Biology Reference
In-Depth Information
- Center the condenser by closing it and moving the octagon to the center of
the field of view. Reopen the diaphragm to encompass the entire field of
view.
- Adjust the light level. High-quality Nomarski optics requires a substantial
amount of light. Optimal settings must be empirically determined.
- Adjust the exposure time, gain, and other settings on the CCD camerawithin
Micro-Manager a final time if needed.
h. Invoke the Micro-Manager 4D acquisition script (
Fig. 2
). This protocol pre-
supposes that a ''favorite'' has been created previously using the script window
in Micro-Manager. This window is invoked using the ''Tools -
>
Script Panel''
menu command in Micro-Manager. When the script window appears, select
''Acquire_4D.bsh'' from the list of favorites. Make sure that the cursor is
blinking within the code of this script. Then click ''Run.'' Enter the desired
parameters for time interval, number of time points, number of focal planes,
and distance between focal planes. Enter the root name for the images that will
be collected. [Note: because most operating systems limit the total length of a
file
'
s name to 32 characters, the root name should be kept short]. If a shutter is
being used, makes sure that the ''Use shutter'' option is selected.
i. Click ''OK.'' The parameters that have been entered will be displayed. If these
are acceptable, click ''OK.'' When prompted for a location to which to save
images, make a new directory that will contain the images from the 4D
sequence. Within the newly created directory, we recommend making two
additional directories: (a) one called ''working'' and (b) one called ''terminal.''
The latter is useful for acquiring a final Z stack of the terminal embryos.
Typical settings for a long overnight movie are the following:
Number of time points: usually 200-300 for an overnight movie
Time interval (s): usually 120-180
Number of shutters: 1
Number of focal planes: 20
Distance between focal planes: 1
m
m
Root name: ''working,'' or a short name of choice
Information for movie: Enter any pertinent information.
j. Once a directory is specified, the computer should start acquiring images.
Status updates will be displayed in the ImageJ main window. To abort, click the
''Stop'' button in the ''Script Panel'' window.
k. When the movie is finished, we recommend collecting a terminal image stack.
To do so, keep the field of view the same. Collect a second movie, specifying
''1'' as the number of time points. Save this movie in the ''terminal'' folder
created previously.
l. To view the movie, there are several options available:
(i) Raw 4D datasets: These can be viewed in one of several ways, including
(a) importing the sequences as a ''Virtual 5D Stack'' or Hyperstack within
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