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Tracking Movement Behavior of Multiple Worms on Food

1. has expired Contact National Instruments to purchase a runtime license Problem Step 5 ii The tracker cannot find the worms Solution Consider the following 1 Make sure there is good contrast between the worm and the background With the optical setup as described contrast should be 50 2 Make sure the lighting is bright but does not saturate the camera 3 Alter the tracking parameters such as intensity threshold within the MWT Problem Step 5 ii The tracker finds many tiny worms as well as the adult worms Solution Consider the following 1 Increase the minimum object size in the MWT 2 Raise the worms at a colder temperature or run the experiment before any eggs have hatched 3 Select and transfer adult worms to the plate instead of waiting for them to grow from eggs Allow at least 2 h to elapse between transfer and recording if possible Worm trackers fall into two general types Single worm trackers follow individual animals at relatively high magnification keeping them in the field of view by moving the camera or a motorized stage Such systems have been used to study aspects of locomotion such as turning and speed Pierce Shimomura et al 1999 as well as body posture and morphology Karbowski et al 2008 MWTs track multiple individuals in populations by necessity at lower magnification and usually focus on locomotion Cite this article as Cold Spring Harbor Protoc 2011 doi 10 1101 pdb prot067025 1485
2. 108 1019 1029 Buckingham SD Sattelle DB 2009 Fast automated measurement of nematode swimming thrashing without morphometry BMC Neuro sci 10 84 Karbowski J Schindelman G Cronin CJ Seah A Sternberg PW 2008 Systems level circuit model of C elegans undulatory locomotion Math ematical modeling and molecular genetics J Comput Neurosci 24 253 276 Pierce Shimomura JT Morse TM Lockery SR 1999 The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis J Neurosci 19 9557 9569 Ramot D Johnson BE Berry TL Jr Carnell L Goodman MB 2008 The Par allel Worm Tracker A platform for measuring average speed and drug induced paralysis in nematodes PLoS One 3 e2208 Roussel N Morton CA Finger FP Roysam B 2007 A computational model for C elegans locomotory behavior Application to multiworm tracking IEEE Trans Biomed Eng 54 1786 1797 Simonetta SH Golombek DA 2007 An automated tracking system for Cae norhabditis elegans locomotor behavior and circadian studies appli cation J Neurosci Methods 161 273 280 Tsechpenakis G Bianchi L Metaxas D Driscoll M 2008 A novel compu tational approach for simultaneous tracking and feature extraction of C elegans populations in fluid environments IEEE Trans Biomed Eng 55 1539 1549 Yemini E Kerr RA Schafer WR 2011a Preparation of samples for single worm tracking Cold Spring Harb Protoc doi 10 1101 pdb prot066993 Yemini E Kerr RA Schafer WR 20
3. 11b Illumination for worm tracking and behavioral imaging Cold Spring Harb Protoc doi 10 1101 pdb prot067009 Cite this article as Cold Spring Harbor Protoc 2011 doi 10 1101 pdb prot067025 1487 Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by Cold Spring Harbor Laboratory Press CSH fon Cold Spring Harbor Protocols Tracking Movement Behavior of Multiple Worms on Food Eviatar Yemini Rex A Kerr and William R Schafer Cold Spring Harb Protoc doi 10 1101 pdb prot067025 Email Alerting Receive free email alerts when new articles cite this article click here Service Subject Browse articles on similar topics from Cold Spring Harbor Protocols Categories Behavioral Assays 47 articles C elegans 38 articles Image Analysis 109 articles Imaging for Neuroscience 278 articles Imaging Microscopy general 545 articles In Vivo Imaging 287 articles Video Imaging Time Lapse Imaging 164 articles To subscribe to Cold Spring Harbor Protocols go to hitp cshprotocols cshlp org subscriptions 2011 Cold Spring Harbor Laboratory Press
4. a with the companion command line Choreography program as described in the MWT user manual i Supply the pixel size measured in Step 2 with the p option e g p 0 024 for a setup with 24 um pixel which is approximately what it should be with the imaging setup described ii Request the output parameters desired e g o Ns will produce a table containing the number of worms and the mean speed of those worms at each time point See the users guide for more detail TROUBLESHOOTING DISCUSSION Problem Steps 4 and 5 ii The worms are obscured by condensation on the lid of the plate Solution Consider the following 1 Check the temperature in the room if the lid of the plate is colder than the base condensation will appear The light source box may need to be moved farther away or downwind instead of upwind the light plate itself should heat the base enough to cause fogging 2 Remove the lid from the Petri dish Be aware however that worms are highly sensitive to drying and long assays gt 1 h will be measuring predominantly a drying related response unless the humidity is very high Problem Step 5 ii The MWT fails to show any image of worms Solution Consider the following 1 Check that the Measurement and Automation Explorer tool can find the camera and view images from it then close the Explorer and try the MWT again 2 The National Instrument Vision Runtime License has not been activated and the trial period
5. et the camera to 8 bit resolution 2 Measure the size of a pixel in millimeters View a ruler with the camera and compute for example number of pixels in 1 in 25 4 3 Prepare worms for tracking i Seed one plate of NGM with 50 pL of NGM media spread to cover the center of the plate ii Leave plate overnight iii Select five young adult hermaphrodites and transfer them onto the NGM plate Incubate them for 3 h to lay eggs and then remove the adult hermaphrodites iv Allow the eggs to grow to young adults 72 96 h 4 Place the plate in position for imaging with the lid on making sure that the worms are in focus with the camera that the brightness across the field of view varies by no more than 40 and that the brightest pixels are below saturation a good target is 200 220 out of 255 See Troubleshooting 5 Run the tracker i In the MWT software select a target directory an experiment title and a duration to record ii Verify in the image window that the MWT is detecting the worms See Troubleshooting iii Press Go and wait for capture to complete Cite this article as Cold Spring Harbor Protoc 2011 doi 10 1101 pdb prot067025 feos Cold Spring Harbor Protocols PROTOCOLS ooo www cshprotocols org Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by Cold Spring Harbor Laboratory Press Multiworm Tracking 6 Analyze the resulting dat
6. fos Cold Spring Harbor Protocols PROTOCOLS ooo www cshprotocols org Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by Protocol Cold Spring Harbor Laboratory Press Tracking Movement Behavior of Multiple Worms on Food Eviatar Yemini Rex A Kerr and William R Schafer MATERIALS Neurobiological research in genetically tractable organisms relies heavily on robust assays for behav ioral phenotypes The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quan titatively Forward genetic screens and screens of drug libraries require high throughput phenotyping a task traditionally incompatible with manual scoring of quantitatively varying behaviors High throughput automated analysis of C elegans movement behavior is now possible with several different tracking software packages The Multiworm Tracker MWT described here is designed for high throughput analysis it can record dozens of worms simultaneously at 30 frames per second for hours or days at a time This is accomplished by performing all image analysis in real time saving only the worm centroid bearing and outline data to the disk To simplify image processing the system focuses only on worms that have moved and detects and discards worms that are touching rather than trying to isolate the
7. ing Harbor Protoc 2011 doi 10 1101 pdb prot067025 1483 feos Cold Spring Harbor Protocols PROTOCOLS www cshprotocols org ooo Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by E Yemini et al METHOD 1484 Cold Spring Harbor Laboratory Press FIGURE 1 Hardware A The stage composed of x and y axis actuators and linear translation stages B the camera C the sample a worm on an agar Petri dish D a diffuser above a collimating Fresnel lens to provide uniform illumination E a high intensity red light emitting diode LED for illumination F coarse and fine focusing knobs to focus the sample in view of the camera G a rigid cage to center the illumi nation above the camera and eliminate vibrations caused by moving the stage Multiworm Tracker software http sourceforge net projects mwt National Instruments NI PCle 1427 image capture card Petri dishes gt 5 cm Plate holder 15 cm above light 30 cm below camera Rodenstock 60 mm f 4 0 Rodagon lens with MFB F mount adapter modular focus block 25 mm M39 x0 75 lens adapter Schott ACE light source with fiber optic backlight 4 x 4 88 in The complete hardware setup including stage camera and light source is shown in Figure 1 1 Set up the recording system the Multiworm Tracker MWT as described in its user manual and use the Measurement and Automation Explorer tool to s
8. m computationally Because the software is entirely automated proto cols can run unattended once the worms have been placed and the software has been started The MWT does not save images for later analysis but behavior can be validated manually with a companion analysis tool that replays recorded body postures This protocol describes a basic basal movement assay on food using the MWT similar protocols apply to related assays and to similar multiple animal track ers The protocol can be extended to a variety of assays ranging from tap response to chemotaxis Reagents Equipment It is essential that you consult the appropriate Material Safety Data Sheets and your institution s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol RECIPE Please see the end of this article for recipes indicated by lt R gt Additional recipes can be found online at http cshprotocols cshlp org site recipes C elegans Bristol N2 adult hermaphrodites Nematode growth medium NGM lt R gt For liquid medium omit the agar OP50 Escherichia coli Computer with at least 2 GB RAM and 1680 x 1050 or 1600 x 1200 monitor Dalsa Falcon 4M30 camera 2352 x 1728 at 31 Hz with M42 to F Mount lens adapter Adapted from Imaging in Neuroscience ed Helmchen and Konnerth CSHL Press Cold Spring Harbor NY USA 2011 2011 Cold Spring Harbor Laboratory Press Cite this article as Cold Spr
9. not automatically detect qualitative phenotypes often scored manually e g kinker so a quantitative proxy must be discovered For example for tap habituation assays reversal distance is a typical metric Broster and Rankin 1994 but is relatively difficult to extract computa tionally However for longer interstimulus tap intervals increase in speed of animals on food is a good alternative metric for response to tap Fig 2A the transient increase in speed due to manipu lation of the plate before the beginning of the experiment is typical When beginning a new assay it is valuable to compare manually scored behaviors with automated results To assist in this the Chor eography program contains a map option that brings up a map browser interface which allows one to replay the movements of the tracked worms Fig 2B The analysis parameters and exclusion cri teria used in the results shown in Figure 2 are listed in Table 1 RECIPE Nematode Growth Medium NGM Amount to Final Reagent add for 1 L concentration Agar 17 0 g 1 7 w v NaCl 2 9g 50 mM Peptone 2 5g 0 25 w v CaCl 1 m 1 mL 1 mM Cholesterol 5 mg mL 1mL 5 pg mL KH PO 1 m 25 mL 25 mM MgSO 1 m 1 mL 1 mM Mix the first three reagents and autoclave After the mixture is cool add the last four reagents REFERENCES Broster BS Rankin CH 1994 Effects of changing interstimulus interval during habituation in Caenorhabditis elegans Behav Neurosci
10. urons The few minor changes this protocol may require involve using higher magnification to view tracked neurons and inverting the binary thresholding to discover bright neurons on a dark background worms are usually imaged in bright field where they are darker than their background A variety of methods for recording many worms simultaneously have been developed recently Image analysis methods used on single worms can be applied to high resolution images of whole plates of worms to give similar data some individual measurements are noisier or impossible but col lecting large sample sizes is much faster Ramot et al 2008 have created the Parallel Worm Tracker a suite of three related open source programs that acquire image data perform basic image processing to identify worms and produce tracks of the worms s centroid positions and analyze those tracks to compute linear and angular velocity Similarly Roussel et al 2007 and Tsechpenakis et al 2008 have created software that analyzes stored image data with more sophisticated algorithms that account for noise and adjacent worms and can provide basic postural data in addition to centroid pos ition Other efforts at simultaneous worm tracking have focused on worms in 96 well plates with no Simonetta and Golombek 2007 or very minimal Buckingham and Sattelle 2009 image processing or on robustly detecting pairs of worms in contact for detailed assays The MWT discussed here is conceptuall
11. www cshprotocols org Sosy Cold Spring Harbor Protocols PROTOCOLS too Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by E Yemini etal 1486 Cold Spring Harbor Laboratory Press gt o N w Speed mm sec gt Time min FIGURE 2 A tap habituation assay performed on the MWT A Average speed of N worms over 10 min with plate tap delivered at 5 min and 3x min thereafter 15 taps total Black line is mean standard error in gray N 45 50 animals per time point one plate B Manual validation of tap response with worm position 1 0 sec before top at center and 1 0 sec after bottom a tap Colored dots are centroid positions colored by speed where red is slower and yellow faster one dot is drawn every 33 msec features Generally speaking single worm trackers allow one to measure a greater number of par ameters whereas MWTs offer much higher efficiency and throughput A discussion of the principles of single worm tracking along with a protocol can be found in Preparation of Samples for Single Worm Tracking Yemini et al 2011a A protocol is also available for Illumination for Worm Track ing and Behavioral Imaging Yemini et al 2011b This protocol describes the use of the software package MWT and discusses possible applications for genetic analyses of behavior Neuronal tracking uses precisely the same algorithm to search for labeled ne
12. y very similar to the trackers described by Ramot et al 2008 and Tsechpenakis et al 2008 save for its focus on real time image processing Multiworm tracking is possible with a wide variety of cameras and imaging speeds For the MWT the primary two requirements are that worms must overlap over at least 50 of their bodies from one frame to the next and must be large enough to cause a clear change in brightness when moving into a new area of the plate Thus frame rates slower than 0 5 Hz for crawling or 10 Hz for swimming because the worm swings side to side will tend to fail because of excessive movements magnifi cations that result in pixels that span more than 60 um also tend to give substandard results TABLE 1 Choreography analysis parameters used in the example results shown in Figure 2 Speed averaging Object min Object min Pixel size window duration movement Output quantities 0 0243 mm sec 0 2 sec 20 sec 2 body lengths ss s speed mean standard deviation number of samples Cite this article as Cold Spring Harbor Protoc 2011 doi 10 1101 pdb prot067025 fess Cold Spring Harbor Protocols PROTOCOLS ooo www cshprotocols org Downloaded from http cshprotocols cshlp org at MRC LAB OF MOL BIOLOGY on February 4 2015 Published by Cold Spring Harbor Laboratory Press Multiworm Tracking Converting a single worm manual assay to an automated assay can be a considerable amount of work The MWT does

Tracking Movement Behavior of Multiple Worms on Food

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