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Volocity SOP

VIEWS: 3 PAGES: 10

									                             VOLOCITY SOP
              Olympus IX81 and Volocity Acqusition.




1   Switch on the λ LS lamp housing. ( Check hours bulb can be used up to 1500hrs )




2   Switch on the Olympus IX2-UCB unit, the Mac 5000 controller, the improvision shutter hub,
    the improvision acquisition hub, the camera control unit and the PC.
                                   VOLOCITY SOP

                                                                                       Mac 5000 controller




                                                                                       Improvision
                                                                                       Acqusition and
                                                                                       shutter hub.

Olympus IX2-
UCB controller.



                                                                                       Camera control
                                                                                       unit.




  2       Select Beatson User and double click on the Volocity software icon on the desktop. Volocity 5
          will open.
  3       Choose Create a new Library or Open an existing Library from the start up dialog window.
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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.

From the top menu select window | Show Video Preview




The image window and acquisition controls will now appear.




 The control panel on the right hand side of the window contains all the settings required to
 capture your images.
VOLOCITY SOP
                                       VOLOCITY SOP
                                                       Timing feedback.
Timelapse/number of
frames/Timepoints time                                 Video feed back- information
                                                       on the grey levels in your
                                                       video preview.

                                                  Information on acquisition protocol
Capture a single image
Start or stop acquisition protocol                Light path manager. Pre saved light
Freeze or resume preview                          paths.


                                                       Auto Exposure
                                                       Set TL mode
                                                       Binning




                                                  Filters in the Olympus internal filter
                                                  wheel.
                                                  1 DAPI dichroic and emission.
               light path to eyes or              2 GFP dichroic and emission
               camera                             3 TR dichroic and emission
                                                  4 GFP/RFP dichroic
                                                  5 Tirple dichroic


     Fluorescent shutter click to
     open and close.                                   Piezo Z controls
     BF shutter.



                                                      External emission filter wheel.

                                                      External excitation filter wheel

                                                      External ND filter wheel
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From the light path manager select the filters and light path required to view your sample with.




Open the fluorescent shutter. Or the BF shutter.




Set the light path to eyes by selecting binocular.




Set the piezo Z stage as follows: reset top and bottom, move to hard center and set zero.
Focus on your sample using the microscope focusing knob as normal.
Change the light path to the camera by selecting side port.




Increase the exposure time until you see an image on the screen. Right mouse click on the light path
manager position you are working on to apply the new exposure time select overwrite. Make sure you
have not altered any filter positions as it will save these changes as well!!!!!!
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From the top menu select Video | Acquisition setup. The acquisition set up window will open.
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Give your protocol a title , add your name and a description of the protocol.




                .

Select the light path channels you wish to use. Add or remove channels using the + or –
If you wish to acquire one channel at a different rate to other channels Click on the timing clock.




If you are acquiring Z sections you must select the appropriate device. Use Ludl Piezo Z or select None.
Select Z spacing or number of slices.
Z spacing option volocity will calculate the number of slices required to move through the range set in
the video preview window.
Using the number of slices volocity will calculate the spacing between slices to capture that number of
slices in your set range.

Scan direction select up. This is recommended as it moves the focus device against gravity for the most
accurate positioning.
                                    VOLOCITY SOP




If you wish to acquire multiple time points select FOR . Enter a number into the text field and select
either timepoints, ms, seconds, minutes, or hours from the drop down menu.

For a single time point select FOR 1 timepoints.

Manage shutters lets you select how the shutters will operate between channels and timepoints.




If you are acquiring multiple channels and Z sections you must decide in which ordere velocity will
acquire your images.




Click save to save your protocol. Click on restore to display a list of saved acquisition protocols.
Highlight the protocol you wish to restore and click restore.




Click on start button on the acquisition control window to run your protocol.




In our offline analysis room ( room 129 ) we have three PC systems with volocity .( PC 1 , 2, and 4 ).
Copy your images to be analysed to the HD of the PC and open the volocity analysis software.

See BAIR handout for analysis.
VOLOCITY SOP

								
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