Another use for AVS
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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

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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Network for preliminary viewing

Network for preliminary viewing

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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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