Lecture 4
The Principles of Confocal Microscopy:
Components of the microscope.
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these
graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material
copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be
used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and
figures in these lecture notes are taken from this text.
J.Paul Robinson - Purdue University Cytometry Laboratories
UPDATED February 1, 2000
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Overview
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Components of a confocal microscope system
Optical pathways
Optical resolution - Airy disk
Other components
J.Paul Robinson - Purdue University Cytometry Laboratories
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Benefits of Confocal Microscopy
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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
J.Paul Robinson - Purdue University Cytometry Laboratories
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Arc Lamp
Fluorescent
Microscope
Excitation Diaphragm
Excitation Filter
Ocular
Objective
Emission Filter
J.Paul Robinson - Purdue University Cytometry Laboratories
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Confocal
Principle
Laser
Excitation Pinhole
Excitation Filter
PMT
Objective
Emission
Filter
Emission Pinhole
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
J.Paul Robinson - Purdue University Cytometry Laboratories
Emission
Filter
Emission Pinhole
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MRC 1024 System
UV Laser
Optical Mixer
Kr-Ar Laser
Scanhead
Microscope
J.Paul Robinson - Purdue University Cytometry Laboratories
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Bio-Rad MRC 1024
J.Paul Robinson - Purdue University Cytometry Laboratories
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MRC 1024 System
Light Path
PMT
J.Paul Robinson - Purdue University Cytometry Laboratories
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Optical Mixer - MRC 1024 UV
Fast Shutter
Argon Laser
353,361 nm
UV
Visibl
e
Filter
Wheels
UV Correction
Optics
ArgonKrypton
Laser
488, 514
nm
488,568,647 nm
Beam Expander
To Scanhead
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
J.Paul Robinson - Purdue University Cytometry Laboratories
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From Scanhead
To Scanhead
J.Paul Robinson - Purdue University Cytometry Laboratories
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Scanning Galvanometers
Point Scanning
x
y
Laser out
To
Microscope
J.Paul Robinson - Purdue University Cytometry Laboratories
Laser in
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The Scan Path of the Laser Beam
Start
767, 1023, 1279
0
0
Specimen
511, 1023
Frames/Sec
# Lines
1
2
4
8
16
512
256
128
64
32
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
J.Paul Robinson - Purdue University Cytometry Laboratories
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Fundamental Limitations of
Confocal Microscopy
From
Source
n1 photons
PIXEL
1
y
VOXEL
x
1
.
z
x,y,z
2
n2 photons
2
To Detector
From: Handbook of Biological Confocal Microscopy.
J.B.Pawley, Plennum Press, 1989
J.Paul Robinson - Purdue University Cytometry Laboratories
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Optical Resolution
• Gray Level
• Pixelation
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!
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.
J.Paul Robinson - Purdue University Cytometry Laboratories
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Pixels
T
J.Paul Robinson - Purdue University Cytometry Laboratories
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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
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)
J.Paul Robinson - Purdue University Cytometry Laboratories
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320x240 x 24
Originals
collected at high
resolution compared to a
low resolution
image magnified
1500x1125x24
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
J.Paul Robinson - Purdue University Cytometry Laboratories
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Raman Scattering
• At an excitation line of 488 nm, Raman scatter
will be at 584 nm or less with increased
concentration of protein, etc.
• Is directly proportional to the power of the laser
light.
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.
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 photoprobe (note: does not mean no possible damage to
specimen)
Problems: optical reflections from components of
microscope
J.Paul Robinson - Purdue University Cytometry Laboratories
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Reflected light images
CD-ROM pits
J.Paul Robinson - Purdue University Cytometry Laboratories
Collagen
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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 reproducibily measure distance through a specimen - tenths of
microns will make a big difference over 50 microns
J.Paul Robinson - Purdue University Cytometry Laboratories
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SUMMARY SLIDE
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Components of a confocal system
Optical pathways
Optical resolution
Sampling rate (Nyquist Theorem)
J.Paul Robinson - Purdue University Cytometry Laboratories
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