Introductory Guide to Light Microscopy - Biomedical Confocal Microscopy
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1 Introductory Guide to Light Microscopy - Biomedical Confocal Microscopy 7 May 2007 Michael Hooker Microscopy Facility Michael Chua microscopy@unc.edu Thurston Bowles Wendy Salmon wendy_salmon@med.unc.edu Thurston Bowles
2 Confocal Microscopy Limitation of wide field microscopy Origins of Confocal Microscopy Confocal Principal Confocal Laser Scanning Resolution X-Z axis scanning Visualization
3 Limitation of wide field microscopy Live Buccal Epithelial cells (DIC) Transmitted Fluorescence FM 1-43 membrane dye
4 Limitation of wide field microscopy Light source Specimen Plane Image Plane Tube Lens Objective Dichroic splitter Emission Filter Out In focus Out of focus Eye
5 Origins Marvin Minsky 1957 Confocal Patent focal Scanning Microscope: U.S.Patent
6 Confocal Principal the confocal pinhole Light source Excitation Pinhole Specimen Plane Tube Lens Image Plane Point image (voxel) Objective Dichroic splitter Emission Filter Confocal Pinhole Eye is not useful
7 Confocal Laser Scanning Microscope Raster Scanning mirrors Frame buffer memory x - axis y - axis Synched to mirror scanning Excitation Pinhole Number stream (pixels) Specimen Plane Image Plane A2D Laser Excitation Filter Tube Lens Objective Dichroic splitter Emission Filter Confocal Pinhole Photo Multiplier Tube (PMT)
8 Confocal Principal Live Buccal Epithelial cells Widefield Confocal FM 1-43 membrane dye
9 Confocal Laser Scanning Microscope PMT Photo Multiplier Tube (PMT) Quantum yield <= 0.3 Gain can be very large >10 7 Gain is exponential function of applied voltage Noise increases disproportionably at high gain Significant dark current
10 Confocal Laser Scanning Microscope Analogue to Digital Converter & Frame Buffer Display I 255 Gain Offset Contrast A2D Pixel Number Stream LUT Offset bit A2D = Analogue to Digital Converter LUT = Look Up Table
11 Confocal Laser Scanning Microscope Raster Scanning mirrors Frame buffer memory x - axis y - axis Synched to mirror scanning Excitation Pinhole Number stream (pixels) Specimen Plane Image Plane A2D Laser Excitation Filter Tube Lens Transmitted light PMT Condenser Objective Dichroic splitter Emission Filter Confocal Pinhole Photo Multiplier Tube (PMT)
12 10 um beads xy view y Confocal fluorescence x y Wide field transmitted light x
13 Scanning modes y x z x Stage vibrated x - y scanning (conventional slice) x - z scanning (vertical slice)
14 10 um bead xz side view z z z x Confocal fluorescence x Wide field transmitted light x Overlay
15 10 um bead confocal xz view axial lateral z x 10x 20x 40x 100x objective numerical aperture
16 Raster Scanning mirrors Airy Disk Excitation Pinhole Specimen Plane Image Plane Tube Lens Subresolution bead (<0.2 um) Objective Dichroic splitter Emission Filter Confocal Pinhole Eye Piece PMT
17 Airy Disks 0.0 µm 3.4 µm 1.1 µm 3.7 µm 2.4 µm 5.2 µm 0.5 µm bead Plan Apo 100x 1.4 NA oil
18 Airy Disk 0.5 um bead Plan Apo 20x 0.7 NA oil
19 Numerical Aperture (NA) - resolution Rayleigh Criterion: 2 points are said to be resolved when there separation causes the center of the Airy pattern of one point to fall on the 1 st minimum of the other Airy disk objective maximum imaging cone angle NA = n.sin (a), a = half cone angle n = refractive index of medium λ = wavelength Wide Field r airy = 0.61 λ / NA (fluorescence) = 0.22 µm r airy.axial =? a Confocal r airy = 0.61 λ / NA / 2 = 0.15 µm r axial-fluor = 1.77 λ / NA 2 = 0.4 µm (for green emission)
20 Components of the Stretch (Myotatic) Reflex Visualization - Example Muscle Spindle Ia afferent α α Ia inhibitory interneuron Muscle spindle Flexor (agonist) muscle Extensor (antagonist) muscle Transmitted light 16x widefield Fluorescence 4x widefield transgenic mouse Thy1-YFG Wide field non confocal Thin slice and z-series Gallery Serial slices Z-axis sampling Orthogonal view Average & Maximum projections Volume render Multiple channels (dyes / fluorophores)
21 Visualization - Live Muscle Spindle (Thy1-YFP) single confocal slice 40x 1.3 NA 230 um wide field
22 Visualization - Focus to Different Planes Serial Sections
23 Pinhole Size versus optical Section
24 Optical Sectioning Step size 0.9 um Optical Section Size 0.9 um Step size 0.45 um z Inadequately Sampled Just Adequately Sampled (Nyquist criterion at least twice frequency, ~2.3x)
25 Visualization - Live Muscle Spindle (Thy1-YFP) single slice serial slices 40x 1.3 NA 230 um
26 Visualization - Orthogonal View
27 Visualization - Extended Focus 40x z y x Maximum projection Brightest pixel in column of stack good for viewing thin structures bad for quantification Average projection Sum corresponding pixels and divide by the number of slices good for quantification
28 Visualization - Extended Focus 40x Maximum projection Average projection
29 Visualization - Volume Renderimg Each voxel intensity * opacity
30 Confocal Multichannel Fluorescence FITC Ex Em Texas Red Ex Em Triple band Filter (Chroma Corp.) -Exciter - Dichroic - Emission
31 Confocal Multichannel Fluorescence Fiber optic No Excitation Filter Emission Filter (11) Dichroic splitter (4, 7, 8, 10)
32 ARC Lamp versus Laser Excitation Laser Lines Argon Laser HBO Lines
33 Acousto-Optical Tunable Filter (AOTF) RF = radio frequency
34 Excitation Light System AOTF Controlled Replaces excitation filters RF modulator To scanhead Fiber optic Piezo Electric Crystal RF = radio frequency Tellurium Oxide 488 nm 561 nm 633 nm Merging Dichroic Mirrors
35 Confocal Multichannel Fluorescence Fiber optic Fluorescence (confocal) 6 = AOTF 11 = dichroic 19 to 21 = PMTs Transmitted light (non confocal)
36 Confocal Multichannel Fluorescence Ab-Neurofilaments 200 (blue) YFP (gray/green) VNaCh (red)
37 Confocal - Multichannel Fluorescence Ab-Neurofilaments 200 (green) YFP (Blue) VNaCh (red)
38 Confocal Microscopy Summary A microscope system which removes out of focus information optically. Better lateral and much improved axial resolution Higher contrast Reduced glare Single voxel scanning permits 3-D reconstructions (DIC) Transmitted (widefield) Fluorescence FM 1-43 membrane dye (confocal) Overlay
39 References Confocal Microscopy Methods and Protocols, in Methods in Molecular Biology, Stephen W. Paddock, 1999 Handbook of Biological Confocal Microscopy, 2 nd ed., James Pawley, 1995 Handbook of Biological Confocal Microscopy, 3 rd ed., James Pawley, 2006 Confocal Microscopy for Biologists, Alan R. Hibbs, 2004 (Missing at UNC, Duke & NC State)
40 Advanced Fluorescence/Confocal May 14 Multi-channel bleed through/cross talk Co-localization Live cell imaging FRAP, fluorescence recovery after photobleaching Deconvolution Your requests?
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