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How to measure lethargus quiescence without - WormBook

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THE WORM BREEDER’S GAZETTE
VOLUME 19 NUMBER 3 | FEBRUARY 2013
How to measure lethargus quiescence without monopolizing the
dissection microscopes in your lab
1
Huiyan Huang1, Chen-Tseh Zhu2 and Anne Hart1
Department of Neuroscience, Brown University, Providence RI, 2Department of Ecology and Evolutionary Biology,
Brown University, Providence RI
Correspondence to: Anne Hart (anne_hart@brown.edu)
During C. elegans lethargus, a sleep-like behavior called quiescence occurs in short bouts during which animals spontaneously pause locomotion and feeding (Raizen et al., 2008). Multiple laboratories are interested in studying quiescence, however monitoring this behavior is problematic as highmagnification images must be captured for individual animals over a long period of time. Using a dissection scope with a camera to measure quiescence is generally expensive and monopolizes microscopes that would normally be used for other purposes. We describe here a set-up that can be used to
follow individual animals without using a dissection microscope. We load up to 6 late-L4 animals into
simple, reusable microfluidic chambers and analyze images captured every 10 seconds throughout lethargus (Singh et al., 2011). MATLAB scripts for acquiring and analyzing the images are available
upon request.
Equipment (total cost: $2072 without computer)
1. 76mm HH Dual Illuminated Track Stand (Halogen/Halogen) with N-Type Adapter from Howard
Electronic Instruments (#HEI-TS-HH-N) for $462. This is a special order that is not available in the
catalog. The sales manager is willing to custom-assemble this stand.
2. AVT Guppy Pro F-503 color CCD camera from Edmund Optics (# NT68-572) for $760.
AVT camera driver compatible with MATLAB needs to be acquired from the AVT website.
3. 1/4-20 Mounting Adapter for Guppy Pro from Edmund Optics (#NT68-585) for $45.
4. 50 mm Fixed Focal Length 10 MegaPixel Lens with Manual Focus and Iris, C-mount, 2/3in. format, F-stop: 2.0, Filter: 30.5, Pitch: 0.5 from Graftek Imaging (# 1-19558) for $747.
5. Schneider C-mount Extension Tube Kit from B&H (B&H # SCCMETK, Mfr# 21-06010) for
$58. The kit includes 0.5/1/5/10/20/40 mm tubes.
6. Computer and adapters:
a) If you are concerned about budget and have plenty of space, a desktop computer with a Firewire
card adapter and operating system that meets MATLAB system requirements will do. The cost for both
the desktop and Firewire card adapter starts at $500.
b) A laptop can be used to save space but is more expensive. Purchase a laptop with an operating
system that meets MATLAB system requirements and has an Expresscard slot. Also, purchase a SIIG
NN-EC2812-S2 2-port Firewire 800 ExpressCard/54 from Newegg (# N82E16839150025) for $78 and
Power adapter(# N82E16839150034) for $22.
Instructions for assembly:
1. Assemble the track stand:
Assemble the track stand according to the instructions. The top lighting does not need to be assembled
since it is not necessary.
2. Assemble the camera and lens:
The minimum focusing distance for the lens is 10 cm and the field-of-view at minimum focusing distance is 14.3 mm Г— 19.1 mm. Currently, our chip for quiescence chamber is 4 mm Г— 6 mm. To achieve
a shorter focusing distance and smaller field-of-view, connect the camera and lens with a combination
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THE WORM BREEDER’S GAZETTE
VOLUME 19 NUMBER 3 | FEBRUARY 2013
of extension tubes (20 mm and 5 mm). You can change extension tubes when working with microfluidic chips of different sizes.
3. Mount the camera to track stand:
Mount the camera to the N-type adapter with the mounting adapter. Make sure the camera and lens are
perpendicular to the track stand base.
4. Connect the camera to a computer with a Firewire cable.
5. Install MATLAB with Image Acquisition Toolbox and AVT camera driver compatible with
MATLAB.
References
Raizen DM, Zimmerman JE, Maycock MH, Ta UD, You YJ, Sundaram MV, Pack AI. (2008) Lethargus is a
Caenorhabditis elegans sleep-like state. Nature 451, 569-472.
Singh K, Chao MY, Somers GA, Kamatsu H, Corkins ME, Larkins-Ford J, Tucey T, Dionne HM, Walsh MB, Beaumont
EK, Hart DP, Lockery SR, Hart AC. (2011) C. elegans Notch signaling regulates adult chemosensory response and
larval molting quiescence. Curr Biol 21, 825-834.
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