Investigating Plant Growth Using AVS Dr. R. P. Fletcher University of York

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Another use for AVS
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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

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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

• 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?

• 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?

• Aribidopsis thalinana
• A member of the brassica family
• Also known as
• Thale cress
• or
• Mouse Eared cress
• Its a weed!

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Just so you know what it looks like
Whole Plant
Flowers
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and theres more ...
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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
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

• 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

• 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

• 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
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Typical System
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The real thing
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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!

• Everybodys 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

• 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
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Pretty, Pretty
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and they can swim
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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
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An Image Stack
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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 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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Network for preliminary viewing
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Using AVS to view along a different axis
tip
Single frame
Back a bit
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Movie view along the axis
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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
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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?
Divide
Root tip cell
Differentiate
Some elongate and grow
Some just grow
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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
Orthoslices
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Animate the orthoslices
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Complex Network
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Add in some real 3D
Volume
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Another View
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Animated volume cutaway
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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

• 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
  N1
• Big data processing effort here needed

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THATS ALL FOLKS