Confocal Microscopy

Michael ChuaCell & Molecular Physiologymicroscopy@unc.eduhttp://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