The technique presented here measures the path of freely swimming microscopic species using single wavelength exposure. C. elegans are used to demonstrate shadow imaging as an inexpensive alternative to costly microscopes. This technique can be adapted to accommodate various orientations, environments and species to measure direction, speed, acceleration and forces.
This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, acceleration and forces involved in the locomotory behavior of nematodes suspended in a column of water in response to single wavelengths of light. We demonstrate how to evaluate the locomotion of a microscopic organism using Single Wavelength Shadow Imaging (SWSI) using two different examples.
The first example is a systematic and statistically viable study of the average descent of C. elegans in a column of water. For this study, we used living and dead wildtype C. elegans. When we compared the velocity and direction of nematode active movement with the passive descent of dead worms within the gravitational field, this study showed no difference in descent-times. The average descent was 1.5 mm/sec ± 0.1 mm/sec for both the live and dead worms using 633 nm coherent light.
The second example is a case study of select individual C. elegans changing direction during the descent in a vertical water column. Acceleration and force are analyzed in this example. This case study demonstrates the scope of other physical properties that can be evaluated using SWSI while evaluating the behavior using single wavelengths in an environment that is not accessible with traditional microscopes. Using this analysis we estimated an individual nematode is capable of thrusting with a force in excess of 28 nN.
Our findings indicate that living nematodes exert 28 nN when turning, or moving against the gravitational field. The findings further suggest that nematodes passively descend in a column of water, but can actively resist the force of gravity primarily by turning direction.
Caenorhabditis elegans is a free-living beneficial soil nematode that is a powerful model organism for studying mechanisms of gene regulation, development and more recently for understanding sensory biology and behavior. Despite having only 302 neurons, C. elegans are capable of complex locomotory patterns, reproductive behaviors, navigation, chemotaxis and many other behaviors. C. elegans possess mechanoreceptors, chemoreceptors and even detect blue wavelengths of light (Ward et al., 2008)1. While much is known about the neural circuitry of sensorimotor function and general locomotory patterns in C. elegans, less is known about the responses to multiple, concurrent stimuli or more complex environmental conditions than can be modeled under a microscope. A few studies have revealed more complex locomotory patterns that are highly plastic2,3,4. Our methodological approach will enable studies of nematodes in solution in real time where we can readily provide multiple environmental conditions simultaneously. This question is difficult to address using conventional microscope-based imaging techniques. We have developed an imaging technique that allows us to place nematodes within a water column to examine locomotory behaviors, as well as determine the capabilities of nematodes to change locomotion in response to different environmental conditions.
Single Wavelength Shadow Imaging (SWSI) is presented in this paper for the first time to address the shortcomings of traditional microscopes. Traditional microscopes are limited to observe species in a horizontal focal plane a few microns in depth5,6. Regarding single wavelength studies, most traditional microscopes use color filters to filter white light very broadly, typically, 50-100 nm. Using a laser for SWSI narrows the wavelength selection to less than 1 nm while maintaining significant light intensity7. Similarly, single wavelengths have been used to measure swimming frequencies of C. elegans in real time8.
For the first demonstration of our method, we monitor the horizontal position, x, and the vertical position, y, of a freely swimming C. elegans in a water column, over a distance of about a centimeter. In particular, we are interested in the vertical movement since gravity also acts vertically. The slope of a linear fit to the vertical position gives the vertical speed, vy, of the nematode as it descends in the water column:
(1)
The root mean square of the error (RMSE)9 indicates the quality of the fit and indicates whether the descending speed is generally constant. The vertical speeds are then averaged for each species and dead worms. Using these results, the drag, which the worms experience can be estimated.
For the second demonstration of our method, we selected C. elegans that did not descend at a constant rate unlike the majority of the worms observed. The selected worms either turned around and swam upwards or hovered for a while before continuing the descent. Physically, this case study shows that the thrust of a swimming microorganism can be calculated. Newton's laws dictate that a body that changes directions accelerates, which implies a net force, , is acting on that body10:
(2)
where is the linear momentum and t is time. The acceleration of the worm is directly proportional to the force acting on the worm since the mass of the worm remains constant. As a result, the vertical net force is:
(3)
where m is the mass of a worm and ay represents the vertical acceleration. The net force in the vertical direction represents then the worm thrust in the same direction. The total thrust can be calculated by taking the horizontal component into account.
The SWSI technique provides an additional way to understand the locomotory capabilities of microscopic organisms like free-living nematodes. With this technique we have distinguished between active locomotion (swimming) and passive drift due to gravity operating on dead nematodes. In addition, when free-swimming nematodes change direction during locomotion in water, we are able to measure the drag forces and angular forces, which are operating on the nematodes and exerted by the nematodes.
Nematodes encounter different environmental conditions within the soil. There are water pockets within soil, as well as solid particles and biological materials of different shapes and textures. In addition, nematodes exist within a gravitational environment that they respond to14. Further, nematodes near the surface of the soil are exposed to different wavelengths of light, changes in temperature and humidity, as well as biological variables like bacteria, predatory fungi and other soil organisms. Nematodes must respond to all these different variables, swimming and crawling in different media, turning and altering navigational strategies. All of these complex computations are carried out by only 302 neurons, a subset of which are involved in locomotion, and 95 body wall muscle cells. Measurements of the sort described by SWSI technique provide important insight into how nematodes accomplish this navigational complexity.
For the first part, we have measured the overall descending rate of wildtype C. elegans in 633 nm light. Using these measurements, we can estimate the drag force a worm encounters.
For the case study of an accelerating nematode, the forces involved change continuously since the drag force changes with speed. There are some statements that we are able to make about the forces acting on the worm. As the worm slows down and tries to swim upwards the vertical component of the drag force decreases until it reaches zero at the low point of the nematode's trajectory. At this point, the worm must have an upward force to swim up.
This method can be modified in several ways. Any microscopic species that navigates in a clear liquid can be tracked using SWSI. Studies can be conducted with any wavelengths that are accessible to digital cameras. Digital cameras will typically pick up wavelengths ranging from the UV to near IR. In addition, horizontal studies can be conducted by directing the laser vertically upward. The species can then be placed on a horizontal transparent surface, like a microscope slide. Adjusting the beam expander or the magnifying lens after the beam expander can sharpen blurry images. The user should be sure to fasten all components to the table to ensure consistent and easy beam alignment.
The method is limited by available laser wavelengths and resolution. In essence the advantages of this method over existing microscopes, which are the flexibility in directions and wavelengths, are also weaknesses since the setup is simple. The unsophisticated optics and speckles of the laser limit the resolution. Some of these drawbacks can certainly be improved in the future by including spatial filter and projecting the image directly onto a CCD camera.
The most critical steps in the protocol can easily be learned as the experiment is performed for the first time. Placing the nematode in the cuvette without creating turbulence is critical. Also, vibrations may disturb the setup and alter the behavior of the worms. Be sure to limit the power, which is used to shadow image. 2 mW for a laser beam that is 1 mm in diameter should be the maximum to avoid heating effects. The setup should be tested for scattering effects when using liquids other than distilled water.
Currently most microscopes operate on a horizontal plane using white light or color filters, which are still very broad in the wavelength range. Microscopes that truly use single wavelengths and have flexibility in the viewing scenario, i.e. horizontal placement, are usually limited to one advantage or the other. Also, these types of microscopes are usually very expensive and still limited to focal planes unlike our method. Our setup can easily be built with an extremely low budget. This method is ready to be used by schools, environmental companies as well as other entities that operate with little funding. In the future, this method can be used in a very sophisticated setup to study real time effects on locomotion and mechanosensation of microscopic species. This method makes single wavelength studies at a wide range of angles and viewing depths easily available.
The authors have nothing to disclose.
We are grateful for the support of the Vassar College Undergraduate Research Summer Institute (URSI), the Lucy Maynard Salmon Research Fund, NASA award No. NX09AU90A, National Science Foundation Center for Research Excellence in Science and Technology (NSF-CREST) award No. 0630388 and the NSF award No. 1058385.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Tunable Helium-Neon laser | Research Electro-Optics | 30602 | Four wavelengths can be selected between 543 nm and 633 nm. |
2 Front Surface Aluminum Mirrors | Thorlabs | PF10-03-F01 | |
High Speed Exilim Camera | Casio | ||
Quartz Cuvette | Starna Cells | 21/G/5 | |
LoggerPro (Software) | Vernier | http://www.vernier.com/products/software/lp/ | |
Mathematica 8 | Wolfram | http://www.wolfram.com/ | |
5x – 10x variable zoom Galilean beam expander | Thorlabs | BE05-10-A | |
Plano-convex lens with a positive focal length of 75 mm | Thorlabs | LA1257 |