Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.
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.2
C. 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.
We have developed a novel approach to the real-time measurement of movement and simple locomotory behaviors in microscopic organisms like nematodes that does not require the use of microscopes.8 This methodological approach could also be utilized for studying numerous microscopic organisms like protists. This method is only limited by the wavelength of light used. The organism should not be smaller than the wavelength of the light. In addition to the cost-savings and portability of the equipment needed, one key advantage of this approach is the ability to measure behavior in real-time and in three dimensions, without the narrow constraints of image planes under a microscope. It is also possible with this technique to examine influences of gravitational forces or numerous other conditions on behavior that cannot be studied using microscope-based approaches.9 Thus, we can achieve a better understanding of microorganism natural locomotory behaviors freed from the confines of microscope slide droplets or specialized microfluidic chambers (Park et al, 2008).10
The lack of phase information in a diffraction pattern does not allow for the direct retrieval of the image corresponding to the diffracting object since the far-field diffraction pattern is proportional to the square of the absolute value of the Fourier transform. We are therefore calculating diffraction patterns from worm images so that they can be matched with the diffraction patterns of freely swimming nematodes (Figure 6).
This method has yielded results for truly freely swimming C. elegans and can be applied to any microscopic species that maneuvers in an optically transparent environment like water or many different ionic solutions. Conventional microscopes only allow studies with a focal depth on the order of micrometers.11 This is due to the limited depth of field when focusing light:
where the f-number N has a reciprocal relationship with the circle of confusion (c) so that a short focal length is associated with a large c.12,13 While this diffraction method is certainly not a replacement for conventional microscopy, it is able to deliver quantitative results quickly so that species can even be manipulated in real time at low cost. The diffraction patterns can be obtained with any laser pointer. The diffraction patterns can be filmed at a reduced temporal resolution using a regular digital camera. While the user may not have a microscope or a photodiode readily available, key parts of this experiment such as measuring thrashing frequencies and evaluating diffraction patterns can be completed at extremely low cost.
The authors have nothing to disclose.
We thank Tzlil Rozenblat, Alexandra Bello and Karl Spuhler for technical assistance. This work was supported by the Vassar College Undergraduate Research Summer Institute (URSI), Lucy Maynard Salmon Research Fund and the NASA award # NX09AU90A, National Science Foundation Center for Research Excellence in Science and Technology (NSF-CREST) award # 0630388 and the NSF award # 1058385.
Name | Company | Catalogue number | Comments (optional) |
543 nm HeNe Laser | Melles Griot | LGX1 | Any laser in the visible range with less than 5 mW can be used. |
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/ |