FRAP Plugin - Volocity Spinning Disk Confocal

Previously in the lab of Dr Vipoir…

Dr Vipoir got quite excited about spinning disk technology. He got even more excited when he learned about the FRAP Plugin available for the Volocity Spinning Disk Confocal and Volocity version 4.2. We’re checking on how much super strong coffee he’s drinking at the moment but let’s join him as he gets to grips with the tools on offer…

I’m interested in using FRAP, Fluorescence Recovery After Photobleaching, to measure the mobility of intracellular molecules. To do this I can use a property of fluorescent labels that we usually try to minimize, namely photobleaching. Normally we label our molecule of interest with a fluorescent marker then image it under low intensity fluorescence light of a wavelength suitable to cause fluorescence excitation, and use the camera to detect the emission. What we do in a FRAP experiment is use a high intensity light source and deliberately bleach an area of the field. If the labeled molecules around the bleached area are able to diffuse, they will do so, and the measured intensity of emission in the bleached area will rise again. We return to the low intensity light source to watch this.

In Volocity we are starting to use this technique and here’s how we do it. First to acquire some images:

Select Show FRAP Preview from the Window menu.

It looks a lot like the Video Preview but has additional controls and tools just for FRAP type experiments.

1

Laser unit controls. The laser is the high intensity light source that does the bleaching.

2

Live Intensity plot. This live plot shows the mean intensity of ROIs (or the mean intensity of the whole image if no ROIs have been selected). Use this to help to monitor the experiment.

3

The Freeze button. When I find my field of interest I click this to ‘freeze’ the preview. The shutter associated with the current light path closes so that my sample isn’t exposed to light, but the last frame from the camera is kept on screen so I can draw my bleach ROI at my leisure.

The FRAP Preview has all the ROI tools I need for drawing the ROIs in the toolbar that goes with the view.

1. I put my specimen on the ‘scope and image it as usual with the spinning disk, I get it in focus and then produce the best possible image without photobleaching or taking too long. The live intensity plot helps with this.

2. I usually have to do a dry run at some point, to get the right intensity for the bleaching laser. I’m aiming for maximum photobleaching without damaging or excessively heating the specimen. I use another sample or a less interesting part of my current sample to do the test run.

3. Once I’ve got all these light intensities and camera settings optimized, I freeze the preview. My sample sits in the dark and I get on with setting the bleaching area.

4. Any ROI tool draws an ROI or I restore an ROI I created for a previous experiment and saved by selecting ROI then Restore… from the Edit menu. When I’m always using an ROI of fixed dimensions I configure the ROI stamp tool so that drawing the ROI becomes a one click job.

5. Next I need an acquisition protocol to perform the imaging.

6. Select Acquisition Setup… from the FRAP menu.

7. The FRAP Setup dialog opens. How this is configured really depends on the exact experiment you want to do. It’s very like the acquisition setup but designed just for the job of FRAP. Everything I need is in one place.

8. Select the bleaching device that you wish to use for the experiment from the “Bleaching device to use” drop down menu.

Select the light path that you wish to use for the bleaching phase of the experiment from the “Bleaching device light path” drop down menu.

9. Select a shutter management option from the “Manage shutters for” drop down menu.

10. In Acquire pre-bleach images for, specify the duration and the frame rate of the pre-bleach period.

11. In Acquire recovery images for, set the duration and frame rate of the recovery phase.

You may wish to set a very high frame rate for initial recovery period, but slow down the acquisition as your recovery approaches plateau in order to reduce photobleaching of your specimen. This can be achieved by setting a number of recovery phases.

To add an additional recovery phase click the “+” button. Set the duration and acquisition rate of the new phase. In my experiment I’ll be looking at an initial recovery period of ten seconds in which images are acquired at maximum speed, followed by a sixty second phase in which images are acquired every four seconds, which is followed by a final recovery period of sixty seconds, in which images are acquired every 10 seconds.

12. Set the Number of bleach/recovery cycles. This will usually be set to 1 for FRAP experiments. With the number of cycles set to 1 the protocol will finish after completing the recovery phases. You may wish to repeatedly bleach your specimen, for FLIP experiments for example. With the number of cycles set to greater than 1, the bleaching and all of the recovery phases will be repeated. Note:the pre-bleach phase is never repeated.

13. When you are happy with your protocol click OK.

14. Click on the “Capture” video control to apply the acquisition protocol that you have just set up. (A Volocity library must be open to store the image sequence that you acquire; you will be prompted to open one, if necessary.)

When acquisition begins, the ROI changes to a colored “Bleach area”, and the intensity within a “Bleach area” is plotted in a matching color on the live intensity plot. If you have set multiple ROIs on the Preview, which become multiple “Bleaching areas”, each is given its own color and plot when acquisition begins.

15. You can study the intensity of the bleached areas in the live intensity plot over the course of the recovery. When the protocol comes to an end the preview will be automatically frozen so that the final image of the recovery and the intensity plot can be reviewed. The plot and the FRAP preview will be overwritten when the preview is “unfrozen”. The bleaching/recovery data is stored as an image sequence in the Volocity library, a channel is recorded for each of the light paths that you selected for the pre-bleach/recovery phases. Volocity also adds a template channel which records the positions of your bleach areas for analysis.

Carrying Out FRAP Analysis

The next bit of the job takes place using the FRAP analysis tools in Volocity Quantitation.

Since this is a quantitative technique I need to prepare the data before I use the analysis tools, so I’m going to use the background correction and photo-bleaching correction tools that are under the Tools menu. These allow me to correct for some of the sources of intensity changes that are not due to the process of FRAP so that I can be more certain that what I am seeing is actually FRAP.

1. I select the image sequences that I’m going to include in the analysis and select FRAP Analysis… from the Tools menu.

2. The channel I want to perform the FRAP analysis on will be the one with the corrections applied.

3. The bleaching mask channel is the one made from the bleach ROI(s), there’s usually only one in the image sequence so this will already be set.

4. Select the appropriate equation. The math bit.

The Single Exponential equation is appropriate for a single binding state (see Sprague and McNally (2005) for further information on single binding states).

The Double Exponential equation is appropriate for two binding states or more complex transfer barriers. However, be aware that this equation will fit almost all recovery curves and does not necessarily indicate two binding states for your molecule of interest (see Spague and McNally (2005) for further information on two independent binding states and the caution required to use this equation).

The Soumpasis analytical solution (Soumpasis1983) provides a solution for pure diffusion.

5. Specify a name for the measurement item in which to store the result and click Analyze. When analysis is complete your measurement item will automatically open in the Chart view.

The Chart view provides the most accessible view of the results.

The FRAP chart show the normalized recovery for each of your bleached areas (each recovery is plotted in its own color and style), as well as the best fit curve (shown with a line joining each point by default).

Parameters that are displayed either graphically or in the legend are shown in the table below.

For a more detailed view of the normalized intensity values from your recoveries as well as the curve fitted recovery values select the Raw tab. Note the Rel Time column; this represents the time relative to the first frame to be acquired after bleaching, which is set to 0.

So we go straight from the sample to the analyzed data. What could be simpler or more powerful? Work in the lab just goes from strength to strength.

Join me next time to find out what other new discoveries I’ve made.