1. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 1 t:/classes/BMS602B/lecture 4 602_B.ppt BME 695Y / BMS 634 Confocal Microscopy: Techniques and Application Module Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine & Department of Biomedical Engineering, Schools of Engineering J.Paul Robinson, Ph.D. Professor of Immunopharmacology & Biomedical Engineering 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. Week 3 Different types of scanning 3D construction & Various Applications

2. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 2 t:/classes/BMS602B/lecture 4 602_B.ppt Lecture summary 1. Line scanning confocal microscopy 2. Slit formation 3. Light sources, advantages and disadvantages 4. 4D confocal imaging 5. Applications of Confocal Microscopy

3. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 3 t:/classes/BMS602B/lecture 4 602_B.ppt DVC Linescanner Emission Filters CCD Camera Laser Fiber Optic Link Computer ocular Scanhead

4. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 4 t:/classes/BMS602B/lecture 4 602_B.ppt DVC 250 Line Scanner Ocular Specimen Laser “galvanometer” descanning mirrors Slit Filters Lens scanning mirror

5. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 5 t:/classes/BMS602B/lecture 4 602_B.ppt Stationary Slit Apertures Illuminated line must be scanned over specimen Emitted light must be descanned Light passing through slit must be rescanned to reconstruct a 2D image on the retina

6. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 6 t:/classes/BMS602B/lecture 4 602_B.ppt Scanning The scanning is performed by oscillating mirrors Rate of oscillation is 25-30 Hz

7. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 7 t:/classes/BMS602B/lecture 4 602_B.ppt Mirrors DVC uses mirrors, not lenses Reduces chromatic aberration

8. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 8 t:/classes/BMS602B/lecture 4 602_B.ppt Slit The confocal slit is variable Smallest size is 20 um Images of excellent resolution can be collected using video cameras using small slit width

9. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 9 t:/classes/BMS602B/lecture 4 602_B.ppt Laser spot to line Beam splitting lens Laser in Laser out

10. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 10 t:/classes/BMS602B/lecture 4 602_B.ppt How the laser scans Scan width can be adjusted

11. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 11 t:/classes/BMS602B/lecture 4 602_B.ppt Light Sources - Lasers ArgonAr488-514 nm Krypton-ArKr-Ar488 - 568 - 647 nm Helium-NeonHe-Ne633

12. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 12 t:/classes/BMS602B/lecture 4 602_B.ppt Light Sources Kr-Ar lasers most common (488, 568, 647 nm) Ar - large (100-200 mW) Coupled to head with single mode optical fiber (these preserve coherence) Fibers usually have 60% efficiency Light is spread over specimen not at point so 25 mW laser produces 3-5 mW at specimen

13. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 13 t:/classes/BMS602B/lecture 4 602_B.ppt Main Advantages Can follow very rapid events Up to 30 frames per second Best when searching over large specimens for specific features For thick specimens provides an intermediate image between fluorescence microscopy and point scanners Systems are small Can be easily changed from upright to inverted scopes Very low level light imaging

14. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 14 t:/classes/BMS602B/lecture 4 602_B.ppt Disadvantages Need higher power lasers because point is spread over line Can bleach specimens significantly Much high precision in slit manufacture (increase in $) Must use camera to detect signal Harder to use UV Cost is significant relative to point scanners

15. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 15 t:/classes/BMS602B/lecture 4 602_B.ppt Image collection CCD Camera (usually cooled) Faster - cooled and intensified camera

16. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 16 t:/classes/BMS602B/lecture 4 602_B.ppt 4D confocal microscopy Time vs 3D sections Used when evaluating kinetic changes in tissue or cells Requires fast 3D sectioning Difficult to evaluate

17. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 17 t:/classes/BMS602B/lecture 4 602_B.ppt 4D Imaging Time 1 2 3 4 5 Time Fluorescence

18. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 18 t:/classes/BMS602B/lecture 4 602_B.ppt 4D Imaging Time 1 2 3 4 5

19. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 19 t:/classes/BMS602B/lecture 4 602_B.ppt 4D Imaging Time 1 2 3 4 5 This could also be achieved using an X-Z scan on a point scanner.

20. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 20 t:/classes/BMS602B/lecture 4 602_B.ppt Software Image analysis –Universal Imaging “Metamorph” –Image Pro-Plus –NIH Image Fluorescence Ratioing “Metafluor”

21. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 21 t:/classes/BMS602B/lecture 4 602_B.ppt 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

22. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 22 t:/classes/BMS602B/lecture 4 602_B.ppt Software available SGI - VoxelView MAC - NIH Image PC – Optimus – Microvoxel – Lasersharp – Confocal Assistant

23. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 23 t:/classes/BMS602B/lecture 4 602_B.ppt Differential Interference Contrast (DIC) (Nomarski) Visible light detector Specimen Objective 1st Wollaston Prism Polarizer DIC Condenser 2nd Wollaston Prism Analyzer Light path

24. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 24 t:/classes/BMS602B/lecture 4 602_B.ppt Confocal Microscopy in the Research Laboratory Applications Live Cell studies Time Lapse videos Exotic applications

25. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 25 t:/classes/BMS602B/lecture 4 602_B.ppt Cellular Function –Esterase Activity –Oxidation Reactions –Intracellular pH –Intracellular Calcium –Phagocytosis & Internalization –Apoptosis –Membrane Potential –Cell-cell Communication (Gap Junctions) Applications

26. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 26 t:/classes/BMS602B/lecture 4 602_B.ppt Applications Probe Ratioing –Calcium Flux (Indo-1, Fluo-3) –pH indicators (BCECF, SNARF) Molecule-probeExcitationEmission Calcium - Indo-1351 nm405, >460 nm Magnesium - Mag-Indo-1351 nm405, >460 nm Calcium-Fluo-3488 nm525 nm Calcium - Fura-2363 nm>500 nm Calcium - Calcium Green 488 nm515 nm Phospholipase A - Acyl Pyrene351 nm405, >460 nm

27. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 27 t:/classes/BMS602B/lecture 4 602_B.ppt Exotic Applications Release of “Caged” compounds FRAP (UV line) Lipid Peroxidation (Paranaric Acid) Membrane Fluidity (DPH)

28. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 28 t:/classes/BMS602B/lecture 4 602_B.ppt “Caged” Photoactivatable Probes Ca ++ : Nitr-5 Ca ++ - buffering: Diazo-2 IP 3 cAMP cGMP ATP ATP-  -S Examples Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm

29. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 29 t:/classes/BMS602B/lecture 4 602_B.ppt Applications Organelle Structure & Function –Mitochondria (Rhodamine 123) –Golgi(C6-NBD-Ceramide) –Actin (NBD-Phaloidin) –Lipid (DPH) –Endoplasmic Reticulum

30. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 30 t:/classes/BMS602B/lecture 4 602_B.ppt Applications Conjugated Antibodies DNA/RNA Organelle Structure Cytochemical Identification Probe Ratioing

31. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 31 t:/classes/BMS602B/lecture 4 602_B.ppt 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

32. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 32 t:/classes/BMS602B/lecture 4 602_B.ppt 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.

33. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 33 t:/classes/BMS602B/lecture 4 602_B.ppt GN-4 Cell Line Canine Prostate Cancer Conjugated Linoleic Acid 200 µM 24 hours 10 µM Hoechst 33342 / PI

34. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 34 t:/classes/BMS602B/lecture 4 602_B.ppt 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

35. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 35 t:/classes/BMS602B/lecture 4 602_B.ppt Applications Organelle Structure Probe ratioing Conjugated antibodies DNA/RNA Cytochemical Identification Oxidative Metabolism Exotic Applications Organelle Structure & Function –Mitochondria (Rhodamine 123) –Golgi(C6-NBD-Ceramide) –Actin (NBD-Phaloidin) –Lipid (DPH)

36. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 36 t:/classes/BMS602B/lecture 4 602_B.ppt Step 1: Cell Culture Step 2: Cell Wash Lab-Tek 1 2 3 4 5 6 7 8 top view side view 170  M coverslip Step 3: Transfer to Lab- Tek plates confocal microscope oil immersion objective 37 o heated stage stimulant/inhibitor added Step 4: Addition of DCFH- DA, Indo-1, or HE Below: the culture dishes for live cell imaging using a confocal microscope and high NA objectives. Live cell studies

37. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 37 t:/classes/BMS602B/lecture 4 602_B.ppt Confocal System Culture System Photos taken in Purdue University Cytometry Labs Photo taken from Nikon promotion material

38. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 38 t:/classes/BMS602B/lecture 4 602_B.ppt Example of DIC and Fluorescnece Human cheek epithelial cells (from JPR!) stained with Hoechst 33342 - wet prep, 20 x objective, 3 x zoom (Bio-Rad 1024 MRC) Giardia (DIC image) (no fluorescence) (photo taken from a 35 mm slide and scanned - cells were live when photographed)

39. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 39 t:/classes/BMS602B/lecture 4 602_B.ppt Fluorescence Microscope image of Hoechst stained cells (plus DIC) Image collected with a 470T Optronics cooled camera

40. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 40 t:/classes/BMS602B/lecture 4 602_B.ppt Use for DNA content and cell viability –33342 for viability Less needed to stain for DNA content than for viability –decrease nonspecific fluorescence Low laser power decreases CVs Measurement of DNA G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events

41. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 41 t:/classes/BMS602B/lecture 4 602_B.ppt PI - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead PI PI PI PI PI PI PI PI PI PI PI PI PI PI Viable CellDamaged Cell

42. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 42 t:/classes/BMS602B/lecture 4 602_B.ppt 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

43. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 43 t:/classes/BMS602B/lecture 4 602_B.ppt Confocal Microscope Facility at the School of Biological Sciences which is 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.

44. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 44 t:/classes/BMS602B/lecture 4 602_B.ppt 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.

45. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 45 t:/classes/BMS602B/lecture 4 602_B.ppt 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.

46. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 46 t:/classes/BMS602B/lecture 4 602_B.ppt 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

47. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 47 t:/classes/BMS602B/lecture 4 602_B.ppt 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

48. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 48 t:/classes/BMS602B/lecture 4 602_B.ppt 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)

49. Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 49 t:/classes/BMS602B/lecture 4 602_B.ppt Summary Linescanning allows faster imaging Usually requires a CCD camera 4D imaging Application of fixed cell imaging Introduction to live cell imaging