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

Abstract

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field. 1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform. 2C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming. 3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated. 4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions. Video LinkThe video component of this article can be found at https://www.jove.com/video/4412/ Protocol C. elegans Preparation for Video Analysis1. Move 10-20 gravid adult nematodes to fresh NGM agar-filled Petri plates using a thin, flattened platinum wire pick. The Petri dishes filled with the NGM agar contain a small circular spot of E. coli (a growth limited strain, OP50, gives the best results) for the nematodes to eat. Allow the nematodes to lay eggs for 3-5 hr and then remove the adults, thus establishing a small, developmentally-synchronized culture of 50-150 nematodes. Allow the nematodes to grow to early adulthood by incubating the Petri plates at 20 °C for 4-5 days. These culture methods are based on established procedures (Stiernagle 2006). 2. On the day of the video analysis, flush a plate of young adult nematodes with 1 ml of deionized, distilled water, thus collecting 50-150 worms from the synchronized culture. A buffer solution like M9 or PBS can also be used. Use a microcentrifuge to spin the worms to the bottom of the tube, or allow them to settle by gravity for about 15 min. Remove the majority of the water from the tube and replace it with 1 ml of deion...

Caenorhabditis elegansExhibits Positive Gravitaxis

“…C. elegans offers important experimental advantages, including a small and simple nervous system, accessibility to rapid genetic manipulation and to other powerful experimental tools, and ease of cultivation. There is only one report suggesting that C. elegans suspended in solution may orient with the gravitational field (3). Since C. elegans is heavier than suspending buffers typically used in laboratories, it settles when suspended in solution (4).…”

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

“…Some previous characteristics have been confirmed using frequency analysis of diffraction signals such as swimming frequencies 2 4 . More importantly, this method can be used as a complementary method to traditional microscopy to observe locomotion in real time on a computer screen as the data are being collected.…”

“…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 .…”

Using flatbed scanners in the undergraduate optics laboratory—An example of frugal science

“…Thus, they cannot be used to image a number of interesting diffraction phenomena that have larger or twodimensional diffraction patterns that are several centimeters in width and height. [36][37][38][39] Commercial equipment to image interference and diffraction patterns for undergraduate teaching laboratories is also available, 40 and while they can image large patterns in one dimension they cannot image twodimensional patterns and are considerably more expensive than the setup we describe here.…”