The multi photon fluorescence. Recovery after photobleaching procedure is easily demonstrated with a freely diffusing sample of fluorescent dye molecules. First, a low intensity laser beam is sent into the sample to generate a reference level of fluorescence from the D molecules within the focal volume of the laser.
Next, a brief high intensity laser flash is sent in photobleaching a portion of the dye molecules within the focal volume. Finally, the laser beam is attenuated to the previous low intensity level and the fluorescence is monitored as photo bleached molecules diffuse outward and are replaced by still fluorescent molecules from outside the focal volume. The resulting fluorescence as a function of time curve is analyzed to yield the diffusion coefficient.
Hi, I'm Kelly Sullivan from the Laboratory of Edward Brown in the Department of Biomedical Engineering at the University of Rochester. Today we'll be showing you how to perform multi photon fluorescence recovery after photobleaching. We use this technique in our laboratory to study diffusion in both in vitro and in vivo biological systems.
So let's get started. To begin set the desired laser wavelength and insert the appropriate filter in front of the photo multiplier tube or PMT. Next to avoid damage to the pcal cell, adjust the laser power to 200 milliwatts or less.
There are two mirrors oriented in a figure four upstream of the pcal cell. These mirrors are necessary and sufficient to align the laser beam through the pcal cell. Set the voltage offset on the pcal cell bias controller to zero, adjust the laser path through the pcal cell such that the laser is centered in the front and back windows.
Use the mirror upstream in the figure four to center the beam in the front window and the mirror downstream in the figure four to center the beam in the back window. Now set the voltage across the pcal cell crystal to the maximum value. The appropriate value is wavelengths dependent and can be found in the pcal cell instruction manual.
Adjust the mirrors in the figure four upstream of the pcal cell until the output power from the pcal cell is maximized. Now switch the voltage across the crystal to zero and adjust the voltage offset on the bias controller to minimize the output power from the pcal cell. Return the laser power incident on the pcal cell to the value required for the experiment.
Now moving over to the microscope, get a good visual focus on a standardized fluorescent sample. Here we are using fixed fluorescent pollen grains. Once focused, seal up the microscope, adjust the laser power to an appropriate level for imaging the sample by changing the voltage across the pcal cell crystal.
Finally, turn off the lights and flip on the PMT. Start a repeat image scan of the standardized sample and adjust the mirrors downstream of the pcal cell until a sharp image of the sample is obtained. Now let's look at determining safe monitor powers folk focus within the sample.
For the purpose of this video, we will use a sample of aqueous dye held within a depression in a glass slide and capped with a cover slip. Attenuate the laser to a low but reasonable power for generating fluorescence within the sample. Seal up the microscope, divert the output from the PMT to the photon counter.
Turn off the lights flip on the PMT and begin a point Scan on the microscope software. Set the photon counter to integrate over a timescale much longer than the expected fluorescence recovery time and record a reasonable number of data points. Increase the power and take another series of photon count data.
Continue increasing the power until the increase in photon counts appears to diminish significantly. With respect to the power increase plot, the log of photon counts as a function of the log of power. Any deviation from a slope of two indicates bleaching.
During the monitor, select a reasonable monitor power that is below this cutoff. Now let's determine the input parameters to the multichannel scaler and the pulse generator. On the pulse generator.
Set the pre-teach time to be one to two times larger than the expected half recovery time and the bleach time to be one 10th the expected half recovery time on the scaler. Set the bin width to approximately one half the bleach duration and set the number of bins per record, such that the product of your selected bin width and bins per record is greater than your expected full recovery time. Plus the preble and bleach times return to the pulse generator and set the frequency of the preble and bleach sequence equal to a value just smaller than the inverse of the product of the bin width times the number of bins per record.
Finally, set the number of records per scan on the scaler based on the signal intensity at your chosen monitor power. Now set the monitor power to a safe level and set the bleach power to around 200 milliwatts above the bleaching cutoff determines during the monitor bleaching test. These powers are both set as different voltages across the al cell crystal seal, the microscope shut off the lights.
Turn on the PMT. Begin a point scan on the imaging software. Then start the pulse generator and scaler.
To analyze the resulting recovery curve, mark three important points. The preble fluorescence, the fluorescence immediately post bleach and the fluorescence at the end of the dataset. Use the fluorescence values pre and immediately post bleach to better estimate the half recovery time.
Now, use the rules outlined in earlier steps to update the values for the preble bleach and begin width durations. Take a new curve and continue tweaking parameters until the resulting curve exhibits a strong bleach and smooth recovery. Check for excitation saturation by plotting the log of the bleach depth parameter as a function of the log of power.
Any deviation from a slope of two indicates excitation saturation. Choose a bleach power that yields a strong bleach but does not cause saturation. Finally, with the appropriate parameters determined, continue taking multi photon fluorescence recovery after photobleaching or MP FP curves of your sample of interest.
Now we'll show representative results of both raw MP frap data at a normalized MP F wrapp curve that has been fitted teel, the diffusion coefficient, the raw MP F frap data exhibits, a strong bleach and smooth recovery. With the full recovery being reported for the latter half of the data to fit the data. We first normalize the recovery to the average preble fluorescence and then perform a non-linear leased squares fit.
With respect to the appropriate model. Both datasets were taken in aqueous fluorescein and yield diffusion coefficients. Consistent with the literature We have just shown you how to perform a multi photon fluorescence recovery after photobleaching experiment.
When performing this procedure, it's important to take care and setting your experimental parameters, in particular, the bleach duration and the bleaching and monitoring powers to the safe limits to defined in the protocol. So thanks for watching and good luck with your experiments.
