Name: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching (Video) | JoVE Journal
Uploaded: 2010-02-26T16:45:00.000+00:00
Duration: 7 min
Description: Learn to measure diffusion coefficients using two-photon fluorescence recovery after photobleaching. This method enables quantitative analysis of molecular dynamics in biological samples through fluorescence recovery curves.

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5 minutes

This work was funded by a Department of Defense Era of Hope Scholar Award (No. W81XWH-05-0396) and a Pew Scholar in the Biomedical Sciences Award to Edward B. Brown III.

1. Align the optics.
The key equipment for an MP-FRAP experiment include: a mode-locked laser source, Pockels Cell (for beam modulation), pulse generator, dichroic, objective lens, fluorescence emission filter, gated photomultiplier tube, and a data recording system (photon counter and multichannel scaler).
2. Determine safe monitor powers.
<ol>
<li>Attenuate the laser to a low, but reasonable, power for generating fluorescence within the sample.</li>
<li>Focus within the sample.</li>
<li>Set a photon counter to integrate over a time scale much longer than the expected fluorescence recovery. With the focal volume held stationary within the sample (on our microscope software this is achieved by a point scan), record a reasonable number of photon count data.</li>
<li>Increase the power and take another series of photon count data. Repeat until the increase in photon counts appears to diminish significantly with respect to the power increase.</li>
<li>Plot log(photon counts) as a function of log(power). A deviation from a slope of two indicates bleaching during the monitor. Choose as your monitor power a power that is below this cut off, but which allows for a reasonable signal to noise ratio.</li>
</ol>
3. Determine input parameters to the pulse generator and multichannel scaler.
<ol>
<li>Determine back-of-the-envelope estimates of the diffusion coefficient and recovery time based on the literature and/or the Stokes-Einstein equation and the diffusion equation.</li>
<li>Set the necessary parameter values on the pulse generator and the scaler.
<ol class="lalpha">
<li>On the pulse generator, set the pre-bleach and bleach times. A good set of rules of thumb is that the pre-bleach should be 1-2 times larger than the expected half recovery time, and the bleach time should be one-tenth the expected half recovery time.</li> 
<li>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 or approximately equal to your expected full recovery plus pre-bleach and bleach times.</li> 
<li>Return to the pulse generator and set the frequency of the pre-bleach/bleach/monitor sequence equal to a value just smaller than the inverse of the product of the bin width times the number of bins per record. *</li> 
<li>Finally, set the number of records/scan on the scaler based on the signal intensity at your chosen monitor power. 
*It is very important that the time for a complete pre-bleach/bleach/monitor sequence (1/frequency) set on the pulse generator is greater than the data collection time (bin width x bins/record) set on the scaler. If this criteria is not met, multiple bleach triggers may appear within a single record on the scaler.</li>
</ol>
</li>
<li>Set the monitor power to an acceptable level, determined previously. Set the bleach power to 200 mW or so above the bleaching cutoff determined during the monitor bleaching test. These powers are both set as different voltages across the PC crystal.</li>
<li>Seal the microscope, shut off the lights, turn on the PMT, begin a point scan on the microscope software, then start the pulse generator and scaler.</li>
<li>Analyze the resulting recovery curve by plotting the curve and marking three important points: 1) the pre-bleach fluorescence, 2) the fluorescence immediately post-bleach, and 3) the fluorescence at the end of the data set. Use the fluorescence values pre- and immediately post-bleach to better estimate the half-recovery time.</li>
<li>With this estimate of the half-recovery time, use the rules of thumb outlined previously to determine new values for the pre-bleach, bleach, and bin width durations. Also, use the pre-bleach fluorescence and fluorescence at the end of the data set to better estimate the time required to achieve full recovery. As a rule of thumb, data should be collected for a time period that allows for the full recovery to be reported for the latter half of the data.</li>
<li>Adjust the parameters on the pulse generator and scaler, take a new curve, and continue tweaking the parameters until your curve exhibits a strong bleach and smooth recovery. Keep in mind that the bleach duration can, and should, be reduced below the rule of thumb, if possible. The bleach power may also be adjusted if too shallow, or too deep, a bleach is achieved.</li>
</ol>
4. Test for excitation saturation.
<ol>
<li>Using appropriate monitor and bleach powers, determined previously, take a series of MP-FRAP curves. Analyze each recovery, normalized to the pre-bleach fluorescence, using the appropriate mathematical form and a non-linear least squares fitting algorithm (see "Data Analysis" below). Record the fitted value of the bleach depth parameter.</li>
<li>Continue taking and analyzing successive series of MP-FRAP curves at increasing values of bleach power.</li>
<li>Plot log(bleach depth parameter) as a function of log(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.</li>
</ol>
5. Continue taking MP-FRAP curves.
<ol>
<li>Continue taking curves, as above, will all the parameters properly set.</li>
</ol>
6. Data Analysis.
<ol>
<li>Remove the pre-bleach and bleach data from the fluorescence data set and adjust the time data such that t = 0 corresponds to the fluorescence value immediately post-bleach.</li>
<li>Normalize the resulting fluorescence recovery with respect to the average pre-bleach fluorescence.</li>
<li>Fit the normalized curve using the appropriate mathematical model and a non-linear, least squares fitting algorithm, such as the lsqcurvefit function in MATLAB. The model presented here accounts for fluorescence recoveries influenced by both diffusion and convective flow: <img src="/files/ftp_upload/1636/1636eq.jpg" alt="Static equilibrium formula, diagram, depicting complex summation for dynamic systems analysis." data-altupdatedat="1788235205000" data-alt="Equation"> where Fo is the pre-bleach average fluorescence, β is the bleach depth parameter, τD is the characteristic diffusive recovery time, R is the ratio of the square of the axial (wz) to radial (wr) widths of the two-photon focal volume, τvx = wr/vx, τvy = wr/vy, τvz = wz/vz, and v2 = vx2 + vy2 + vz2 is the speed of the convective flow.
For flow confined to the imaging plane, τvz → ∞ and 1/ τvx + 1/ τvy can be replaced with 1/ τvr. In the absence of convective flow, both exponentials in the numerator go to 1 and the expression is greatly reduced, to only two fitting parameters, β and τD.
The diffusion coefficient is easily calculated as D = wr2/8τD.</li>
</ol>
7. Representative Results.
<img src="/files/ftp_upload/1636/1636fig1.jpg" alt="Photon count vs Time graph; photon emission analysis; spectral measurement results." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Representative bleach and recovery curve for FITC-BSA. A good recovery curve exhibits a strong bleach and smooth recovery, with the fluorescence at full recovery being reported for the latter half of the data.
<img src="/files/ftp_upload/1636/1636fig2.jpg" alt="Normalized fluorescence vs. time graph showing exponential fit, used in fluorescence lifetime analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Normalized recovery curve for FITC-BSA (red) and the associated least-squares fit (black). This fit yields a diffusion coefficient of 52.9 um2/s, consistent with the literature.

<ol><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+laser+scanning+fluorescence+microscopy%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li>
<li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li>
<li>Sullivan, K. D., Sipprell, W. H. B. rown, Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li>
</ol>

The power of multi-photon fluorescence recovery after photobleaching lies in its ability to probe thick samples with 3D resolution. Since its development in the 1990's, MP-FRAP has been used to determine the diffusion coefficient (or analogous transport parameters) in cell bodies, ex vivo thick tissue slices, and in vivo tissue and interstitium. In this article, we presented the equipment necessary to run an MP-FRAP experiment, as well as the proper procedure for aligning the beam path, setting experime...

<table><tbody><tr><td>Ti:sapphire laser</td><td>Newport Corp.</td><td>Mai Tai</td><td></td></tr><tr><td>Pockels Cell</td><td>Conoptics</td><td>350-80</td><td></td></tr><tr><td>Laser Scanning Microscope</td><td>Olympus Corporation</td><td>Fluoview</td><td></td></tr><tr><td>Short pass dichroic mirror</td><td>Chroma Technology Corp.</td><td>670 DCSX-2P</td><td></td></tr><tr><td>Photomultiplier tube</td><td>Hamamatsu Corp.</td><td>HC125-02</td><td></td></tr><tr><td>Photon counter</td><td>Stanford Research Systems</td><td>SR 400</td><td></td></tr><tr><td>Multi-channel scaler/averager</td><td>Stanford Research Systems</td><td>SR 430</td><td></td></tr><tr><td>Pulse Generator</td><td>Stanford Research Systems</td><td>DG 535</td><td></td></tr><tr><td>FITC-BSA</td><td>Invitrogen</td><td>--</td><td></td></tr></tbody></table>

measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).

In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

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.

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