1. Confocal Microscopy M. Chua Cell & Molecular Physiology, UNC Michael Hooker Microscopy Facility BME 2003-04-02

2. A microscope system which removes out of focus information optically. Consists of a high quality microscope, light source & electronic detector. Confocal Microscope Origins Principal of operation Imaging examples

3. Common use in Biomedical Science SECOND SENIOR AGENT: With all due respect, Agent Scully... it looks like something from the Hubble telescope. SCULLY: Actually, it's an image created by what's called a confocal microscope. You're looking at a three-dimensional picture of the location of a protein. In this case, cowpox structural protein number six. It's one of the inoculants used in smallpox vaccines. X-Files, 1997, 20th Century Fox

4. 1957 Confocal Scanning Microscope: U.S.Patent 3013467 History – Marvin Minsky

5. Confocal Patent

6. Standard Microscope Focal Plane Image Plane Eye Piece Tube Lens Lamp Condenser Objective

7. Standard Microscope Transmitted light microscopy Sample needs to be mostly transparent Dyes give contrast/color Reflected light microscopy Sample needs to be reflective See surface Exciting light adds background – reduces contrast

8. Fluorescence Emitted light different color to exciting light See through transparent sample S’1 S1 photon Energy huex huem photon ground state t (ns)

9. FITC Fluorescence Microscopy Em Ab Arc Lamp (white light) Image Plane Specimen Plane Excitation Filter Dichroic Split Emission Filter Eye Piece Tube Lens Objective

10. Fluorescence – Live Buccal Epithelial cells Transmitted Nomarski (DIC) Fluorescence FM 1-43 membrane dye

11. Fluorescence Epifluorescence Label sample with a fluorescent dye Exciting light causes emission of light of different color See through mostly transparent sample Reflected light filtered out with color selective filter Fluorophore Absorbs exciting light Emits light of long wavelength (Stokes shift) Can also see sub resolution structures - See emitted light without resolving it - like light house in fog

12. Confocal Principal 1 – Limitation of wide field Light source Image Plane Specimen Plane Tube Lens Dichroic splitter EmissionFilter Eye Objective in Out Out

13. Confocal Principal 2 – Confocal pinhole Light source Excitation Pinhole Image Plane Specimen Plane Tube Lens Dichroic splitter Confocal Pinhole Eye is no good EmissionFilter Objective

14. Raster Scanning mirrors Confocal Principal 3 – Laser Scanning Excitation Pinhole Image Plane Specimen Plane Tube Lens Dichroic splitter Confocal Pinhole EmissionFilter PMT Objective

15. Raster scan frame buffer Raster Scanning mirrors Confocal Principal 4 – LSCM Excitation Pinhole Image Plane A2D Specimen Plane Tube Lens Dichroic splitter Confocal Pinhole EmissionFilter PMT Objective

16. Pacinian Corpuscle 10X NA 0.3 FM 1-43 Wide Filed versus Confocal PC Wide field ~150 um thick view Glare Out of focus Low resolution Confocal ~10 um thick High contrast Low background Good resolution

17. Raster Scanning mirrors Airy Disk Excitation Pinhole Image Plane Specimen Plane Tube Lens Subresolution bead (<0.2 um) Dichroic splitter Confocal Pinhole Eye Piece EmissionFilter PMT Objective

18. Airy Disk 0.0 mm 3.4 mm 1.1 mm 3.7 mm 2.4 mm 5.2 mm 0.5 mm bead Plan Apo 100x 1.4 NA oil

19. Airy Disk 0.5 um bead Plan Apo 20x 0.7 NA oil

20. 10 um beads – xy view Confocal fluorescence Wide field transmitted light

21. 10 um bead – xz side view Confocal fluorescence Wide field transmitted light

22. Numerical Aperture - resolution objective maximum imaging cone angle NA = n.sin (a) , a = half cone angle Wide Field rairy = 0.61 l / NA = 0.22 mm Confocal rairy = 0.61 l / NA / Ö2 = 0.15 mm a Confocal raxial-fluor = 1.77 l / NA2 = 0.4 mm

23. 10 um bead – confocal xz view axial lateral 10x 20x 40x 100x objective 0.3 0.7 1.25 1.4 numerical aperture

24. Muscle Spindle Transmitted light 16x widefield Fluorescence 4x (insert 20x) widefield Transgenic mouse Thy1-YFG

25. Live Muscle Spindle (Thy1-YFP) single slice serial slices 40x 1.3 NA 230 um

26. Extended Focus 40x Sum Maximum projection Widefield 20x

27. Volume Render

28. Orthogonal View

29. Texas Red FITC Confocal Multichannel Fluorescence Ex Em Em Ex • Exciter • Dichroic • Emission Chroma Dual band Filter

30. Confocal Multichannel Fluorescence

31. Confocal Multichannel Fluorescence YFP Ab-TTX-VNaCh Overlaid

32. Ab-Neurofilaments 200 (blue)YFP (gray/green)VNaCh (red) Confocal Multichannel Fluorescence

33. Ab-Neurofilaments 200 (green)YFP (Blue)VNaCh (red) Confocal - Multichannel Fluorescence

34. A microscope system which removes out of focus information optically. • Consists of a high quality microscope, light source & electronic detector. • Better lateral and axial resolution • High contrast • Reduced glare Confocal Microscopy - Summary Fluorescence – Reflection – Transmission Fixed or live cells/tissues Protein location in cell Membrane/Lipid location Fluid compartments IC inspection. material analysis, etc.

35. Biology – Zeiss 510 – Zeiss 410 – 2x Perkin Elmer spinning disk Cell Biology – Zeiss 410 – Zeiss 510 2 Photon – Home made 2 photon Cell & Molecular Physiology – LeicaTCS-NT Neuroscience Center – Zeiss 510 2 Photon – Zeiss Pascal Pharmacology – Olympus Michael Hooker Microscopy – Zeiss 510 meta – LeicaSP2 abos – Perkin Elmer spinning disk – Leica-4D Pathology – Zeiss 110 Confocals at UNC

36. Confocal – further reading • Pawley, J. B. ed. (1995) Handbook of Biological Confocal Microscopy, 2nd Edition, Plenum, NY • Yuste, R., Lanni, F. & Konnerth, A (2000) Imaging Neurons, A Laboratory Manual, Cold Spring Harbor, NY

37. The End Hydroethidine R. Zucker, EPA