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J.Paul Robinson - Purdue University Cytometry Laboratories Slide 1 t:/powerpnt/course/ /524lect4.ppt
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
UPDATED February 1, 2000
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.
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Overview
• Components of a confocal microscope system
• Optical pathways
• Optical resolution - Airy disk
• Other components
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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
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Fluorescent Microscope
Objective
Arc Lamp
Emission Filter
Excitation Diaphragm
Ocular
Excitation Filter
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Confocal Principle
Objective
Laser
Emission Pinhole
Excitation Pinhole
PMT
EmissionFilter
Excitation Filter
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Fluorescent Microscope
Objective
Arc Lamp
Emission Filter
Excitation Diaphragm
Ocular
Excitation Filter
Objective
Laser
Emission Pinhole
Excitation Pinhole
PMT
EmissionFilter
Excitation Filter
Confocal Microscope
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MRC 1024 System
UV Laser
Kr-Ar Laser
Optical Mixer
ScanheadMicroscope
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Bio-Rad MRC 1024
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MRC 1024 System
Light Path
PMT
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Optical Mixer - MRC 1024 UVArgon Laser
Argon-KryptonLaser
Fast Shutter
UV CorrectionOptics
FilterWheels
To Scanhead
UV Visible
353,361 nm
488, 514 nm
488,568,647 nm
Beam Expander
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MRC 1024 Scanhead
From Laser
To and from Scope
32
1PMTGalvanometers
EmissionFilterWheel
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To Scanhead
From Scanhead
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Scanning Galvanometers
xy
Laser in
Laser out
Point Scanning
ToMicroscope
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The Scan Path of the Laser Beam767, 1023, 1279
511, 1023
00Start
Specimen
Frames/Sec # Lines1 5122 2564 1288 6416 32
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How a Confocal Image is Formed
CondenserLens
Pinhole 1 Pinhole 2
ObjectiveLens
Specimen
Detector
Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989
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Fundamental Limitations of Confocal Microscopy
FromSource
To Detector
. x,y,z
2
n2 photons2
1
n1 photons
1
z
y
xVOXEL
PIXEL
From: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989
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Optical Resolution
• Gray Level
• Pixelation
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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!
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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.
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Pixels
T
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320x240 x 24
Zoom x 2Zoom x 8
Zoom x 4
Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.
Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.
The final image appears to be very “boxy” this is known as “pixilation”.
The final image appears to be very “boxy” this is known as “pixilation”.
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541x600x8(2,596,800) 1.5x)
361x400x8
(1,155,200) 2x
180x200x8
(288,000) 1X
Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!
Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!
Socrates?….well perhaps not...
Socrates?….well perhaps not...
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320x240 x 24
1500x1125x24
Originals collected at high resolution - compared to a low resolution image magnified
Originals collected at high resolution - compared to a low resolution image magnified
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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
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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.
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Digital Zoom
1 x1024 points
2 x1024 points
4 x1024 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.
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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
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Reflected light imagesCD-ROM pits 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
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SUMMARY SLIDE
• Components of a confocal system
• Optical pathways
• Optical resolution
• Sampling rate (Nyquist Theorem)