BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Lecture 6: Components of the microscope
Department of Basic Medical Sciences,
School of Veterinary Medicine
Weldon School of Biomedical Engineering
Purdue University
Bartek Rajwa, Ph.D.
Bindley Bioscience Center
Discovery Park, Purdue University
J. Paul Robinson, Ph.D.
SVM Professor of Cytomics
Professor of Immunopharmacology & Biomedical Engineering
Director, Purdue University Cytometry Laboratories, Purdue University
See also Microscopy Encyclopedia chapter
http://www.cyto.purdue.edu/flowcyt/research/pdfs/micro.pdf
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This lecture was last updated in January, 2007
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© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Learning Objectives
Understand:
• Components of a confocal microscope system
• Optical pathways used in systems
• Optical resolution - Airy disks
• Reflected light/backscatter imaging
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Benefits of Confocal
Microscopy
• Reduced blurring of the image from light
scattering
• Increased effective resolution
• Improved signal to noise ratio
• Clear examination of thick specimens
• Z-axis scanning
• Depth perception in Z-sectioned images
• Magnification can be adjusted electronically
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Wide-field microscopy
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Arc Lamp
Fluorescent
Microscope
Excitation Diaphragm
Excitation Filter
Ocular
Objective
Emission Filter
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Confocal microscopy
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Confocal
Principle
Laser
Excitation Pinhole
Excitation Filter
PMT
Objective
Emission
Filter
Emission Pinhole
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Fluorescent Microscope
Confocal Microscope
Arc Lamp
Laser
Excitation Diaphragm
Excitation Filter
Excitation Pinhole
Excitation Filter
Ocular
PMT
Objective
Objective
Emission Filter
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
Emission
Filter
Emission Pinhole
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Wide-field vs confocal vs 2-p
Drawing by P.
D. Andrews, I.
S. Harper and
J. R. Swedlow
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Bio-Rad MRC
1024 at Purdue
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Radiance 2100MP
at Purdue
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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MRC 1024 System
UV Laser
Optical Mixer
Computer
analysis
Kr-Ar Laser
Scanhead
Microscope
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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MRC 1024 System
Light Path
PMT
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Optical Mixer - MRC 1024 UV
Argon Laser
353,361 nm
UV
Fast Shutter
Visib
le
UV Correction
Optics
ArgonKrypton
Laser
Filter
Wheels
488, 514
nm
488,568,647 nm
Beam Expander
To Scanhead
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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MRC 1024 Scanhead
3
2
Emission
Filter
Wheel
From Laser
PMT
1
Galvanometers
To and from Scope
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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From Scanhead
To Scanhead
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Scanning Galvanometers
Point Scanning
x
y
Laser out
To
Microscope
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
Laser in
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The Scan Path of the Laser Beam
Start
0
767, 1023, 1279
0
Specimen
511, 1023
Frames/Sec
# Lines
1
2
4
8
16
512
256
128
64
32
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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How a Confocal Image is
Formed
Pinhole 1
Pinhole 2
Specimen
Detector
Condenser
Lens
Objective
Lens
Modified from: Handbook of Biological Confocal
Microscopy. J.B.Pawley, Plennum Press, 1989
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Fundamental Limitations of
Confocal Microscopy
From
Source
PIXEL
2D space
n1 photons
1
1
y
VOXEL
3D space
x
.
z
x,y,z
2
n2 photons
2
To Detector
From: Handbook of Biological Confocal
Microscopy. J.B.Pawley, Plennum Press, 1989
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Gray Level & Pixelation
• Analogous to intensity range
For computer images each pixel is assigned a value. If the
image is 8 bit, there are 28 or 256 levels of intensity If the
image is 10 bit there are 1024 levels, 12 bit 4096 levels etc.
• The intensity analogue of a pixel is its grey level which
shows up as brightness.
• The display will determine the possible resolution since on
a TV screen, the image can only be displayed based upon
the number of elements in the display. Of course, it is not
possible to increase the resolution of an image by
attributing more “pixels” to it than were collected in the
original collection!
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Pixels
• Pixels & image structure
Hardcopy usually compromises pixel representation. With
20/20 vision you can distinguish dots 1 arc second apart
(300 m at 1 m) so 300 DPS on a page is fine. So at 100
m, you could use dots 300 mm in size and get the same
effect! Thus an image need only be parsimonius, i.e., it
only needs to show what is necessary to provide the
expected image.
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Pixels
T
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Magnification beyond acceptable limits
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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320x240 x 24
Zoom x 4
Magnifying with
inadequate
information.
This is known as
“empty
magnification”
because there are
insufficient data
points.
Zoom x 2
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
The final image appears to be very
“boxy” this is known as “pixilation”.
Zoom x 8
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Socrates?….well
perhaps not...
180x200x8
(288,000) 1X
Magnifying with
adequate
information. Here,
the original image
was collected with
many more pixels so the magnified
image looks better!
361x400x8
(1,155,200) 2x
541x600x8
(2,596,800) 1.5x)
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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320 x 240 x 24
Originals
collected at
high resolution
- compared to
a low
resolution
image
magnified
1500 x 1125 x 24
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Sampling Theory
• The Nyquist Theorem
– Nyquest theory describes the sampling frequency (f) required to
represent the true identity of the sample.
– i.e., how many times must you sample an image to know that your
sample truly represents the image?
– In other words to capture the periodic components of frequency f
in a signal we need to sample at least 2f times
• Nyquist claimed that the rate was 2f. It has been
determined that in reality the rate is 2.3f - in essence you
must sample at least 2 times the highest frequency.
• For example in audio, to capture the 22 kHz in the
digitized signal, we need to sample at least 44.1 kHz
(unless of course you can’t hear 22k Hz and then you don’t
need 44.1 kHz!!!!)
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Digital Zoom
1x
1024 points
2x
1024 points
4x
1024 points
Note that we have reduced the
field of view of the sample
Note #2: There will only be a single zoom value where
optimal resolution can be collected.
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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3D imaging using CLSM
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Reflection Imaging
Backscattered light imaging
Same wavelength as excitation
Advantages: no photobleaching since not using a
photo-probe (note: does not mean no possible
damage to specimen)
Problems: optical reflections from components of
microscope
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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CLSM can be employ autofluorescence!
Example: topography of SIS
Raw image
Deconvolved image
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Backscattered light imaging – another
CLSM modality
Collagen gel – an optical
section
Collagen gel (ECM model) visualized by
BSL-confocal microscopy. Volume- (left)
and surface-rendering (right) of a
confocal dataset.
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Backscattered light: tissue imaging
Left: Optical sections of SIS
visualized with BSL-confocal
technique. Bottom: color-coded
height map revealing the
topography of height map of SIS
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Example: BSL and AF signals
combined
HepG2 cells grow embedded
within a collagen matrix
(animation)
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Backscattered light and autofluorescence
signals combined: collagen gel & HepG2
cells
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Reflected light images
CD-ROM pits
imaged on a 1024
Bio-Rad confocal
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
Collagen imaged by
a 1024 Confocal
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Imaging spectroscopy
1400
1200
800
600
Intensity [AU]
1000
400
200
617
609
601
593
586
579
572
565
558
552
546
540
534
528
522
517
511
506
501
496
491
0
Wavelength [nm]
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Spectral CLSM (Leica)
Fluorescence signal is dispersed on a
prism, to provide spectral analysis
capability
Excitation via acousto-optical
tunable filter:
•
Free selection of excitation
ines
•
Individual line attenuation
•
Less crosstalk
•
Less fading
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Spectral CLSM (Zeiss)
Multianode PMT
Zeiss LSM 5 LIVE
Dispersion grating
A new system concept of optics and electronics
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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Issues for good confocal imaging
• Axial Resolution
– Must determine the FWHM (full width half maximum) intensity values of a
vertical section of beads
• Field Flatness
– Must be able to collect a flat field image over a specimen - or z-axis
information will be inaccurate
• Chromatic Aberration
– must test across an entire field that emission is constant and not collecting
radial or tangential artifacts due to chromatic aberration in objectives
• Z-drive precision and accuracy
– must be able to reproducibility measure distance through a specimen - tenths
of microns will make a big difference over 50 microns
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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SUMMARY SLIDE
•
•
•
•
•
Components of a confocal system
Optical pathways
Optical resolution
Sampling rate (Nyquist Theorem)
Reflection imaging
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
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