confocal microscopy - mmmmphysiology.med.unc.edu/images/2006-5-talks/4-confocal.pdf · confocal...
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Confocal Microscopy
Michael ChuaCell & Molecular [email protected]://microscopy.unc.edu6007 Thurston Bowles843-3268
2006 Oct 30, Nov 6, 13, 20 & 27
Michael Hooker Microscopy Facility
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Confocal Microscopy• Origins of Confocal Microscopy• Limitation of wide field microscopy• Confocal Principal• Confocal Laser Scanning• Resolution• X-Z axis scanning• Visualization
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History – Marvin Minsky
1957 Confocal Patent focal Scanning Microscope: U.S.Patent 3013467
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Fluorescence – Live Buccal Epithelial cells
(DIC) Transmitted FluorescenceFM 1-43 membrane dye
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Limitation of wide field microscopy
Out of focus
Objective
SpecimenPlane
Image Plane
TubeLens
Dichroic splitter Emission
FilterEye
Light source
In focus
Out
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Dichroic splitter
Confocal Principal – confocal pinhole
Objective
SpecimenPlane
Image Plane
TubeLens
EmissionFilter
Eye is no good
ConfocalPinhole
Light source
Excitation Pinhole
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Confocal Laser Scanning Microscope
Objective
SpecimenPlane
Image Plane
TubeLens
Raster Scanning mirrors
EmissionFilter
Photo Multiplier Tube (PMT)
Excitation Pinhole
ConfocalPinhole
A2D
Raster scanframe buffer
Dichroic splitter
Laser ExcitationFilter
x - axisy - axis
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Confocal Laser Scanning Microscope – PMTPhoto Multiplier Tube (PMT)
• Quantum yield ~= 0.3• Gain can be very large >108
• Gain is exponential function of applied voltage
• Noise increases disproportionably at high gain
• Large dark current
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Image Representation - Digitally• Intensity values of pixels in a 2 D array for monochrome – f(x,y)• Raster scan – left to right then top to bottom. • Numbers typically 8 bit binary (values 0 to 255 ) – good for
confocal with only a few dozen photons per pixel
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Wide Field versus Confocal
PC
Pacinian Corpuscle 10X NA 0.3 Label FM 1-43 (lipid)
Confocal~10 um thick1 mm wideHigh contrastLow backgroundGood resolution
Wide field~150 um thick view1 mm wideGlareOut of focus Low resolution
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10 um beads – xy view
Confocal fluorescence Wide field transmitted lightx
y
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10 um bead – xz side view
Wide field transmitted light OverlayConfocal fluorescence
x
z
x
z
x
z
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Numerical Aperture (NA)
Numerical aperture is loosely related to resolving power.High NA leads to smaller working distance.u = half angle of cone of illuminationn = refractive index of medium
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10 um bead – confocal xz view
10x 20x 40x 100x objective0.3 0.7 1.25 1.4 numerical aperture
axial
lateral
x
z
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Numerical Aperture (NA) - resolution
NA = n.sin (a) , a = half cone anglen = refractive index
of mediumλ = wavelength
aWide Field rairy = 0.61 λ / NA(fluorescence) = 0.22 μm
Confocal rairy = 0.61 λ / NA / √2= 0.15 μm
raxial-fluor = 1.77 λ / NA2
= 0.4 μm
objectivemaximum imagingcone angle
rairy.axial = ?
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Airy Disk
Objective
SpecimenPlane
Image Plane
EyePiece
TubeLens
Raster Scanning mirrors
EmissionFilter
PMT
Excitation Pinhole
ConfocalPinhole
Subresolutionbead (<0.2 um)
Dichroic splitter
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Airy Disks
0.5 μm bead Plan Apo 100x 1.4 NA oil
0.0 μm
1.1 μm
2.4 μm 5.2 μm
3.7 μm
3.4 μm
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Visualization - Muscle Spindle Example
Wide field – non confocalThin slice and z-seriesGallerySequence of slicesOrthogonal viewAverage & Maximum projectionsVolume renderMultiple channels (dyes / fluorophores)
α
αIa a ffe ren t
“Ia ” inh ib ito ryin te rneu ron
E x tenso r (an tag on is t) m usc leF lexo r (agon is t) m usc le
M usc le sp ind le
C o m p o n en ts o f th eS tre tch (M yo ta tic )
R eflex
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Muscle Spindle
Transmittedlight 16xwidefield
Fluorescence 4x widefield(Insert 20x)Transgenic mouse Thy1-YFG
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Live Muscle Spindle (Thy1-YFP)
single confocal slice 40x 1.3 NA 230 um
wide field
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Serial Sections
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Live Muscle Spindle (Thy1-YFP)
single slice
serial slices40x 1.3 NA 230 um
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Orthogonal View
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Extended Focus 40x
Maximum projectionBrightest pixel in column of stack
Average projectionSum corresponding pixels and divide by the number of slices
x
z
y
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Extended Focus 40x
Maximum projection
Average projection
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Volume RenderEach voxel – intensity * opacity
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Confocal Multichannel Fluorescence
FITC TexasRed
Triple bandFilter (Chroma Corp.)
- Exciter- Dichroic- Emission
Em EmEx
Ex
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Confocal Multichannel Fluorescence
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Confocal Multichannel Fluorescence
Transmitted light(non confocal)
Fluorescence(confocal)
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Confocal Multichannel FluorescenceAb-Neurofilaments 200 (blue) YFP (gray/green) VNaCh (red)
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Confocal - Multichannel FluorescenceAb-Neurofilaments 200 (green) YFP (Blue) VNaCh (red)
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Other Modes of Confocal Microscopy
Spinning disk – faster acquisition, less light (photobleaching)
Reflection mode – surface height, e.g. minerals, cell surface
2 Photon – less prone to scattered light depth penetration
Ratio Imaging – e.g. pH, Ca2+
Spectral scanning
FRAP – fluorescence recovery after photobleaching
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Confocal Microscopy – Summary
Fluorescence – ReflectionFixed or live cells/tissuesProtein location in cellMembrane/Lipid locationFluid compartmentsMaterial surface analysis, e.g. integrated circuit
• 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
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Viewed Intensity Depends On:
1. Focus 2. Laser excitation power3. Fluorophore concentration4. Confocal pinhole size5. PMT gain
• Photobleaching• Fluorescence saturation• Incorrect filters• Fluorescence channel cross talk• Light scattering• Reflection• Sample absorption• and many other factors……. Next week…….,
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Scratched the surface of confocal microscopy.
• Confocal Microscopy Methods and Protocols, in Methods in Molecular Biology, Stephen W. Paddock (1999)
• Handbook of Biological Confocal Microscopy, 2nd ed., James Pawley, 1995
• Handbook of Biological Confocal Microscopy, 3rd ed., James Pawley, 2006