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iv. Problem: After several hundred time points Micro-Manager reports an error
from which it cannot recover.
Solution: Some users have reported errors under Micro-Manager when using
USB-to-serial port adapters. This is known issue with version of Micro-Manager
prior to 1.4. Using a PCI-based serial port card appears to alleviate this problem.
Alternatively, acquire several shorter movies. We have successfully used this
script under Micro-Manager for acquiring up to 150 time points with 25 focal
planes/time point. We have had success acquiring long movies with Micro-
Manager 1.46, Mac OS X 10.7, and a KeySpan USB-to-serial adapter.
Discussion
This procedure will result in the production of 4D datasets in the form of a series of
consecutively named TIFF files that can be read by many different programs,
including ImageJ, especially when supplemented with appropriate plugins. The
reduced costs of such a system make this basic system feasible for teaching labo-
ratories and research laboratories within limited funds. While we have described the
use of such a setup for imaging C. elegans embryos, this apparatus is well suited to
acquiring images of any transparent specimen.
3. Introducing Pharmacological Agents During 4D Acquisition
(Adapted from Williams-Masson et al., 1997 )
Introduction
This procedure assumes a fairly standard laser ablation setup. Many C. elegans
laboratories use a nitrogen laser to pump a tunable dye laser routed through the
epifluorescence light path of the microscope. The dye cuvette typically contains
Coumarin 440 dye, which can be obtained from Sigma or other suppliers, and is
reconstituted in methanol at a concentration of 5 mM in methanol (see the protocol in
Walston and Hardin, 2010 for further details). A common version of this setup is sold
by Photonic Instruments, a division of Andor Corp., under the trade name
Micropoint.
Methods
1. Creating the mount
i. Cut gravid hermaphrodites at the vulva in water. Mouth pipette embryos in a
small volume (approx. 3 m L) of water onto coverslips precoated with 1 mg/
mL poly-L-lysine. Poly-L-lysine is typically applied from a premixed stock
solution in distilled water (Sigma).
ii. Allow embryos to settle for 30 s and then treat for 2 min with 100 m gof
FITC-conjugated poly-L-lysine (Sigma). FITC-poly-L-lysine enhances the
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