november 8th 1999uauug birmingham1. investigating plant growth using avs presentation to the uk avs...
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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
November 8th 1999 UAUUG Birmingham 3
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
November 8th 1999 UAUUG Birmingham 4
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?)
November 8th 1999 UAUUG Birmingham 5
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?
November 8th 1999 UAUUG Birmingham 6
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!
November 8th 1999 UAUUG Birmingham 7
Just so you know what it looks likeJust so you know what it looks like
FlowersFlowers
Whole PlantWhole Plant
November 8th 1999 UAUUG Birmingham 9
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
November 8th 1999 UAUUG Birmingham 10
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”
November 8th 1999 UAUUG Birmingham 11
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)
November 8th 1999 UAUUG Birmingham 12
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
November 8th 1999 UAUUG Birmingham 13
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
November 8th 1999 UAUUG Birmingham 17
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
November 8th 1999 UAUUG Birmingham 18
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
November 8th 1999 UAUUG Birmingham 19
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).
November 8th 1999 UAUUG Birmingham 23
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
November 8th 1999 UAUUG Birmingham 26
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
November 8th 1999 UAUUG Birmingham 27
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
November 8th 1999 UAUUG Birmingham 28
Network for preliminary viewingNetwork for preliminary viewing
November 8th 1999 UAUUG Birmingham 29
Using AVS to view along a Using AVS to view along a different axisdifferent axis
Single frameSingle frame
tiptip
Back a bitBack a bit
November 8th 1999 UAUUG Birmingham 31
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
November 8th 1999 UAUUG Birmingham 33
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
November 8th 1999 UAUUG Birmingham 34
Cell fate?Cell fate?
Root tip cellRoot tip cell
DivideDivide
DifferentiateDifferentiate
Some elongate and growSome elongate and growSome just growSome just grow
November 8th 1999 UAUUG Birmingham 35
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”
November 8th 1999 UAUUG Birmingham 36
Preliminary 3D InvestigationPreliminary 3D InvestigationOrthoslicesOrthoslices
November 8th 1999 UAUUG Birmingham 42
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
November 8th 1999 UAUUG Birmingham 43
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