Analysis of Freely Swimming C. elegans Using Laser Diffraction

Abstract

Soil and aquatic multicellular microorganisms play a critical role in the nutrient-cycling and organismal ecology of soil and aquatic ecosystems. These organisms live and behave in a complex three-dimensional environment. Most studies of microorganismal behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field. We report on a novel analytical approach that provides real-time analysis of freely swimming C. elegans without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light onto nematodes in a cuvette. We measured oscillation frequencies for freely swimming nematodes in cuvettes of different sizes to provide different physical constraints on their swimming. We compared these frequencies with those obtained for nematodes swimming within a small droplet of water on a microscope slide, a strategy used by microscope-based locomotion analysis systems. We collected data from diffraction patterns using two methods: video analysis and real time data acquisition using a fast photodiode. Swimming frequencies of nematodes in a droplet of ionic solution on a microscope slide was confirmed to be 2.00 Hz with a variance of 0.05 Hz for the video analysis method and 0.03 Hz for the real time data acquisition using a photodiode; this result agrees with previously published estimates using microscope-based analytical techniques. We find the swimming frequency of unconstrained worms within larger cuvettes to be 2.37 Hz with a variance of 0.02 Hz. As the cuvette size decreased, so did the oscillation frequency, indicating a change in locomotion when physical constraints are introduced.

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

“…3 Using this diffraction method, the average swimming frequencies of a C. elegans, which is confined to a microscope slide, has been found to match the previously published value of 2 Hz. 1,7 Following procedures 3.) and then 6.)…”

“…Transfer nematodes in water or buffer to a quartz cuvette using a micropipette. It is important to work with a small number, about [5][6][7][8][9][10] nematodes in total at one time or even fewer so that one nematode at a time is likely to be illuminated by the laser. If the number of nematodes on the culture plates is too large, dilute the worms in water or buffer until a desired density is reached.…”

Fourier-Based Diffraction Analysis of Live <em>Caenorhabditis elegans</em>

“…This method allows for quantitative real-time analysis and provides complementary information to images/videos obtained with traditional microscopes. Fraunhofer diffraction, also called far-field diffraction, provides the basis for obtaining live diffraction data 1 2 . The light intensity at any single point in the diffraction pattern is the result of superimposing light from every point in the outline of the nematode 3 .…”

Dissection of C. elegans behavioral genetics in 3-D environments

“…X-ray microtomography 5 or laser diffraction analysis 6 , reported for quantification of the worm movement in 3-D environments, is not appropriate for behavioral study of the worm. Scanning time is extremely slow (~80 minutes) in X-ray microtomography and merely one parameter (oscillation frequency) is affordable in laser diffraction analysis.…”