1. Microscopy I Fluorescence Confocal Multiphoton Flow Cytometry By Luis Filgueira

2. For Wednesday Tutorial Please Activate you PHEME account If not yet done

3. For next week’s labs please put your name down on list at the white board

4. Fluorescence Three-stage process 1. Exitation Photon of energy h EX 2. Exitation-state Lifetime (1-10x10 -9 seconds) 3. Emission Photon of energy h EM

5. Difference in energy = h EX – h EM Differerence in vave length

6. Exitation and Emision

8. polyaromatic hydrocarbons heterocycles Fluorophore = fluorescent dye

9. Binding specifically to biochemical structures Protein DNA RNA Lipids Ca K H +

10. Fluorophore = fluorescent dye Fluorescence labelled macromolecules Antibodies Lipids Nucleotides Oligo-DNA Oligo-RNA Peptides Lectines Phalloidin

11. Fluorophore = fluorescent dye Detection of Organelles Nucleus Golgi Lysosomes RER Mitochondria Cytoskeleton Membranes

12. Fluorophore = fluorescent dye Enzymes and Enzyme Substrates and Cell Function Glycosidases Phosphatases Proteases/Peptidases Peroxidases Lipases Membrane potential

13. Applications for fluorescent dyes Microscopy Flow Cytometry Molecular Biology Spectrofluorometric Assays

14. Molecular Biology Gel-Electrophoresis Real time/quantitative RT-PCR DNA RNAProtein

15. Spectrofluorometric Assays Cell Viability Cytotoxicity Enzymatic Assays Ca+ Measurement

16. Flow Cytometry Cell Cycle Analysis Cell Viability Cytotoxicity Quantification of Surface marker expression Intracellular Protein expression

17. Microscopes AxialInvert

18. Fluorescence Microscopy Mercury Vapor lamp = Arc Lamp

19. Filters

20. Confocal Microscopy Ar UV 351, 364nm HeCd 442nm Ar 457nm, 488nm, 514nm ArKr 488nm Kr 568nm HeNe 633nm

21. Comparison

22. Confocal Microscopy optical sectioning <> electronic reconstruction

23. Axial optical sectioning of the object is possible in two ways. Either by - direct acquisition of x/z- or y/z-optical cross sections, or - calculation of vertical optical sections using the normal x/y-image stack (as shown here).

24. Rotation and volume rendering. Rotation of the image stack and projection into one plane allows viewing the object from different angles.

25. Stereoscopic imaging. The combination of a left and a right image either as - stereo pairs or - red/green anaglyphs allows easy visualisation of 3D-structures! Use red/green glasses for stereoscopic viewing of this red/green anaglyph.

26. Multi-photon Fluorescence Microscopy

28. Flow Cytometry Measurement and quantification of optical properties of particles in suspension Beads Bacteria Cells

29. Charging electrode Sample injection tube Sheat tube Vent tube Laser light Deflection plates Collection tubes Flow Cytometry

30. Flow Cytometry

31. Flow Cytometry

33. Flow Cytometry

34. Flow Cytometry