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.

1. C. elegans Preparation for Video Analysis
<ol>
 <li>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 &deg;C for 4-5 days. These culture methods are based on established procedures (Stiernagle 2006).5</li>
 <li>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 deionized, distilled water in order to wash any adhering bacteria from the nematodes.</li>
 <li>Transfer nematodes in water or buffer to a quartz cuvette using a micropipette. It is important to work with a small number, about 5-10 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. It is also possible to pick individual nematodes to a drop of water or buffer on a fresh agar plate to wash them and transfer individual worms to the cuvette. In our study, we used 1 mm, 2 mm and 5 mm cuvette sizes to examine the effects of physical constraints on swimming behavior.</li>
</ol>
2. Optical Setup for the Video Analysis
<ol>
 <li>Perform setup as shown in Figure 1 using a 543 nm (green) Helium-Neon (HeNe) laser, two front-surface aluminum mirrors, a cuvette holder, a projection screen, and a high-speed camera (Casio High Speed Exilim) capable of filming at ~240 fps. The two mirrors are used to steer the laser beam through the cuvette. Different sizes of cuvettes can be used depending on the nature of the study. As an example, we used cuvettes that are 5 mm thick to show the effects of spatial constraints on swimming frequencies. The laser beam must fulfill the requirements for oversampling; i.e., the organism should obstruct no more than one third of the laser beam. For C. elegans, the laser beam should be about 2 mm in diameter when it interacts with a nematode. The laser beam should not be larger than 5 mm in diameter since the diffraction pattern will be difficult to locate. The distance from the diffracting organism to the screen should be much larger than the organism itself.</li>
 <li>Place parafilm over the top of the cuvette and invert it to mix the nematodes into the water column. Place the cuvette in a cuvette holder so that worms are initially near the top of the cuvette. Using the mirrors, steer the laser beam through the center of the cuvette. Because the nematodes are denser than water, they will slowly fall to the bottom of the cuvette, while at the same time swimming within the water column.</li>
 <li>On the projection screen, color the spot corresponding to the transmitted laser beam black to reduce scattering as the transmitted beam meets the projection screen (Figure 2). Alternatively put an opening in the projection screen to allow the transmitted beam to pass through the screen. Eliminating or reducing scattering from the transmitted beam will keep the CCD array of the camera from saturating due to the transmitted beam.</li>
 <li>To capture a measure of the size of the diffraction pattern, draw a line of about 5 cm on the projection screen next to the transmitted laser beam spot without interfering with the diffracted light image.</li>
 <li>Start recording with the camera while the room light is on so that the 5 cm line is on the same footage as the diffraction images. Turn the room light off. Record diffraction images on the screen with the camera as worms pass through the laser beam.</li>
</ol>
3. Video Data Preparation
<ol>
 <li>Install video analysis program (Logger Pro: <a href="http://www.vernier.com/products/software/lp/" target="_blank">http://www.vernier.com/products/software/lp/</a>) on the computer. Import the video into video analysis program</li>
 <li>Set the origin to coincide with the transmitted laser spot. Set the scale using the 5 cm line on the projection screen.</li>
 <li>Track the angular displacement of the diffraction image formed by each nematode using the video analysis software (Figure 3)</li>
 <li>To find the angular displacement by taking the arctan (y/x), copy and paste data into a spreadsheet (Excel) and add 180 deg or &pi; for all negative angles to produce a continuous graph (Figure. 4).</li>
</ol>
4. Real Time Data Acquisition for Instant Observation of Swimming Frequencies
<ol>
 <li>Using the same setup as for the video analysis, place a photodiode (DET10A from ThorLabs) with a small area (~1 mm2) off center in the diffraction pattern right in front of the projection screen.</li>
 <li>Connect the photodiode to the digital oscilloscope (PicoScope made by Pico Technology) that connects to the computer via USB port. Observe the thrashing patterns on the computer screen.</li>
 <li>Save acquired data sets in ASCII or text format.</li>
</ol>
5. Data Analysis
<ol>
 <li>Import the data acquired using video or the photodiode into a data analysis program capable of fitting waveforms using chi-square minimization. The program used here is Origin (<a href="http://www.originlab.com/" target="_blank">http://www.originlab.com/</a>). Fit a sinusoidal curve to determine thrashing frequency (Figures 4 and 5).</li>
 <li>Average swimming frequencies from various samples and determine variance. For this study, data were analyzed statistically using a single-factor ANOVA followed by the Bonferroni multiple comparisons test. A p-value of &lt; 0.05 was considered statistically significant.</li>
</ol>
6. Model Diffraction Patterns using Mathematica as an Example
Note: Diffraction patterns can be modeled using many different computational tools. This procedure will differ for different computational tools such as MatLab, Excel, Origin etc.
<ol>
 <li>Create Image: Take images of nematodes on a microscope slide using a traditional microscope.</li>
 <li>Binarize image: Import the worm image 'Worm' into Mathematica by dragging the image into Mathematica. Convert the image into a black and white image (binarize). This can be achieved with the 'Binarize' command in Mathematica: BlackandWhiteImage=Binarize[Worm]. The image can then be viewed as a black and white image. 
 Alternatively, use the 'EdgeDetect' command to create a BlackandWhiteImage: EdgeDetect[Worm,6.0,0.035], where the two trailing numbers control the coarseness of the resulting edges.</li>
 <li>Convert image into a matrix of zeroes and ones: This can be achieved with the 'ImageData' command in Mathematica: Matrix=ImageData[BlackandWhiteImage].</li>
 <li>Fourier transform matrix: Use the 'Fourier' command in Mathematica to Fourier transform the matrix using the indicated FourierParameters: FTransform = Fourier[Matrix, FourierParameters -&gt; {0, -2}].</li>
 <li>Square modulus of matrix to obtain the diffraction matrix and improve image contrast: Square the absolute value of the matrix and view the resultant image, which is the diffraction pattern corresponding to the original image. Also, use the 'Log' function to scale the contrast of the image: ImageContrast = Image[Log[1 + (Abs[FTransform])^2]/0.5].</li>
 <li>Resize image for easy viewing: Crop the central diffraction pattern and resize as desired: ImageResize[ImageContrast [y, 100], 500].</li>
 <li>Compare the modeled diffraction patterns with diffraction patterns obtained from freely swimming worms. (Figure 6)</li>
</ol>

<ol>
	<li>Korta, J., Clark, D. A., Gabel, C. V., Mahadevan, L., Samuel, A. D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensation+and+mechanical+load+modulate+the+locomotory+gait+of+swimming+C.+elegans.">Mechanosensation and mechanical load modulate the locomotory gait of swimming C. elegans.</a> J. Exp. Biol. 210, (2007).</li><li>James, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A student's guide to Fourier transforms with applications in physics and engineering. , (1995).</li><li>Pierce-Shimomura, J. T., Chen, B. L., Mun, J. J., Ho, R., Sarkis, R., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc. Natl. Acad. Sci. USA. 105, 20982-20987 (2008).</li><li>Li, W., Feng, Z., Sternbert, P. W., Xu, X. Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+stretch+receptor+neuron+revealed+by+a+mechanosensitive+TRP+channel+homologue.">A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue.</a> Nature. 440, 684-687 (2006).</li><li>Stiernagle, T., ed, W. o. r. m. B. o. o. k. ,. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Eells, R., Lueckheide, M., Magnes, J., Susman, K., Rozenblat, T., Kakhurel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Diffraction+Analysis+and+the+Transformation+of+C.+elegans.">Dynamic Diffraction Analysis and the Transformation of C. elegans.</a> , (2011).</li><li>Magnes, J., Raley-Susman, K. M., Melikechi, N., Sampson, A., Eells, R., Bello, A., Lueckheide, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Freely+Swimming+C.+elegans+using+Laser+Diffraction.">Analysis of Freely Swimming C. elegans using Laser Diffraction.</a> O. J. Biphys. , (2012).</li><li>Magnes, J., Raley-Susman, K. M., Sampson, A., Eells, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotive+Analysis+of+C.+Elegans+through+Diffraction,+Control+number:+348+Session:+JTuA+-+Joint+FiO/LS+Poster+Session+I+Pres.+number:+JTuA39.">Locomotive Analysis of C. Elegans through Diffraction, Control number: 348 Session: JTuA - Joint FiO/LS Poster Session I Pres. number: JTuA39.</a> , (2010).</li><li>Kim, N., Dempsey, C. M., Kuan, C. J., Zoval, J., O'Rourke, E., Ruvkun, G., Madou, M. J., Sze, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gravity+fource+transduced+by+the+MEC-4/MEC-10+DEG/EnaC+channel+modulates+DAF-16/FoxO+activity+in+Caenorhabditis+elegans.">Gravity fource transduced by the MEC-4/MEC-10 DEG/EnaC channel modulates DAF-16/FoxO activity in Caenorhabditis elegans.</a> Genetics. 177, 835-845 (2007).</li><li>Park, S., Hwang, H., Nam, S. -. W., Martinez, F., Austin, R. H., Ryu, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Caenorhabditis+elegans+locomotion+in+a+structured+microfluidic+environment.">Enhanced Caenorhabditis elegans locomotion in a structured microfluidic environment.</a> PLOS One. 3, e2550 (2008).</li><li>Keller, H. E., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+Lenses+for+Confocal+Microscopy.">Objective Lenses for Confocal Microscopy.</a> Handbook of Biological Confocal Microscopy. , 145-161 (2006).</li><li>Martin, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TECHNICAL OPTICS. II, (1950).</li></ol>

No conflicts of interest declared.

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:
<img alt="Equation 1" src="/files/ftp_upload/4412/4412eq1.jpg" /> 
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.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>543 nm HeNe Laser</td>
 <td>Melles Griot</td>
 <td>LGX1</td>
 <td>Any laser in the 			visible range with less than
5 mW can be used.</td>
 </tr>
 <tr>
 <td>2 Front Surface Aluminum Mirrors</td>
 <td>Thorlabs</td>
 <td> PF10-03-F01</td>
 <td></td>
 </tr>
 <tr>
 <td>High Speed Exilim Camera</td>
 <td>Casio</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Quartz Cuvette</td>
 <td>Starna Cells</td>
 <td>21/G/5</td>
 <td></td>
 </tr>
 <tr>
 <td>LoggerPro (Software)</td>
 <td>Vernier</td>
 <td></td>
 <td><a href="http://www.vernier.com/products/software/lp/" target="_blank">http://www.vernier.com/products/software/lp/</a></td>
 </tr>
 <tr>
 <td>Mathematica 8</td>
 <td>Wolfram</td>
 <td></td>
 <td><a href="http://www.wolfram.com/" target="_blank">http://www.wolfram.com/</a></td>
 </tr>
 </tbody>
</table>

quantitative locomotion study of freely swimming micro-organisms using laser diffraction

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.

As an example, we studied C. elegans in a quartz cuvette 1 cm wide, 5 mm thick and 4 cm tall cuvette. Sampling a single worm using video analysis, the average swimming frequency obtained from video analysis in a 5 mm thick cuvette is about 2.5 Hz (Figure 4). Similarly, sampling a single worm using the real time data acquisition method, we obtain a swimming frequency of about 2.7 Hz (Figure 5), using the digital oscilloscope (PicoScope).This procedure can be repeated for many worms. A detailed study of freely swimming worms revealed an average swimming frequency of 2.37 Hz in a 5 mm cuvette.6 As expected, the swimming frequency is higher than that for a crawling worm (~.8 Hz).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.) allows for the modeling of swimming diffraction patterns with the help of worm images obtained with a conventional microscope. The modeled diffraction patterns are used to simulate a swim cycle of the C. elegans (Figure 6). A successful model consists of physically feasible successive swim patterns matching the swimming frequencies. The worm should be in the same shape at the end of a swim cycle as it was in the beginning of a swim cycle.
<img alt="Figure 1" src="/files/ftp_upload/4412/4412fig1.jpg" /> 
Figure 1. A green HeNe laser was used to create a dynamic diffraction pattern using live C. elegans. This diffraction pattern was filmed at 240 fps.
<img alt="Figure 2" src="/files/ftp_upload/4412/4412fig2.jpg" /> 
Figure 2. Drawing a black dot increases absorption of the transmitted beam. Saturation of the camera due to scattered light is reduced and the diffraction pattern becomes visible.
<img alt="Figure 3" src="/files/ftp_upload/4412/4412fig3.jpg" /> 
Figure 3. Screen shot of the video analysis software (Logger Pro) with a worm diffraction pattern that is being tracked. <a href="https://www.jove.com/files/ftp_upload/4412/4412fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4412/4412fig4.jpg" /> 
Figure 4. Video data corresponding to the swim cycle of a nematode in a 5 mm cuvette. The curve fit reveals a swimming frequency of ~ 2.5 Hz.
<img alt="Figure 5" src="/files/ftp_upload/4412/4412fig5.jpg" /> 
Figure 5. Real time data corresponding to the swim cycle of a nematode in a 5 mm cuvette. The curve fit reveals a swimming frequency of ~ 2.7 Hz.
<img alt="Figure 6" src="/files/ftp_upload/4412/4412fig6.jpg" /> 
Figure 6. The top row represents the actual diffraction patterns and is matched to the modeled diffraction patterns in the bottom row. The modeled diffraction patterns were produced using worms on a microscope slide (middle row).

Watch this Scientific Journal Video about Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction at JoVE.com

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

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 ...

quantitative-locomotion-study-freely-swimming-micro-organisms-using

Research

JoVE Journal

Bioengineering

11.4K Views.  Vassar College. 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.

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

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...