1. Slide 1 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Lecture 12 Applications of Confocal Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” 1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine UPDATED March 2000 J.Paul Robinson, Ph.D. Professor of Immunopharmacology Director, Purdue University Cytometry Laboratories These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text.

2. Slide 2 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Creating Stereo pairs z x y Pixel shifting -ive pixel shift for left +ive pixel shift for right

3. Slide 3 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories 3D images

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8. Slide 8 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Software available SGI - VoxelView MAC - NIH Image PC – Optimus – Microvoxel – Lasersharp – Confocal Assistant

9. Slide 9 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Methods for visualization Hidden object removal –Easiest methods is to reconstruct from back to front Local Projections –Reference height above threshold –Local maximum intensity –Height at maximum intensity + Local Kalman Av. –Height at first intensity + Offset Local Ht. Intensity Artificial lighting Artificial lighting reflection

10. Slide 10 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Visualization Issues Volume rendering is a computer graphics technique whereby the object or phenomenon of interest is sampled or subdivided into many cubic building blocks, called voxels (or volume elements.) A voxel is the 3-D counterpart of the 2-D pixel and is a measure of unit volume. Each voxel carries one or more values for some measured or calculated property of the volume (such as intensity values in the case of LSCM data) and is typically represented by a unit cube. The 3-D voxel sets are assembled from multiple 2-D images (such as the LSCM image stack), and are displayed by projecting these images into 2-D pixel space where they are stored in a frame buffer. Volumes rendered in this manner have been likened to a translucent suspension of particles in 3-D space. In surface rendering, the volumetric data must first be converted into geometric primitives, by a process such as isosurfacing, isocontouring, surface extraction or border following. These primitives (such as polygon meshes or contours) are then rendered for display using conventional geometric rendering techniques. http://www.cs.ubc.ca/spider/ladic/volviz.html

11. Slide 11 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Additional Material Applications Live Cell studies Time Lapse videos exotic applications

12. Slide 12 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Cellular Function –Esterase Activity –Oxidation Reactions –Intracellular pH –Intracellular Calcium –Phagocytosis & Internalization –Apoptosis –Membrane Potential –Cell-cell Communication (Gap Junctions) Applications

13. Slide 13 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Applications Conjugated Antibodies DNA/RNA Organelle Structure Cytochemical Identification Probe Ratioing

14. Slide 14 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Flow Cytometry of Apoptotic Cells G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events PI - Fluorescence # Events Normal G0/G1 cells Apoptotic cells

15. Slide 15 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Flow Cytometry of Bacteria: YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores. mixture BG E.coli BG E.coli Scatter Fluorescence Simultaneous In Situ Visualization of Seven Distinct Bacterial Genotypes Confocal laser scanning image of an activated sludge sample after in situ hybridization with 3 labeled probes. Seven distinct, viable populations can be visualized without cultivation. Amann et al.1996. J. of Bacteriology 178:3496-3500.

16. Slide 16 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories GN-4 Cell Line Canine Prostate Cancer Conjugated Linoleic Acid 200 µM 24 hours 10 µM Hoechst 33342 / PI

17. Slide 17 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Differential Interference Contrast (DIC) (Nomarski) Visible light detector Specimen Objective 1st Wollaston Prism Polarizer DIC Condenser 2nd Wollaston Prism Analyser Light path

18. Slide 18 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Flow-karyotyping of DNA integral fluorescence (FPA) of DAPI-stained pea chromosomes. Inside pictures show sorted chromosomes from regions R1 (I+II) and R2 (VI+III and I), DAPI-stained; from regions R3 (III+IV) and R4 (V+VII) after PRINS labeling for rDNA (chromosomes IV and VII with secondary constriction are labeled) A-B): metaphases of Feulgen-stained pea (Pisum sativum L.) root tip chromosomes (green ex), Standard and reconstructed karyotype L-84, respectively. C) and D): flow-karyotyping histograms of DAPI-stained chromosome suspensions for the Standard and L-84, respectively. Capital letters indicates chromosome specific peaks, as assigned after sorting

19. Slide 19 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Confocal Microscope Facility at the School of Biological Sciences which located within the University of Manchester. These image shows twenty optical sections projected onto one plane after collection. The images are of the human retina stained with Von Willebrands factor (A) and Collagen IV (B). Capturing was carried out using a x16 lens under oil immersion. This study was part of an investigation into the diabetic retina funded by The Guide Dogs for the Blind.

20. Slide 20 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Examples from Bio-Rad web site Paramecium labeled with an anti-tubulin-antibody showing thousands of cilia and internal microtubular structures. Image Courtesy of Ann Fleury, Michel Laurent & Andre Adoutte, Laboratoire de Biologie Cellulaire, Université, Paris-Sud, Cedex France. Whole mount of Zebra Fish larva stained with Acridine Orange, Evans Blue and Eosin. Image Courtesy of Dr. W.B. Amos, Laboratory of Molecular Biology, MRC Cambridge U.K.

21. Slide 21 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Examples from Bio-Rad Web site Projection of 25 optical sections of a triple-labeled rat lslet of Langerhans, acquired with a krypton/argon laser. Image courtesy of T. Clark Brelje, Martin W. Wessendorf and Robert L. Sorenseon, Dept. of Cell Biology and Neuroanatomy, University of Minnesota Medical School. This image shows a maximum brightness projection of Golgi stained neurons.

22. Slide 22 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories Confocal Microscope Facility at the School of Biological Sciences which located within the University of Manchester. The above images show a hair folicle (C) and a sebacious gland (D) located on the human scalp. The samples were stained with eosin and captured using the slow scan setting of the confocal. Eosin acts as an embossing stain and so the slow scan function is used to collect as much structural information as possible. References Foreman D, Bagley S, Moore J, Ireland G, Mcleod D, Boulton M 3D analysis of retinal vasculature using immunofluorescent staining and confocal laser scanning microscopy, Br.J.Opthalmol. 80:246-52

23. Slide 23 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories SINTEF Unimed NIS Norway The above image shows a x-z section through a metallic lacquer. From this image we see the metallic particles lying about 30 microns below the lacquer surface. The above image shows a x-y section in the same metallic lacquer as the image on the left. http://www.oslo.sintef.no/ecy/7210/confocal/micro_gallery.html

24. Slide 24 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories http://www.vaytek.com/ Material from Vaytek Web site The image on the left shows an axial (top) and a lateral view of a single hamster ovary cell. The image was reconstructed from optical sections of actin-stained specimen (confocal fluorescence), using VayTek's VoxBlast software. VoxBlast Image courtesy of Doctors Ian S. Harper, Yuping Yuan, and Shaun Jackson of Monash University, Australia. (see Journal of Biological Chemistry 274:36241-36251, 1999)

25. Slide 25 of t:/classes/BMS524/lectures2000/524lec12.ppt Purdue University Cytometry Laboratories http://www.vaytek.com/vox.htm