Biology Reference
In-Depth Information
alternatives, in that it is being continuously updated to support new hard-
ware. The Micro-Manager program can be obtained at the following URL:
http://www.micro-manager.org/ . The remainder of this section will describe
the basic use a Beanshell script written within the Micro-Manager environ-
ment as an example of how to acquire 4D data.
Methods
This protocol describes the use of a custom script within Micro-Manager ' s
Beanshell scripting language. The script is helpful to reduce the number of shutter
open/close events during 4D acquisition. Alternatively, we have used the standard
Micro-Manager package successfully to acquire 4D footage, but this cannot be
customized, and does not permit custom file naming conventions and directory
structure. The script is available at http://worms.zoology.wisc.edu/4d/4d.html
i.
Setting up a 4D acquisition sequence
a. Turn on the Z-axis and shutter control boxes and the CCD camera. Turn on the
light switch on the microscope. Find a group of embryos using the 10
objective, prior to oiling the coverslip.
b. If a high NA condenser is present, place a drop of oil on the condenser (for
upright microscopes) or the bottom of the slide (inverted microscopes).
Carefully position the condenser so that it contacts the oil and spreads it
uniformly between the condenser and microscope slide.
c. Focus the condenser. The simplest method for achieving good condenser focus
is to stop down the condenser using the iris diaphragm, closing it almost
completely. Then the height of the condenser can be adjusted at high magni-
fication until the octagonal outline of the diaphragm is in focus. When done,
open the condenser.
d. Once embryos have been located at 10 and the condenser has been focused,
swing the 10 objective out of the way and add a drop of immersion oil to the
coverslip (upright microscope) or the 60 objective lens (inverted micro-
scope). We find that Type DF oil works well.
e. Carefully slide the 60 or 100 objective into place (it should just clear the
sealant on the slide, as long as it is not too thick). Make sure the correct
condenser setting is selected to match the lens.
f. Refocus on the embryos, and refocus the condenser.
g. Open Micro-Manager. Use the ''Live'' button in the main Micro-Manager
Studio window to display an image from the camera. If the ''Autoshutter''
option is not checked, click the ''Open'' button to open the shutter. Otherwise it
should open when the ''Live'' button is clicked. Optimize the positioning of
embryos in the field using the stage controls on the microscope, and/or by
rotating the CCD camera gently by hand (if the mount supports this). Optimize
the Nomarski optics through a combination of the following:
Search WWH ::




Custom Search