Acquiring reference images

Do you remember the days when, if you wanted to acquire a reference image alongside your 3D sequences, you had to capture images at every z-plane within your reference channel? Well, with Volocity 5.3 those days are gone! Now you can acquire a channel with only one z-plane to act as a reference channel in your final dataset, saving you time during your acquisition. Volocity can support a mixture of 2D and 3D data in a sequence where reference images are single planes within a 3D sequence.

But why do I need a reference image I hear you ask? Reference images can be used to show overall morphology or orientation information where the detail of 3D is not required. For example, a brightfield reference image could be used to show cellular membranes to confirm the total number of cells in a field.

Reference channels can be created in Volocity using the Reference tab in the Acquisition Setup dialog.

Acquiring reference Images in Volocity 5.3

Make sure that before you set-up your reference channel, you have configured the light paths for the experiment in the Channels/Z tab in the Acquisition Setup dialog. Remember that these light paths should include the one that you want to use for your reference channel. Also ensure that you have configured the ‘change focus using’ option to capture a stack.

Acquiring reference Images in Volocity 5.3

To configure your reference channel, select the Reference tab and choose the channel and the Z position that you require. The Z position of the reference image relates to the current stack dimensions, which depend on the top and bottom positions set in the focus control and the slice number (as set in the Channels/Z tab).  If stack dimensions are changed between experiments, the top, middle and bottom positions will always honor the current stack size. If another plane is selected and this number plane is not available in the current stack the nearest available number plane will be used.  

Acquiring reference Images in Volocity 5.3

The order in which channels are acquired (including the reference image) is dictated by the order in the Channels/Z tab. If you are running a time resolved experiment, you can use the timing options for each channel, also in the Channels/Z tab, to capture the reference channel at a different frequency to the other channels.

Acquiring reference Images in Volocity 5.3

Your reference channel can then be visualized with your 3D data in any of the Image view modes. For example, using the 3D Opacity image view mode, you can visualize your single plane reference channel within the volume. The Reference channel will be shown at a single plane with a white outline in the position at which they were acquired.

I am sure that you will agree that this is a very useful thing to be able to do!

Happy imaging!