1. Advanced Fluorescence & Confocal Microscopy 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology UCT Medical School Room 6.10.1 Phone: 406-6419 E-Mail: DIRK.LANG@UCT.AC.ZA

2. Fluorescence Stokes Shift –is the energy difference between the peak of the absorbence and the peak of the emission spectrum 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescnece Intensity Wavelength

3. Fluorescent Microscope Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination

4. Problems and Challenges Blur: Out of focus light decreases resolution Bleaching: Excited fluorophores react to become nonfluorescent Phototoxicity: Light can harm cells Background/Autofluorescence: Cells have fluorophores too. May look like the ones you want to examine! Bleedthrough: Broad peaks cross over into one another. One fluorophore comes off in 2 channels

7. Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter Confocal Microscope

8. Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines

9. How a Confocal Image is Formed Condenser Lens Pinhole 1 Pinhole 2 Objective Lens Specimen Detector Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

10. Optical Sectioning

11. Z-Projection

14. Laser Confocal Scanning Microscopy ProsCons Allows for higher resolutionLimited EX peaks on lasers Allow collection of stacks of image planes and 3D reconstruction Phototoxicity (up to a 40degree C temp jump at focal point) Laser penetrates somewhat thick sectionsLoss of image intensity Better control for bleedthrough/autofluorescenceFairly expensive Faster than deconvolutionProne to Photobleaching Precise Laser Positioning (FRAP) Pros and Cons of Confocal Microscopy No Pin Hole 50um Pin Hole

15. Two-Photon Confocal Microscopy

16. Two-photon excitation S0S0 S’ 1 Energy S1S1 hv ex hv em Two photons of half the necessary excitation energy (double the wavelength) arrive simultanously Excitation, as with a single photon of high energy (short excitation wavelength)

18. Applications

19. Release of “Caged” Compounds UV Beam Release of “Cage” Culture dish

20. FRAP Intense laser Beam Bleaches Fluorescence Recovery of fluorescence 10 seconds 30 seconds Zero time Time %F

21. Energy Transfer: FRET Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor

22. Fluorescence Resonance Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

23. Applications of FRET

24. Fluorescent Proteins GFP - Green Fluorescent Protein –GFP is from the chemiluminescent jellyfish Aequorea victoria –excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm –contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence –Major application is as a reporter gene for assay of promoter activity –requires no added substrates –now modified forms available: yellow, red, cyan and blue fluorescent proteins –Often used in FRET

26. Multiphoton Microscopy in Neuroscience:  Axon pathfinding in embryogenesis and regeneration  Signal transduction in growing axons  Analysis of brain cytoarchitecture  Intravital imaging of neuronal signalling

27. Analysis of axon pathfinding in live zebrafish brain Micropipette (antibodies or morpholinos) Hindbrain

28. Injection of Function-blocking Antibodies on the Retinotectal Projection:

29. Signal Transduction in Neuronal Growth Cones: Does Nogo-A Receptor (red) signal through raft domains (green)? FRET

30. Analysis of Brain Cyto- Architecture: Deep z-stack acquisition and stereology in live or fixed brain tissues allows detailed analysis of developmental or pathological changes:  Cell numbers  Neurone morphology and connectivity  Subcellular changes (e.g. synaptic spines)

31. Multiphoton microscopy of brain in vivo: Alzheimer plaques and vascularisation (Christie et al., 2001)

32. Miniaturization of multiphoton microscope for brain imaging of live animals (Helmchen et al., 2001)

33. Analysis of Neuronal Signalling: Rapid scanning techniques in the study of brains or live brain slices allow intravital analysis of physiological processes:  Synaptic structure and physiology  Calcium dynamics  Receptor mapping and trafficking

34. Non-Confocal Methods of Removing „Blur“

35. Deconvolution Microscopy operates on the principle of a point spread function (PSF). As one moves away from focal plane in which an object lies the light from it will spread in a predictable manner

36. Image Deconvolution

38. Image Deconvolution can produce optical sections of near-confocal quality on a conventional fluorescence microscopy system

39. Effect of image deconvolution: Immunolabelled axons in a wholemount nerve preparation

40. Deconvolution Microscopy ProsCons Allows for higher ResolutionComputationally intense if iterative Allow collection of stacks of image planesMinimal penetration of thick sections Hg Bulb allows for variety of filter combinations Sensitive to spherical aberrations Can get superior sensitivityCan amplify noise, produce artifacts Cheaper than confocalRequires 3D data set Pros and Cons of Deconvolution Microscopy RawDeconvolved

41. Structured Illumination – “Apotome”

43. High-Resolution Fluorescence Microscopy

44. Resolution Limits of Fluorescence Microscopy (Jaiswal & Simon, 2007)

45. Total Internal Reflection Fluorescence (TIRF)

46. Pushing the Resolution Limit… (Hell, 2007)

47. Pushing the Resolution Limit… (Donnert et al., 2006)