Biology Reference
In-Depth Information
ImageJ, using the ''Virtual 5D Stack'' plugin or ImageJ itself, available on
the ImageJ web site, or (b) using the ''Browse4D'' plugin available at
(ii) Compressed movies: Movies can also be compressed to save disk space
and viewed using QuickTime and the ''QT4D Writer'' and ''QT4D
Player'' plugins available at the same URL. The advantage of postac-
quisition compression is that the storage requirements are greatly
reduced, and, if QuickTime is used, the entire dataset does not need
to be imported into RAM or imported as a virtual stack. Unfortunately,
Quicktime for Java has been deprecated by Apple Computer, Inc. If
QuickTime compression is being used, we typically save the movie
using the same root name as the raw files, with the word ''movie''
appended in the same directory created for the experiment. Although
many compression algorithms are available, we typically use ''Photo/
JPEG,'' compression, ''gray scale,'' and ''Medium'' quality. This
approach can compress movies 30 fold. To play compressed movies,
use the ''QT4D Player'' plugin. Select the desired movie. A graphical
interface with clickable buttons or the arrow keys on the numeric
keyboard can be used to navigate through movies. The up and down
arrow keys can be used to scroll up and down through the focal planes;
the left and right arrow keys scroll backwards and forwards in time.
Once the movie has been successfully compressed and its quality ver-
ified, for routine purposes it is now fine to delete the original files.
Troubleshooting
i. Problem: No light appears to be reaching the camera.
Solution: Make sure the slider that diverts light from the microscope to the
camera port is in the proper position, and that the power supply to the camera is
on. If the shutter has an external toggle switch, make sure that it is in the correct
position. If the exposure time is set to too low a value, increase the exposure time
using the controls in the Main Micro-Manager window.
ii. Problem: The plane of focus drifts systematically over time.
Solution: This often occurs in the first few minutes after making an agar mount.
For this reason, it is advisable to check the focus several times during the first
15-20 min of acquisition. To reset the focus, open the shutter and use the coarse
focus on the microscope to refocus on the top focal plane.
iii. Problem: Temperature variation in the room results in inconsistent time course
of development or variable phenotypes.
Solution: For best results, filming should take place in a room held at constant
temperature, approximately 20
C. Make sure the air conditioner is on and that
the door remains closed.
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