November 8th 1999UAUUG Birmingham1. Investigating Plant Growth using AVS Presentation to the UK AVS...

November 8th 1999 UAUUG Birmingham 1

Investigating Plant GrowthInvestigating Plant Growth

using AVSusing AVSPresentation to thePresentation to the

UK AVS and Uniras User Group MeetingUK AVS and Uniras User Group Meeting

University of BirminghamUniversity of Birmingham

November 8th 1999November 8th 1999

Dr. R. P. FletcherDr. R. P. Fletcher

University of YorkUniversity of York


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

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

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

• … … in collaboration within collaboration with

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

• ……and meand me


OutlineOutline

• Which part of plant growth?Which part of plant growth?

• Which plant?Which plant?

• Why?Why?

• How?How?

• How we use How we use AVSAVS

• What we want to do What we want to do (with (with AVS?)AVS?)


Which part of the plant?Which part of the plant?

• Above or below ground?Above or below ground?

• For us … belowFor us … below

• This means the ROOTSThis means the ROOTS

• Specifically …Specifically …

• How do the root cells differentiate?How do the root cells differentiate?

• Which cells elongate and why?Which cells elongate and why?


Which Plant?Which Plant?

• Aribidopsis thalinanaAribidopsis thalinana

• A member of the brassica familyA member of the brassica family

• Also known as:Also known as:

• Thale cressThale cress

• or …or …

• Mouse Eared cressMouse Eared cress

• It’s a weed!It’s a weed!


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

FlowersFlowers

Whole PlantWhole Plant


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


Why use this weed?Why use this weed?• Small size and rapid life cycleSmall size and rapid life cycle

• Prolific seed productionProlific seed production

• Simple genome Simple genome

• Many mutants and transformed populationsMany mutants and transformed populations

• Perturb the behaviour of targeted cells Perturb the behaviour of targeted cells

• Monitor phenotypic expressionMonitor phenotypic expression


The goalThe goal

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


How we acquire the dataHow we acquire the data

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

• Also known as Confocal Scanning Laser Also known as Confocal Scanning Laser Microscopy (CSLM)Microscopy (CSLM)


Quick tutorial on CLSMQuick tutorial on CLSM

• A scanning laser beam is focussed onto a A scanning laser beam is focussed onto a fluorescent specimenfluorescent specimen

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


Tutorial continuedTutorial continued• Arranged so only the emitted light enters Arranged so only the emitted light enters

the photo-multiplierthe photo-multiplier

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

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


PrinciplesPrinciples


Typical SystemTypical System


The real thingThe real thing


Interesting problemInteresting problem?

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

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

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

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


The SolutionThe Solution!

• Everybody’s buzzword these daysEverybody’s buzzword these days

• Genetic Modification!Genetic Modification!

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

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


Obtaining the GeneObtaining the Gene

• Plenty of naturally fluorescent plants and Plenty of naturally fluorescent plants and animals out thereanimals out there

• The oceans are full of themThe oceans are full of them

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

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


Wibbly Wobbly JellyfishWibbly Wobbly Jellyfish


Pretty, PrettyPretty, Pretty


… … and they can swimand they can swim


Getting the Gene into the PlantGetting the Gene into the Plant• A quick tutorial about genetic modificationA quick tutorial about genetic modification

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

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


A Single ImageA Single Image


An Image StackAn Image Stack


Getting this Stack into AVSGetting this Stack into AVS

• The old nutshell!The old nutshell!

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

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

• Got some code, but was developed for Got some code, but was developed for ALPHAALPHA

• Had “endian” problemsHad “endian” problems


Fix the code and develop Fix the code and develop Visualisation ModulesVisualisation Modules

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

• Amount of data can be a problemAmount of data can be a problem

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

• Hope the decimation modules in Version 5 Hope the decimation modules in Version 5 will help herewill help here

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


Network for preliminary viewingNetwork for preliminary viewing


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

Single frameSingle frame

tiptip

Back a bitBack a bit


Movie view along the axisMovie view along the axis


What are we actually seeing?What are we actually seeing?• GFP fluorescing in the cell wallsGFP fluorescing in the cell walls

• The higher the intensity the more GFPThe higher the intensity the more GFP

• Would be better to invert the imagesWould be better to invert the images


Inverted Image StackInverted Image Stack


Non-invasive non-lethalNon-invasive non-lethal

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

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

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

• Time dimensionTime dimension


Cell fate?Cell fate?

Root tip cellRoot tip cell

DivideDivide

DifferentiateDifferentiate

Some elongate and growSome elongate and growSome just growSome just grow


Need to see 3D viewNeed to see 3D view

• 3D reconstruction from “cloud of points”3D reconstruction from “cloud of points”

• Need to “cut away”Need to “cut away”

• Need to “identify” cellsNeed to “identify” cells

• Need to track “fate”Need to track “fate”


Preliminary 3D InvestigationPreliminary 3D InvestigationOrthoslicesOrthoslices


Animate the orthoslicesAnimate the orthoslices


Complex NetworkComplex Network


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

VolumeVolume


Another ViewAnother View


Animated volume cutawayAnimated volume cutaway


So just how useful is AVS?So just how useful is AVS?• Using AVS can really help to see the dataUsing AVS can really help to see the data

• Reconstructing different orthogonal viewsReconstructing different orthogonal views

• Volume visualisation will helpVolume visualisation will help

• Data volume is a problem on “small” Data volume is a problem on “small” systemssystems

• Decimation routines will be welcomeDecimation routines will be welcome


Future WorkFuture Work• Need to work out how to mark cell volumes Need to work out how to mark cell volumes

in order to track specific cellsin order to track specific cells

• Create new fields from marked dataCreate new fields from marked data

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

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

• Big data processing effort here neededBig data processing effort here needed


THAT’S THAT’S ALL ALL

FOLKSFOLKS

November 8th 1999UAUUG Birmingham1. Investigating Plant Growth using AVS Presentation to the UK AVS...

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