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

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.

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

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.

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.

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

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

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

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

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

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.

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

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

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

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.

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

Fig20: Fluorescent image taken using UV lamp& Dinolite

Fig21: Image taken using Red light

Fig22: Binary Image

Fig23: Threshold Image

Fig24: Setup

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

Fig27: Parameters measured by Image J, wormtrack plugin

Fig28: Aspect ratio vs frame and worm locomotion track

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