This document describes a micro project to develop a C. elegans tracking system using a digital microscope and tracking software. The project aims to analyze the locomotion of C. elegans to better understand the mechanisms underlying its forward movement. A Dino-Lite digital microscope will be used to record videos of C. elegans on an agar plate. Tracking software like WormLab and ImageJ will then analyze the videos to track the worm's movement and calculate metrics like number of bends and directional changes. The goal is to help elucidate the neural control of C. elegans locomotion and how it mediates the worm's foraging and avoidance behaviors.

1. C. Elegans tracking system
Micro project report
16/9/2015
Guide: Dr. Anoop Kumar T.
Scientist F
Molecular Medicine Lab
Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum
By
Yanamala Vijay Raj
Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum

2. ABSTRACT:
The popular model organism Caenorhabditis elegans is a tiny nematode worm
with a largely invariant nervous system, consisting of exactly 302 neurons with
known connectivity. The worm is capable of a surprisingly rich repertoire of
behaviors including navigation and foraging, mating, learning, and even
rudimentary social behavior. Indeed, this humble worm provides us with the first
tangible possibility of understanding the complex behaviors of an organism from
the genetic level, right up to the system level.
Moreover, the behavioral roles of many of these neurons can be uncovered using
experimental setup that can track the locomotion of worm. Despite its small size
and the apparent simplicity of the underlying nervous system, The focus of this
project is on the locomotion tracking system which is motivated part by the fact
that most, if not all, of the worm’s behaviors are mediated by some form of
locomotion. The main objective of this project is to help elucidate the mechanisms
underlying C. elegans forward locomotion.

3. CONTENT TABLE
1) Introduction
1.1 C-Elegans
1.2 Types of Microscopy
2) Methods and Materials
2.1 Nematodes
2.2 Dino-capture Digital Microscope
2.3 List of tracking Software
2.4 Experiment set up
3) Results and Discussions
3.1 Problems identified
3.2 Possible solutions
3.3 Image J
3.4 Conclusions

4. 1. INTRODUCTION
1.1 C-Elegans:
Model organisms are certain species of organisms that are widely used for
research purpose in biological field. Since they are easy to breed and maintain
most of experimental studies are done on them. Certain fields like toxicology,
Neurology, genetics direly need model organisms to test and experiment on their
hypothesis.
The nematode C-Elegans (Caenorhabditis elegans) is widely used for studies of
nervous system function and developmental biology. It is approximately 1 μm in
length and feeds on bacteria like E-coli. It has a simple nervous system which is
well characterized. It has just 302 neurons, thereby makes it a attractive model
organism for neurological studies. Despite its anatomical simplicity, the C. elegans
nervous system mediates diverse and intricate patterns of behavior. The sense
organs of C. elegans are capable of perceiving and responding to a wide range of
environmental conditions, including heavy and light touch, temperature, volatile
odorants, food and other nematodes.
Fig1: GFP tagged C-Elegans
Thank to this humble creature we now have tangible possibility of understanding
the complex behaviors of an organism from the genetic level, right up to the
system level.

5. 1.2 Types of Microscopy:
Microscopes are biomedical instruments that are mostly used for examination of
specimens that are invisible to naked eye. From light microscopy to latest
fluorescent microscopy, microscopes have seen tremendous changes in it mode of
operation. By looking at different types of microscope and its mode of operation,
we will conclude which mode of visualization should be deployed to visualize
microorganisms like C-Elegans.
Light microscopy:
The type of microscopes which uses light as source of energy to visualize
specimens are called light microscopy.
Antonie van Leeuwenhoek was first to pioneer in using microscopy techniques to
biology. He is credited with father of microbiology for his tremendous contribution
in improving microscope.
The single-lens microscopes of Van Leeuwenhoek were relatively small devices.
They are used by placing the lens very close in front of the eye, while looking in
direction of the sun. The other side of the microscope had a pin, where the sample
was attached in order to stay close to the lens. There were also three screws that
allowed to move the pin, and the sample, along three axes.
Fig2: Antonie van Leeuwenhoek Microscope
The light microscope, is a type of microscope which uses visible light and a
system of lenses to magnify images of small samples.

6. Fig3: Optical microscope
Simple microscopes are microscopes that makes use of single convex lens to create
an enlarged virtual image. They are not capable of high magnification.
Digital microscopes are variants of simple microscope with camera mounted on it
to visualize sample on computers. USB digital microscopes are also available.
Fig4: USB Microscope

7. Electron Microscopy:
With the advancement in physics, few scientists started wondering, why
electrons can’t be used for imaging purpose. This though gave rise to electron
microscopy. Images are based on secondary X-rays emission from samples.
Transmission electron microscopy and scanning electron microscopy are the main
types of electron microscopy.
Phase Contrast Microscopy:
For deep visualizing of transparent organisms, traditional microscopes can’t be
used. New techniques have been constantly employed to increase the contrast of
the sample.
Among them fluorescent techniques and phase contrast techniques have gained
importance. The phase contrast optics of a microscope is able to convert the
differences in the refractive index into a difference in brightness.
Right light or green light is often used in phase contrast microscopy.
Fig5: Image contrast enhanced by Fluorescent techniques
Fig6: Image contrast enhanced by inverse phase contrast microscopy

8. The above pictures clearly shows the difference between bright field and phase
contrast microscopy images. Now gaining sufficient theoretical knowledge in
microscopy we planned to build an experimental setup for analyzing the
movements of C-Elegans.
2) Methods and Materials
2.1 Nematodes:
Standard methods for classifying the behavioral patterns of mutant
Caenorhabditis elegans rely on human observation and are therefore subjective.
Behavioral assays in this organism, particularly in more complex behaviors such as
locomotion, are often highly imprecise and important
The main objective of this micro project is to build an experimental setup to
visualize and record the worm on petri plate, and later analyze the video with
software thereby elucidating locomotion of it. While the underlying neural control
obviously plays a major role in locomotion.
The worm’s locomotion is of particular interest in project, due to its involvement
in higher level behaviors, as well as the fact that it is directly observable and easily
quantifiable. Despite the small underlying circuit, locomotion is an adaptive
behavior that changes significantly depending on the worm’s environment and
allows it to navigate effectively. In the laboratory C. elegans worms are typically
grown in petri dishes containing a layer of agar gel. The gel is quite firm, and
worms tend to lie on the surface rather than burrowing into it. The locomotion
behavior observed under these conditions is referred to as crawling.
Fig7: Image of c elegans on media

9. BEHAVIOR OF WORM TOWARDS STIMULUS AND IT LOCOMATION:
Despite its small size and relative anatomical simplicity, C. elegans is capable of
a remarkably rich repertoire of behaviors which, although simpler, have close
parallels in larger animals. Like all animals the worm exists to reproduce. Since it
is hermaphrodite, it takes no active role in mating, and thereby its primary goal
becomes survival which, in turn, necessitates eating and threat avoidance.
The worm exhibits chemotaxis towards chemicals usually associated with food,
while exhibiting a strong avoidance response to certain chemical repellents that are
associated with danger. The worm also has a thermotaxis behavior, which
manifests as a preference for temperatures at which the worm was previously fed
and an avoidance of temperatures at which it was starved. This is also an example
of associative learning in C. elegans.
Similar tactics could be employed to teach worm and experiment on its
associative learning. The no of bends and the directions of its locomotion gives a
good statistical data about it behavior which is directly related to its associated
memory.
Fig8: Direction change

10. 2.2 Dino-capture Digital Microscope:
Dino-Lite are digital microscopes which are basic optical microscopes. It has
USB extension that can be connected to computers. Dinelite software installed on
PC serves as platform to capture and record videos of samples by Dino-Lite
camera microscope.
Fig9: Dino-Lite digital microscope
AM5216ZT Dino-Lite Edge is used in our project. It comes handy with a
movable stand. It has magnification of about 20x to 200x. It also has ultraviolent
light and normal light for sample illumination purpose.
The recorded videos are saved in digital microscope folder in C drive. Generally
videos are saved in flv, wmv formats. Images are saved in various available format

11. 2.3 List of tracking Software
Various software are available in market. While some are free, some are pro
version. As part of project we had evaluated different softwares and shortlisted few
based on experimental set up we had.
The list of softwares which are predominately used for nematode tracking are
1) WormLab (exe),
2) Worm Tracker (Matlab),
3) Worm Tracker 2.0 (labview),
4) Image J,
5) Nemo (Matlab),
6) Maggot tracker.
WormLab:
Womlab software was developed by MBF bioscience lab. It is most used
software cited in research journals. Algorithms deployed automatically detects
worm and created visual matrix overlapping worm and calculates the locomotion
of worm based on the motion analysis of matrix.
Fig10: Wormlab
File is exported by clicking on import image sequence. Brightness of file is
adjusted so that a clear contrast is seen between worm and media. Click on detect
and track button for the software to automatically detect the worm and set the
parameters for tracking.
After the file is executed the results are displayed on Analyze Data tab.

12. NEMO:
Nemo was an algorithm based on Matlab that has GUI interface. It tracks the
motion of worm and gives parameters of location of worm.
Fig11: Nemo
Worm Track:
This software too is matlab based and has user friendly GIU interface. It can
track the locomotion and give statistical data about it.
Fig12: Wormtracker

13. Image J:
It is java based image processing software, which is one of the most famous free
software available on internet. On this software interface many plugins like
WrmTr, Bio-formats, Neuron J can be added and image processing can be done.
Fig13: Image J
Fig14: Import image sequences in Image J

14. 2.4 Experiment set up
With sufficient knowledge of microscope & software to analyze motion
experimental setup is designed.
Salient features of work tracking setup includes
i. Illuminator that can help in imaging worm by creating phase contrast.
ii. Digital microscope to record motion of worm.
iii. Adjust stand to move microscope in three axis.
iv. Stand to hold the sample.
v. Filter for equal intensity of illumination.
vi. Closed environment if possible.
Fig15: Experimental Setup

15. 3. Results and Discussions
3.1 Problems identified
i. Worms are transparent and imaging them need right focus and
magnification.
ii. Proper experiment setup.
iii. Enhancing contrast of the image.
iv. Principle of microscopy to be deployed.
v. User-friendly software for worm tracking.
vi. Source of illumination to use.
3.2 Possible solution
Of all the problems identified enhancing contrast of image is a major constrain. It
is planned to increase contrast by either using fluorescence techniques or refractive
index techniques.
Fluorescence techniques:
Fig16: Fluorescent microscopy
Dino lite microscope comes with infrared light illuminator. Hence the same
camera could be used for imaging fluorescence worms. Thereby glass strip with
thin layer of agar coating was thought to be used for increased magnification of
worm (ie) dinolite microscope gives maximum magnification at closer range of
sample.

16. Fig17: Glass strip setup
Refractive index based:
It is noticed by using red light as source of light in microscopy, the contrast of
image can be drastically improved. As red light has maximum wavelength in
visible spectrum, it can increase contrast of image by phase shift, taking advantage
of refractive index difference between worm and media.
Fig18: Phase shift
By applying basic physics principle to experimental setup imaging with high
contrast is achieved.
Fig19: Normal image of worm on medium

17. Fig20: Fluorescent image taken using UV lamp& Dinolite
Fig21: Image taken using Red light
Fig22: Binary Image

18. Fig23: Threshold Image
Fig24: Setup

19. 3.3 Image J:
It is finalized to use image J for video processing and locomotion tracking of worm
by wormtrack plugin.
Fig25: Image J
o Recorded video in dinolite camera are in flv, wkv formats, which can’t be
imported directly by imageJ.
o The video should be either in uncompressed avi format or image sequence
(JPEG) formats.
o Format-factory software was used to convert videos to avi format which is in
compressed form.
o Therefore virtual dub software was used to convert compressed avi to
uncompressed avi.
o There is a provision to export video as image sequence too.
o Image sequence imported in image J
o Subtraction of pixel intensity done to reduce image noise.
o Image converted to binary image by thresholding.
o Scale is set and wormtrack plugin loaded. Required paramaters are filled in
and run.
Fig 26: Image J tracking results

20. Fig27: Parameters measured by Image J, wormtrack plugin
Fig28: Aspect ratio vs frame and worm locomotion track

21. CONCLUSIONS:
o C.elegans locomotion studied
o Image constrast can be increased by making use of simple physics
techniques.
o Image J and wormlab are finalized to be user-friendly.
o Error in loading file on Image J was solved using virtual dub.
o Converting image to binary image and thresholding of it was learned.
o Locomotion was tracked by wormtracker plugin of ImageJ