Confocal Microscopy and Related
Techniques
Chau-Hwang Lee（李超煌）
Associate Research Fellow
Research Center for Applied Sciences, Academia Sinica
128 Sec. 2, Academia Rd., Nankang, Taipei 11529, Taiwan
E-mail: clee@gate.sinica.edu.tw
1
Imaging Microscopy
2
Light
Light path
path in
in an
an optical
optical imaging
imaging microscope
microscope
Images are from http://micro.magnet.fsu.edu/
3
Image
Image formation
formation
1 1 1
+ =
do di f
Images are from http://micro.magnet.fsu.edu/
4
Specifications
Specifications of
of an
an objective
objective
Images are from http://micro.magnet.fsu.edu/
5
Achromatic
Achromatic
Images are from http://micro.magnet.fsu.edu/
6
Types
Types of
of objectives
objectives
Objective
Type
Achromat
Spherical
Aberration
1 Color
Chromatic
Aberration
2 Colors
Field
Curvature
No
Plan Achromat
Fluorite
1 Color
2-3 Colors
2 Colors
2-3 Colors
Yes
No
Plan Fluorite
3-4 Colors
2-4 Colors
Yes
Plan Apochromat
3-4 Colors
4-5 Colors
Yes
Images are from http://micro.magnet.fsu.edu/
7
Resolution
Resolution
Without resolution, magnified images cannot
provide detailed information.
Images are from http://micro.magnet.fsu.edu/
8
Numerical
Numerical aperture
aperture
Images are from http://micro.magnet.fsu.edu/
9
Numerical
Numerical aperture
aperture and
and resolution
resolution
Rayleigh criterion:
resolution ~ 0.61λ /NA
0.61λ /NA
For dry samples, NA < 1.0
clearly resolved
resolution limit
Ref: M. Born and E.Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1980), Chap. 8.
10
Depth
Depth of
of field
field
d = λn/(NA)2
Images are from http://micro.magnet.fsu.edu/
11
Contrast
12
Fluorescence
Fluorescence microscopy
microscopy
False color images. Usually a monochrome
camera is used to capture the images, and
color is added in the digital image files.
(emission)
Images are from http://micro.magnet.fsu.edu/
13
Fluorescence
Fluorescence microscopy
microscopy
Images are from http://micro.magnet.fsu.edu/
14
Differential
Differential interference
interference contrast
contrast (DIC)
(DIC)
The contrast is from the
gradient of the optical paths,
not the optical paths.
15
Orientation
Orientation in
in DIC
DIC
Ref: D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging
16
(Wiley-Liss, New York, 2001).
Comparison
Comparison between
between phase
phase contrast
contrast and
and DIC
DIC
17
Fluorescence
Fluorescenceresonance
resonanceenergy
energytransfer
transfer(FRET)
(FRET)Microscopy
Microscopy
Images are from http://micro.magnet.fsu.edu/
18
Confocal Microscopy
19
Confocal
Confocal microscopy
microscopy
Conventional fluorescence microscopy
Confocal microscopy
Images are from Scientific American, August 1994, p. 34.
20
Confocal
Confocal images
images
Improved depth resolution
Images are from http://micro.magnet.fsu.edu/
21
Three-dimensional
Three-dimensional point-spread
point-spread function
function
Ref: Carl Zeiss, Confocal Laser Scanning Microscopy
22
Effect
Effect of
of the
the pinhole
pinhole diameter
diameter and
and NA
NA
Ref: Carl Zeiss, Confocal Laser Scanning Microscopy
23
Scanning
Scanning system
system
Images are from http://micro.magnet.fsu.edu/
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Nanometer
Nanometerdepth
depthresolution:
resolution:differential
differentialconfocal
confocalmicroscopy
microscopy
When signal light is from a single surface
zero derivative
1.0
signal
0.8
0.6
linear region
0.4
0.2
0.0
-4
-2
0
2
4
axial displacement (µm)
Typical slope in the linear region = 1/µm
10 nm displacement = 1% signal variation
Ref: C.-H. Lee and J. Wang, Opt. Commun. 135, 233 (1997).
25
Sample
Sample images
images of
of DCM
DCM
70-nm deep H-trench on InGaAs
human red blood cell
80
40
0
0
glass slide
20
40
60
80
cell surface
The center recess is 570 nm.
400
height (nm)
fused silica
(R = 4%)
aluminum
(R = 80%)
200
100
0
Ref: C.-W. Tsai, C.-H. Lee, and J. Wang, Opt. Lett.
24, 1732 (1999).
profiled by DCM
profiled by AFM
300
0
20
40
60
distance (µm)
80
26
Digital Images
27
AA digitized
digitized image
image
Images are from http://micro.magnet.fsu.edu/
28
Charge-coupled
Charge-coupled device
device (CCD)
(CCD)
29
Specifications
Specifications of
of CCD
CCD cameras
cameras
ƒ pixel size (8 µm; 23 µm)
ƒ pixel resolution (640 x 480; 1024 x 1024)
ƒ spectral response (300 nm to 1000 nm)
ƒ well depth (> 300,000 e-)
ƒ dark current ( 50 pA/cm2 at 20 oC)
ƒ dynamic range (> 85 dB)
ƒ digital or analog
ƒ bit depth (10 bit; 12 bit; 14 bit...)
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Signal
Signal digitization
digitization
pixels
31
Sufficient
Sufficient sampling
sampling
Sampling frequency ≥ 2 x signal bandwidth
For CCD cameras, the pixel size on the image should be smaller
than half the optical resolution.
From Carl Zeiss, Confocal Laser Scanning Microscopy
32
Related Technologies
33
Multiphoton
Multiphoton Microscopy
Microscopy
IR light can
penetrate deeper
into the tissues.
Femtosecond laser
pulses are required to
perform two-photon
excitation.
Images are from http://micro.magnet.fsu.edu/
34
Widefield
Widefield optically
optically sectioning
sectioning microscopy
microscopy
Homodyne detection principle
spatial phase shift: 2π/3
Ip =
( I1 − I 2 ) + ( I1 − I 3 ) + ( I 2 − I3 )
2
2
2
Axial response curve:
focal plane
sample
Ref: M. A. A. Neil, R. Juskaitis, and T. Wilson,
Optics Letters 22, 1905 (1997).
Carl Zeiss, ApoTome
Sectioned
Sectioned fluorescence
fluorescence images
images without
without scanning
scanning
Fluorescence
Optically sectioned
The
Theconcept
conceptof
ofdifferential
differentialconfocal
confocalmicroscopy
microscopy
When signal light is from a single surface
zero derivative
1.0
signal
0.8
0.6
linear region
0.4
0.2
0.0
-4
-2
0
2
4
axial displacement (µm)
Typical slope in the linear region = 1/µm
10 nm displacement = 1% signal variation
Ref: C.-H. Lee and J. Wang, Opt. Commun. 135, 233 (1997).
37
The
TheNIWOP
NIWOPtechnique
technique
70 nm trench on InGaAs
14 bit CCD camera
stabilized
Band pass filter
lamp
(350-610 nm)
Water-immersion
objective
All the components are added
outside a bench-top microscope.
Height (nm)
grid pattern
250
200
150
AFM
this technique
100
50
0
5
10 15 20 25 30 35
Distance (µm)
This technique is called non-interferometric widefield optical profilometry
(NIWOP).
C.-H. Lee, H.-Y. Mong, and W.-C. Lin, Optics Letters 27, 1773 (2002).
38
Observation
Observationof
ofmembrane
membraneripples
ripplesof
ofaaliving
livingcell
cell
10 µ m
Bright field image
nm
The ripples are moving away from the cell edge with an
average speed about 1.3 µm/h.
C.-C. Wang, J.-Y. Lin, and C.-H. Lee, Optics Express 13, 10665 (2005).
39
Highlighted in Virtual Journal for Biomedical Optics (January 2006)
40
Stimulated
Stimulated emission
emission depletion
depletion (STED)
(STED) microscopy
microscopy
Ref: G. Donnert et al., Proc. Natl. Acad. Sci. USA 103, 11440 (2006).
41
Confocal
Confocal microscopy
microscopy for
for single
single molecules
molecules
Blinking
Ref: W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
42
X-ray
X-ray microscopy
microscopy
The resolution of a zone plate is
almost equal to the smallest
(outermost) zone width. With current
e-beam lithography, the smallest
zone width can be ~15 nm.
Ref: C. Jacobsen, Trends Cell Biol. 9, 44 (1999).
Ref: W. Chao et al, Nature 435, 1210 (2005).
43
Compact
Compact soft
soft x-ray
x-ray microscope
microscope
Resolution ~ 100 nm
Image of diatom
Rmax @ 3.37 nm
Ref: M. Berglund et al., J. Microsc. 197, 268 (2000).
44
X-ray
X-ray microtomography
microtomography
Commercial product available
Vesicles inside a cell
http://www.microphotonics.com
Capacitor
Resolution ~ 250 nm
Ref: Y.Wang et al., J. Microsc. 197, 80 (2000).
Resolution < 10 µm
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