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Confocal Microscopy

Michael Chua
Cell & Molecular Physiology
microscopy@unc.edu
http://microscopy.unc.edu
6007 Thurston Bowles
843-3268
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
Fluorescence
FM 1-43 membrane dye
Limitation of wide field microscopy
O
u
t

o
f

f
o
c
u
s
Objective
Specimen
Plane
Image
Plane
Tube
Lens
Dichroic
splitter
Emission
Filter
Eye
Light source
I
n

f
o
c
u
s
O
u
t
Dichroic
splitter
Confocal Principal confocal pinhole
Objective
Specimen
Plane
Image
Plane
Tube
Lens
Emission
Filter
Eye
is no
good
Confocal
Pinhole
Light source
Excitation Pinhole
Confocal Laser Scanning Microscope
Objective
Specimen
Plane
Image
Plane
Tube
Lens
Raster Scanning mirrors
Emission
Filter
Photo
Multiplier
Tube (PMT)
Excitation Pinhole
Confocal
Pinhole
A
2
D
Raster scan
frame buffer
Dichroic
splitter
Laser
Excitation
Filter
x - axis
y - axis
Confocal Laser Scanning Microscope PMT
Photo Multiplier Tube (PMT)
Quantum yield ~=0.3
Gain can be very large >10
8
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
PacinianCorpuscle 10X NA 0.3 Label FM 1-43 (lipid)
Confocal
~10 um thick
1 mm wide
High contrast
Low background
Good resolution
Wide field
~150 um thick view
1 mm wide
Glare
Out of focus
Low resolution
10 um beads xyview
Confocal fluorescence Wide field transmitted light
x
y
10 um bead xz side view
Wide field transmitted light Overlay
Confocal 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 illumination
n =refractive index of medium
10 um bead confocal xz view
10x 20x 40x 100x objective
0.3 0.7 1.25 1.4 numerical aperture
a
x
i
a
l
lateral
x
z
Numerical Aperture (NA) - resolution
NA =n.sin (a) , a =half cone angle
n =refractive index
of medium
=wavelength
a
Wide Field r
airy
=0.61 / NA
(fluorescence) =0.22 m
Confocal r
airy
=0.61 / NA / 2
=0.15 m
r
axial-fluor
=1.77 / NA
2
=0.4 m
objective
maximum
imaging
cone angle
r
airy.axial
=?
Airy Disk
Objective
Specimen
Plane
Image
Plane
Eye
Piece
Tube
Lens
Raster Scanning mirrors
Emission
Filter
PMT
Excitation Pinhole
Confocal
Pinhole
Subresolution
bead (<0.2 um)
Dichroic
splitter
Airy Disks
0.5 m bead Plan Apo100x 1.4 NA oil
0.0 m
1.1 m
2.4 m
5.2 m
3.7 m
3.4 m
Visualization - Muscle Spindle Example
Wide field non confocal
Thin slice and z-series
Gallery
Sequence of slices
Orthogonal view
Average & Maximum projections
Volume render
Multiple channels (dyes / fluorophores)

Ia afferent
Ia inhibitory
interneuron
E xtensor (antagonist) muscle
F lexor (agonist) muscle
Muscle spindle
Co m p o n en t s o f t h e
St r et c h (My o t at i c )
Ref l ex
Muscle Spindle
Transmitted
light 16x
widefield
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 slices
40x 1.3 NA 230 um
Orthogonal View
Extended Focus 40x
Maximum projection
Brightest pixel in column of
stack
Average projection
Sum corresponding pixels and
divide by the number of slices
x
z
y
Extended Focus 40x
Maximum projection
Average projection
Volume Render
Each voxel intensity * opacity
Confocal Multichannel Fluorescence
FITC Texas
Red
Triple band
Filter (ChromaCorp.)
- Exciter
- Dichroic
- Emission
Em
Em
Ex
Ex
Confocal Multichannel Fluorescence
Confocal Multichannel Fluorescence
Transmitted light
(non confocal)
Fluorescence
(confocal)
Confocal Multichannel Fluorescence
Ab-Neurofilaments200 (blue) YFP (gray/green) VNaCh(red)
Confocal - Multichannel Fluorescence
Ab-Neurofilaments200 (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, Ca
2+
Spectral scanning
FRAP fluorescence recovery after photobleaching
Confocal Microscopy Summary
Fluorescence Reflection
Fixed or live cells/tissues
Protein location in cell
Membrane/Lipid location
Fluid compartments
Material 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 power
3. Fluorophore concentration
4. Confocal pinhole size
5. 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, 2
nd
ed.,
J ames Pawley, 1995
Handbook of Biological Confocal Microscopy, 3
rd
ed.,
J ames Pawley, 2006