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Another use for AVS

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

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  1. Another use for AVS UAUUG Birmingham

  2. 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

  3. 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 UAUUG Birmingham

  4. Outline • Which part of plant growth? • Which plant? • Why? • How? • How we use AVS • What we want to do (with AVS?) UAUUG Birmingham

  5. 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? UAUUG Birmingham

  6. Which Plant? • Aribidopsis thalinana • A member of the brassica family • Also known as: • Thale cress • or … • Mouse Eared cress • It’s a weed! UAUUG Birmingham

  7. Just so you know what it looks like Whole Plant Flowers UAUUG Birmingham

  8. … and there’s more ... UAUUG Birmingham

  9. 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 UAUUG Birmingham

  10. The goal “To understand the genetical and cellular interactions that co-ordinate the development of the root meristem” UAUUG Birmingham

  11. How we acquire the data • Roots are visualised using Laser Scanning Confocal Microscopy (LSCM) • Also known as Confocal Scanning Laser Microscopy (CSLM) UAUUG Birmingham

  12. 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 UAUUG Birmingham

  13. 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 UAUUG Birmingham

  14. Principles UAUUG Birmingham

  15. Typical System UAUUG Birmingham

  16. The real thing UAUUG Birmingham

  17. 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 UAUUG Birmingham

  18. 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 UAUUG Birmingham

  19. 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). UAUUG Birmingham

  20. Wibbly Wobbly Jellyfish UAUUG Birmingham

  21. Pretty, Pretty UAUUG Birmingham

  22. … and they can swim UAUUG Birmingham

  23. 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 UAUUG Birmingham

  24. A Single Image UAUUG Birmingham

  25. An Image Stack UAUUG Birmingham

  26. 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 UAUUG Birmingham

  27. 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 UAUUG Birmingham

  28. Network for preliminary viewing UAUUG Birmingham

  29. Using AVS to view along a different axis tip Single frame Back a bit UAUUG Birmingham

  30. Movie view along the axis UAUUG Birmingham

  31. 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 UAUUG Birmingham

  32. Inverted Image Stack UAUUG Birmingham

  33. 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 UAUUG Birmingham

  34. Cell fate? Divide Root tip cell Differentiate Some elongate and grow Some just grow UAUUG Birmingham

  35. Need to see 3D view • 3D reconstruction from “cloud of points” • Need to “cut away” • Need to “identify” cells • Need to track “fate” UAUUG Birmingham

  36. Preliminary 3D Investigation Orthoslices UAUUG Birmingham

  37. Animate the orthoslices UAUUG Birmingham

  38. Complex Network UAUUG Birmingham

  39. Add in some “real” 3D Volume UAUUG Birmingham

  40. Another View UAUUG Birmingham

  41. Animated volume cutaway UAUUG Birmingham

  42. 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 UAUUG Birmingham

  43. 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 UAUUG Birmingham

  44. THAT’S ALL FOLKS UAUUG Birmingham

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