Dr. Vipoir demonstrates ratiometric acquisition

I was chatting to one of my old PhD students last week who has recently set up his own lab and is studying the dynamics of cytosolic calcium within cells. He is using ratiometric imaging, which takes advantage of physiological fluophores which show different fluorescent behavior when bound to their target molecules. He is currently performing his ratio imaging experiments in 2D however I told him that with Volocity, he could perform his ratio imaging in 3D, which would provide him with more biologically relevant information. He was very interested and asked me for a demonstration of how I go about setting up a ratiometric experiment using Volocity. This is what I told him…

Volocity Ratiometric Acquisition is designed specifically for online ratiometric imaging and allows you to easily create acquisition protocols that can capture ratio image pairs at high speed and in Z.

1. Select Ratiometric Preview from the library view or choose Show Ratiometric Preview from the window menu. This live preview window is shown below:

Volocity Ratiometric acquisition

The Ratiometric preview mode selection buttons allow live previews of the 2 captured channels (channels A and B), the ratio A/B channel or the Intensity Modulated Display (IMD) channel to be displayed either sequentially or simultaneously in a 4 panel preview. At the bottom of the screen there is a live ratio plot which can show the mean intensity values of ROIs (or the mean intensity of the whole image if no ROIs are present) of channel A, channel B and the ratio plot A/B if these respective channels are being previewed.

Volocity Ration Imaging

The live ratio plot is very useful as it allows you to follow the course of your experiment in real time and monitor events to make sure that your protocol is working correctly. The live plot will rescale continuously to show the full time course of the experiment.

The Ratiometric Acquisition controls are displayed on the right-hand side of the ratiometric preview window in addition to your standard video preview device controls. The Ratiometric Acquisition setup pane contains dropdown menus for the selection of the correct light paths to be used for channel A and B.

Volocity Ratiometric Acquisition Controls

2. The next step is to configure the light paths that you require for ratiometric acquisition. You will need one light path for channel A and another for channel B. Remember that the ratio is always calculated as channel A/channel B; therefore it is important to make sure that you have specified the channels correctly in the ratiometric dialog.

3. You should also configure any additional light paths that you will need to image during the experiment.

4. A constant background value may be subtracted from each voxel in every image of channel A and/or channel B. This can be done by either entering a value in the subtract field or by drawing an ROI in the background of the live preview image of either channel and clicking the ‘Get from ROI’ icon tool next to the subtract field (shown above). Volocity will use the mean intensity from that ROI for each channel as the value to subtract.

5. Thresholding allows the removal of unwanted signals, setting a ratio value to 0 when one of the source voxels has an intensity value at or below the threshold. Threshold values can be set for channel A and /or channel B by entering a value in the Threshold field. This value can be determined by using the Voxel Spy tool.

6. The Ratio value range can be set by entering expected or known values for the experiment in the Rmin and Rmax fields. This sets the range of ratio values over which the rainbow look up table will be spread.

These settings will be used for this experiment, however data acquired using the ratio acquisition may also be reprocessed using the ratio dialog in Volocity Quantitation. I find that after performing several experiments I can determine values for Rmin and Rmax which I can then use for all experiments to allow them to be visually compared without the need for post processing.

7. You can now create your ratio acquisition protocol. Select Acquisition Setup… from the Ratio menu and the ratio setup dialog opens.

Ratio Acquisition Protocol

Tick the appropriate channel check boxes to acquire the ratio channel and IMD channel. You cannot collect an IMD channel without capturing the ratiometric channel.

Ratiometric record buttonWhen you have configured the ratio acquisition protocol and have checked it, click OK. You can now click the record button in the ratio preview to apply your ratio acquisition protocol.

After Ratio acquisition, a measurement item is generated which automatically charts the intensity values from the ROIs selected as well as providing the raw data for further analysis. I can then reanalyze my image data as required using the measurement features in Volocity Quantitation. In addition, the charts and data can be exported and used in other packages and, of course, in publications.

Well, as you can see for yourself, this has all been very intuitive and my colleague was able to set up his own protocol in a very short time. He was excited about being able to view his ratio images in 3D and he went away very impressed!