample is uniformly dispersed in the region between the cover slip and the microscope slide, place a small drop of immersion oil on the surface of the cover slip. Now fasten the microscope, slide to the XY, Z translation stage and translate the slide and stage in the optical axis direction By manually adjusting the linear actuator, translate the slide until the microscope objective comes into contact with the drop of immersion oil, switch the camera to a video mode of data acquisition so the admitted light striking the CCD array is displayed on the computer screen in real time.

Turn off all ambient room lights in order to decrease the background noise detected at the CCD array. Slowly adjust the micrometer controlling the translation stage in the optical axis direction to bring the sample into the focal spot of the laser beam. When the fluorescein sample is in focus, the emission will appear as a sharp line on the CCD array.

Download the readout of the pixel intensities from the CCD array to the computer. Measure the pixel intensity as a function of position on the CCD array by opening the downloaded matrix of intensity values in image J and drawing a line through the fluorescent region. Use the image day command.

Analyze plot profile to create a plot illustrating the emission spectrum of the fluorescein sample. Adjust the incremental parameter of the mirror, which controls the movement of the laser. Focus in the Y direction, such that adjacent y positions within the sample result in the movement of the peak of the fluorescent spectra by exactly one pixel along the spectral dimension on the CCD array.

To monitor this, download the fluorescein spectra intensity for two different Y positions in the sample. Then open the intensity images with image J and find the peak pixel position for each of the fluorescent spectra. Using the image J cursor, leaving the shutter of the CCD open scan the laser focus across the sample in the X direction.

The light emitted from each voxel along the line of the scan should fall incident upon the CCD array at the end of each line scan store the data from the CCD array obtained for this line scan and then clear the pixels of the CCD array. Move the position of the laser. Focus in the Y direction by the amount determined earlier.

Then scan the laser beam in the X direction across the sample. Once again, leaving open the shutter of the CCD store the data. Repeat this line scanning procedure until a physical area around 50%larger than the dimensions of a single biological cell has been illuminated by laser light.

The relationship between row number of a particular line scan image and wavelength should be used to reconstruct the images to obtain multiple ex-wife fluorescent intensities facial maps at different wavelengths. To obtain the ex-wife fluorescent submission image for a particular wavelength, find the row number on the image obtained from the first line scan that corresponds to this wavelength. Then the adjacent row of the subsequent line scan image corresponds to the next row of the fluorescent emission image.

For that particular wavelength stack all the image rows that correspond to this wavelength to obtain the XY fluorescent intensity spatial maps of the sample at that wavelength. Repeat this procedure for all other obtainable wavelengths to collect data on your samples. First, remove from the incubator the plates with transformed yeast colonies.

There should be at least three types of transformed cells. Cells expressing the proteins of interest tagged with both types of fluorescent probes. Cells expressing only proteins tagged with the donor fluorescent probes and cells expressing only proteins tagged with the acceptor fluorescent probes.

Add 100 microliters of 100 millimolar potassium chloride to a micro centrifuge tube. Then using a micro pipette tip, scrape three to five yeast colonies off of the plate of cells expressing proteins tagged with both donor and except a fluorescent probes and a inoculate the 100 millimolar potassium chloride. With these cells, remove 10 microliters of the cell suspension and dispense it on a fresh microscope.Slide.

Cover the droplet with a cover slip and place a droplet of oil on the surface of the cover slip. Next, manually close the shutter in the path of the laser beam to block the laser light from reaching the microscope objective. Fasten the microscope slide to the XY, Z translation stage and translate the slide stage in the optical axis direction until the microscope objective comes into contact with the drop of immersion oil.

Now turn on the wide field light illumination and go to the camera. Video mode of data collection. The wide field image of the sample will be severely blurred because of the presence of the transmission grating in the emission path.

Therefore, place a band pass filter with a small full width half maximum in the optical path preceding the transmission grating. Slowly translate the translation stage in the optical axis direction while viewing the image of the cells on the camera screen until the cells are brought into focus. Translate the stage in either the X or the Y direction in order to bring a single cell to the location of the laser beam focus.

Turn off the widefield illumination source and remove the band path filter from the emission path. Unblock the laser beam for a short time while simultaneously viewing the signal received at the CCD array. If a fluorescent signal is detected on the CCD array during the time the laser beam is incident upon the cell, then perform a full fluorescent data acquisition scan of this cell.

It is vital to use the same scanning parameters, specifically number of lines and why increment as determined in the calibration procedure demonstrated earlier. Repeat the cell location and fluorescence data acquisition process for a large number of cells expressing protein attached to both donor tags and acceptor tags. After accumulating enough fluorescence images of cells expressing proteins tagged with both receptors repeat the entire process for both cells expressing only proteins tagged with the donor fluorescent probes and cells expressing only proteins tagged with the acceptor fluorescent probes.

Finally, reconstruct all scans to obtain spectrally resolved. XY fluorescent intensity spatial maps for each set of data using the procedure described previously illustrated in these three figures are the resultant spatial maps computed from spectrally resolved two photon microscopy measurements performed on a yeast cell expressing the sterile to alpha factor receptor. Two dimensional maps of donor and acceptor signals were obtained from analyzing the spectral emission profiles obtained from each location of the cell.

Fluorescence intensities are assigned false colors according to their values, and the scale is shown as an inset. A two dimensional map of the apparent fret efficiencies was then computed using the KDA and KAD images using the values extracted from the apparent fret efficiencies map of the cell just shown a histogram plot was prepared. The measured EAPP values of the cell are represented by circles.

The red solid line represents the best fit to the measured data using the sum of the individual calcium functions represented by solid green lines. Analysis of the histogram, which is described in the protocol text confirms that the sterile two alpha factor receptor all ligament complex assumes the form of a RBA shaped tetramer in vivo. Once mastered, this technique can be performed in 12 hours if performed properly.

While doing this experiment, it is important to keep the power of excitation source low. It is important to avoid multiple excitation of donor by a single pulse.