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    • CommentAuthorjuruble
    • CommentTimeJan 31st 2008 edited

    This protocol explains how to: aqcuire a time-lapse movie on stereomicroscope using ImagePro+.

    Protocol revised: 11/27/2011

    Protocol written by: Barbara Lom

    1. Turn on the microscope light (black switch at the back left of the base).  Focus on your sample through the microscope eyepieces with the dial on the upper right of the microscope set to "Bino" (which directs the light to your eyes).  You can use the 1X objective to find the general area of the sample and then carefully switch to a higher objective.  When the sample is in focus, switch the dial to "Photo," directing the light to the camera.

    2. Log into Windows as you normally do and then right click anywhere on the desktop.  Select “Properties.”  Under the “Themes” tab change to “Windows Classic” mode from the drop down choices.  Select the “Screen Saver” tab.  Set the screen saver wait time to something longer than your movie time   Also make sure the “On resume password protect” box is unclicked in the screen saver windows. 


    3. Open ImagePro+.  Select the “Complete” option and click “OK”.  (Ignore the registration request and close the macro player window if it pops up.

    4. Click on the camera icon to open the Nikon camera controller window.  (Ignore the VPF savings warning if it pops up.)

    5. Click on “basic dialog” button in the bottom of the camera window to reset any previous settings.

    6. Adjust the lighting and/or focus as necessary (the camera may have slightly different focus and/or sensitivity than your eyes).

    7. Click on the “Calc Auto Exp” button and note the exposure time.

    8. Click on the "Auto Set" button to open the white balance box and then click on the "Calculate" button.


    9. Click on the "More>>" button at the bottom of the camera controller window and change the "Capture Depth" setting to "8-bit mono."


    10. Leave the "Live Preview" window open, but you may want to move it to the lower right corner of your monitor.

    11. Click on the “Advanced” button at the bottom of the camera window and go to the “Image” tab.  Make sure that “Sequence,” “Display sequence during acquire”, and “Enable Multiple Image Capture” buttons are each checked.  “Use minimal possible interval box” should be unchecked.  Set the total number of images you want to collect (usually around 15) and the duration of time between images (usually about 2 minutes).  Click on the “Acquire” tab and then set the exposure time to the exposure duration that you just noted.  Click on the “Camera Features” button and set the color cast to grayscale. 



    12. Make sure your Live Preview Window is open and make readjustments to focus/lighting of the image if necessary. 

    13. Click "Snap" to begin the movie.  

    14. Note that the preview window will not update while the camera is collecting an image (and that image collection takes a few seconds), so lighting/focus changes or changes in your specimen will not always immediately show on the screen.

    15. When the movie is finished the “Sequence Toolbar” will come up. Click > (solid arrow) to play movie or I> (line behind arrow) to play the individual images.  To change the speed of the movie click “Options” and “Change frame rate” (the larger the number of the slower the movie will play) and make sure the “Automatically adjust rate” button is unclicked.

    16. To save your movie first make sure the “Apply To” button for multiple images (above/right) is selected (not the single image button above/left).  Then go to the “File” menu and select “Save As” and give your movie a name and select the .seq file option.  Note: ImagePro will not be able to save movies with over 120 frames in the .seq format





    Here is an example of a time-lapse movie. This movie shows RGC growth cones extending from a cultured Xenopus eyebud:
    • CommentAuthorvibeamer
    • CommentTimeSep 30th 2011
    If you have completed a time lapse video, and the changes of your subject look too drastic, shorten the length of time between each capture. For example if a change occurs within 2 minutes, but you have the captures set for every 5 minutes; decrease the intervals.
    • CommentAuthoralfleming
    • CommentTimeOct 1st 2011
    When changing the focus and the lighting during the video , the video will continue to take images. The changes will not be reflected in the video until you are done changing the lighting and focus of the microscope. Also make sure you make the changes by looking through the microscope and not just using the preview image on the computer screen.
    • CommentAuthorjeroth
    • CommentTimeOct 1st 2011
    Make sure the computer does not go to sleep while you are recording a time-lapse movie; if it does, image acquisition will stop. To prevent this calamity, open the Start menu and select Control Panel. Next go to Performance and Maintenance, then Power Options. Under the Power Schemes tab, find the drop-down menu next to System standby and change the setting to Never. Click Apply and then OK.