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 high-magnification 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 × 19.1 mm. Currently, our chip for quiescence chamber is 4 mm × 6 mm. To achieve a shorter focusing distance and smaller field-of-view, connect the camera and lens with a combination 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.
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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