Log Onto the Computer As Normal

VOLOCITY SOP

RESTORATION

Log onto the computer as normal.

From the start menu select | Programmes | Volocity | Volocity.

From the license Server window select the version of volocity you require

Volocity Classification

Volocity Visualization

Volocity Restoration

The Password is VØLØC1TY.

Choose Create a new Library or Open an existing Library from the start up dialog window.

A library is where the collection of images you are working with will be held. It can also be used as an index with links to where the images are held ie: a CD-Rom. The images are stored in a multi file library folder along with a data folder. The library files and the data folder MUST be kept together in the same folder or the application will not be able to display the images.

Select Create a new Library | Give the library a name and save it to the Hard drive.

Select Action | Create New | Image Sequence.

Drag and Drop your files into the New Untitled Image Sequence Window. Make sure your images are already on the PC hard drive. Make sure you have a backup copy of the original data. Do not work with the files over the network or from a disc. Volocity will open Tiff files and Olympus oif files

From the Add Planes to Sequence window select the number of channels/ Z steps /Timepoints.

The images will now be added to the Untitled Image Sequence Window.

If the image is displayed in the wrong color .Highlight the image | Select Tools from top menu | change color | then change to the color you require.

Select the Ctrl and I keys. The properties window will appear.

The untitled image sequence can now be named . µm/pixel should be set for X Y and Z. With oif files this information should be transferred with the image and will already appear in the properties window. This information is essential for scale bar measurements in volocity.

Applying Fast Restoration

Select the image sequence tab from the untitled image sequence window. Highlight the required sequence by clicking on title of sequence; a blue border will appear on highlighted sequence.

In order to apply restoration a PSF (Point Spread Function) item must be created for each fluorescent marker in your sequence. A PSF is used to model the behaviour of light in the optical system and must be appropriate for the data which is being restored ( i.e. deconvolved). PSF can be created in 2 ways:-

Calculated PSF - based on values entered into the Calculate PSF.. dialogue box ( i.e. the parameters used when acquiring the image).

Measured PSF – based on a volume, typically from a fluorescent bead, acquired under the same conditions as the experiment data.

We shall concentrate on Calculated PSF.

Firstly, highlight the required image sequence as shown above; go to TOOLS and select Calculate PSF

The Calculate PSF window appears; enter the appropriate data (if known) i.e. type of microscopy e.g. widefield , confocal, etc; medium refractive index; numerical aperture of objective and emission wavelength of fluorochrome.

Once data has been added click CALCULATE.

When the PSF has been created it will appear in your library.

This process is repeated for each of your image stacks.

To begin Restoration , from your image sequence, highlight all of your omage stacks; go to TOOLS and select Fast Restoration.

The Fast Restoration window opens up. Here you choose which PSF from your library is relevant for each of your fluorochromes; in our example Untitled Widefileld PSF was created for the green fluorochrome and is chosen first followed by Untitled Widefield PSF2 for the red fluorochrome.

Then click on START.

When restorstion is completed click on DONE and the deconvolved stacks will appear on your Image Sequence. To confirm, hover over images and pop up information will tell you.

From your Image Sequence window click on the 3D tab and a composite image of all stacks will be displayed; and on the right hand side of your screen the NAVIGATION window opens.

From the navigation window you can hide/show which channels you would like to view. In this case channel 3 and 4 are the restoration channels therefore click on channel 1 and 2 hide/show buttons to hide these stacks. From navigation window you can use the brightness, contrast and black level sliders to adjust your image.

These images can then be made into movies, snapshots captured and/or cropped as normal.

Comparison of Original/Restored Image

The images below are from a cropped image:-

Original

Restored.