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Another use for AVS. Investigating Plant Growth using AVS. Presentation to the UK AVS and Uniras User Group Meeting University of Birmingham November 8th 1999 Dr. R. P. Fletcher University of York. A report on work done by:. Dr. S. M. Bougourd, University of York

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Another use for AVS

UAUUG Birmingham


Investigating plant growth using avs

Investigating Plant Growthusing AVS

Presentation to the

UK AVS and Uniras User Group Meeting

University of Birmingham

November 8th 1999

Dr. R. P. Fletcher

University of York


A report on work done by
A report on work done by:

  • Dr. S. M. Bougourd, University of York

  • Dr. C. L. Wenzel, University of York

  • … in collaboration with

  • Dr. J . Haseloff, MRC Laboratory of Plant Science, Cambridge

  • …and me

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Outline
Outline

  • Which part of plant growth?

  • Which plant?

  • Why?

  • How?

  • How we use AVS

  • What we want to do

(with AVS?)

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Which part of the plant
Which part of the plant?

  • Above or below ground?

  • For us … below

  • This means the ROOTS

  • Specifically …

  • How do the root cells differentiate?

  • Which cells elongate and why?

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Which plant
Which Plant?

  • Aribidopsis thalinana

  • A member of the brassica family

  • Also known as:

  • Thale cress

  • or …

  • Mouse Eared cress

  • It’s a weed!

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Just so you know what it looks like
Just so you know what it looks like

Whole Plant

Flowers

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And there s more
… and there’s more ...

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Why use this weed
Why use this weed?

  • Small size and rapid life cycle

  • Prolific seed production

  • Simple genome

  • Many mutants and transformed populations

  • Perturb the behaviour of targeted cells

  • Monitor phenotypic expression

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The goal
The goal

“To understand the genetical and cellular interactions that co-ordinate the development of the root meristem”

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How we acquire the data
How we acquire the data

  • Roots are visualised using Laser Scanning Confocal Microscopy (LSCM)

  • Also known as Confocal Scanning Laser Microscopy (CSLM)

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Quick tutorial on clsm
Quick tutorial on CLSM

  • A scanning laser beam is focussed onto a fluorescent specimen

  • Mixture of reflected and emitted light is captured by a photo-multiplier via beam splitter

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Tutorial continued
Tutorial continued

  • Arranged so only the emitted light enters the photo-multiplier

  • A confocal aperture (pin-hole) placed in front of the photo-multiplier

  • The effect is to only allow emitted light from the “in focus” area to pass into the photo-multiplier

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Principles
Principles

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Typical system
Typical System

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The real thing
The real thing

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Interesting problem
Interesting problem?

  • Its all very well staining specimens so that they fluoresce, but ...

  • We need to see whole root tip, not just sections and ...

  • We need same level of staining throughout, but ...

  • Normal stains kill the cells and are bleached by the laser scanning process

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The solution
The Solution!

  • Everybody’s buzzword these days

  • Genetic Modification!

  • The idea is to get the plant to manufacture its own fluorescent stain

  • So, we will borrow a gene from somewhere else in the natural world

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Obtaining the gene
Obtaining the Gene

  • Plenty of naturally fluorescent plants and animals out there

  • The oceans are full of them

  • The jellyfish, Aequorea victoria, from the Pacific Ocean has been used.

  • They produce the protein, Green Fluorescent Protein (GFP).

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Wibbly wobbly jellyfish
Wibbly Wobbly Jellyfish

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Pretty pretty
Pretty, Pretty

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And they can swim
… and they can swim

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Getting the gene into the plant
Getting the Gene into the Plant

  • A quick tutorial about genetic modification

  • … gene extracted ... put in vector, a soil bacterium … isolate “infected cells” and regenerate whole plants.

  • Can even link “instructions” to the GFP gene to make the plant only produce the fluorescent protein in certain parts of the plant

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A single image
A Single Image

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An image stack
An Image Stack

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Getting this stack into avs
Getting this Stack into AVS

  • The old nutshell!

  • First, find out the format of the Bio-Rad PIC files.

  • Hunt round for some “v” … IAC maybe?

  • Got some code, but was developed for ALPHA

  • Had “endian” problems

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Fix the code and develop visualisation modules
Fix the code and develop Visualisation Modules

  • Fix the “v” code to read the correct “endian-ness” of the data

  • Amount of data can be a problem

  • 512 * 768 * stack size (loadsa data!)

  • Hope the decimation modules in Version 5 will help here

  • Even running on 350Mhz PC or SGI 02, both with 128 Mb of memory, AVS is slow

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Using avs to view along a different axis
Using AVS to view along a different axis

tip

Single frame

Back a bit

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Movie view along the axis
Movie view along the axis

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What are we actually seeing
What are we actually seeing?

  • GFP fluorescing in the cell walls

  • The higher the intensity the more GFP

  • Would be better to invert the images

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Inverted image stack
Inverted Image Stack

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Non invasive non lethal
Non-invasive non-lethal

  • The use of the GFP means we can study the plant root growth “in vivo”

  • The aim is to understand the fate of the different root tip cells

  • Need to find a way to “tag” cells from one image stack to another

  • Time dimension

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Cell fate
Cell fate?

Divide

Root tip cell

Differentiate

Some elongate and grow

Some just grow

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Need to see 3d view
Need to see 3D view

  • 3D reconstruction from “cloud of points”

  • Need to “cut away”

  • Need to “identify” cells

  • Need to track “fate”

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Preliminary 3d investigation
Preliminary 3D Investigation

Orthoslices

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Animate the orthoslices
Animate the orthoslices

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Complex network
Complex Network

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Add in some real 3d
Add in some “real” 3D

Volume

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Another view
Another View

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Animated volume cutaway
Animated volume cutaway

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So just how useful is avs
So just how useful is AVS?

  • Using AVS can really help to see the data

  • Reconstructing different orthogonal views

  • Volume visualisation will help

  • Data volume is a problem on “small” systems

  • Decimation routines will be welcome

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Future work
Future Work

  • Need to work out how to mark cell volumes in order to track specific cells

  • Create new fields from marked data

  • Visualise these “new” fields with time “n” images

  • Difference frames may help from time “n” to time “N+1”

  • Big data processing effort here needed

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That s all folks
THAT’S ALL FOLKS

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