Confocal Microscopy - MMMMphysiology.med.unc.edu/images/2006-5-talks/4-Confocal.pdf · Confocal...
Transcript of Confocal Microscopy - MMMMphysiology.med.unc.edu/images/2006-5-talks/4-Confocal.pdf · Confocal...
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
Confocal Microscopy• Origins of Confocal Microscopy• Limitation of wide field microscopy• Confocal Principal• Confocal Laser Scanning• Resolution• X-Z axis scanning• Visualization
History – Marvin Minsky
1957 Confocal Patent focal Scanning Microscope: U.S.Patent 3013467
Fluorescence – Live Buccal Epithelial cells
(DIC) Transmitted FluorescenceFM 1-43 membrane dye
Limitation of wide field microscopy
Out of focus
Objective
SpecimenPlane
Image Plane
TubeLens
Dichroic splitter Emission
FilterEye
Light source
In focus
Out
Dichroic splitter
Confocal Principal – confocal pinhole
Objective
SpecimenPlane
Image Plane
TubeLens
EmissionFilter
Eye is no good
ConfocalPinhole
Light source
Excitation Pinhole
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
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
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
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
10 um beads – xy view
Confocal fluorescence Wide field transmitted lightx
y
10 um bead – xz side view
Wide field transmitted light OverlayConfocal fluorescence
x
z
x
z
x
z
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
10 um bead – confocal xz view
10x 20x 40x 100x objective0.3 0.7 1.25 1.4 numerical aperture
axial
lateral
x
z
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 = ?
Airy Disk
Objective
SpecimenPlane
Image Plane
EyePiece
TubeLens
Raster Scanning mirrors
EmissionFilter
PMT
Excitation Pinhole
ConfocalPinhole
Subresolutionbead (<0.2 um)
Dichroic splitter
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
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
Muscle Spindle
Transmittedlight 16xwidefield
Fluorescence 4x widefield(Insert 20x)Transgenic mouse Thy1-YFG
Live Muscle Spindle (Thy1-YFP)
single confocal slice 40x 1.3 NA 230 um
wide field
Serial Sections
Live Muscle Spindle (Thy1-YFP)
single slice
serial slices40x 1.3 NA 230 um
Orthogonal View
Extended Focus 40x
Maximum projectionBrightest pixel in column of stack
Average projectionSum corresponding pixels and divide by the number of slices
x
z
y
Extended Focus 40x
Maximum projection
Average projection
Volume RenderEach voxel – intensity * opacity
Confocal Multichannel Fluorescence
FITC TexasRed
Triple bandFilter (Chroma Corp.)
- Exciter- Dichroic- Emission
Em EmEx
Ex
Confocal Multichannel Fluorescence
Confocal Multichannel Fluorescence
Transmitted light(non confocal)
Fluorescence(confocal)
Confocal Multichannel FluorescenceAb-Neurofilaments 200 (blue) YFP (gray/green) VNaCh (red)
Confocal - Multichannel FluorescenceAb-Neurofilaments 200 (green) YFP (Blue) VNaCh (red)
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
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
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…….,
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