Biology Reference
In-Depth Information
2. Acquisition of 4D Nomarski Images During Morphogenesis
Introduction
Acquisition of stacks of images throughout the thickness of the embryo over time
is a crucial method for identifying the positions and contacts between cells. Such
''four-dimensional'' (4D) microscopy is a routine tool in laboratories that study early
C. elegans development.
Imaging Setup
i. Microscopy/camera hardware: This protocol assumes a basic high numerical
aperture (NA) microscope equipped with oil immersion objectives and, option-
ally, an oilable, high NA condenser from any of the major microscope manu-
facturers. We typically acquire 4D movies using a 60-63
, 1.4-1.45 N.A.
PlanApo objective. Older Newvicon video cameras, coupled to the video port
on the microscope, are adequate for many applications, especially if they are
equipped with a zoomable video lens attachment, such as those sold by Nikon
Corporation. In this case, an AG-5 digitizing board (Scion Corporation) or
similar video frame grabber can be used to digitize the video signal.
Alternatively, modern cameras are almost exclusively CCD cameras, and have
much higher spatial resolution than older video formats. We have successfully
used cameras from Scion Corporation and QImaging Corporation. The mount-
ing hardware for such devices differs depending on the microscope being used.
ii. Cooled environment:WehavefoundthatC. elegans embryos can be imaged for
long periods of time if the ambient temperature is reduce to approximately 20
C.
iii.
Z-axis controller/shutter/serial port: Avariety of Z-axis controllers are available
from commercial sources (e.g., Prior, Ludl, ASI). In addition, a shutter to block
the transmitted light path between time points is strongly encouraged to mini-
mize exposure of embryos to light and heat. A number of shutters are available.
We have used shutters from Ludl and Vincent Associates.
iv.
Software: Many commercial software packages can be used to acquire 4D
footage. If an inexpensive alternative is desired, I have written free software
as an alternative. Several options are available:
(a) Legacy acquisition plugins for ImageJ: These plugins are available free of
charge at the following URL:
http://worms.zoology.wisc.edu/research/4d/
4d.html
. Full documentation of the plugins and detailed instructions for
installation of ImageJ and Quicktime for Java are available at the same URL.
Image J can be obtained at
http://rsbweb.nih.gov/ij/
(b) Micro-Manager: The public domain program Micro-Manager, which is
built on top of ImageJ, supports a variety of CCD cameras, Z motors, and
shutters, and, with some effort, novices can extend its functionality using the
Beanshell scripting language or through the Java plugin architecture sup-
ported by ImageJ. Micro-Manager has a major advantage over other free
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