Download Optical Microscopy and 4 Pi Microscopy

Optical Microscopy and 4
Pi Microscopy
Carolyn A. Sutton
PH 464
Overview
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The Optical
Microscope
• Resolution
• Advantages and
Disadvantages
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Confocal
Microscopy
4 Pi Microscopy
Optical Microscope for Metallography
Optical Microscope: Origins
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Invented by either
Hans and
Zacharias Janssen
or Galileo Galilei
Galileo’s compound
microscope
Huygen’s two lens
ocular system
achromatically
corrected
A Modern Optical Microscope
Optical Microscope: Components
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Eyepiece: used to
focus and view
subject
Objective lens:
used to enlarge
subject
Stagepiece: used
to hold subject
Illumination
Source: used to
light subject
Parts of a Simple Optical Microscope
Optical Microscope: Optics
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Objective lens
close to subject
creates enlarged
inverted image
inside tube.
Compound
eyepiece then
focuses rays into
virtual image for
viewing
Optics in Simple Compound Microscope
Optical Microscope: Advantages
and Disadvantages
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Advantages:
• Relatively easy to
use
• Samples analyzed
in water or air
• Samples shown in
natural color
• Magnifications are
100 – 1000X.
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Disadvantages:
• Depth of focus
decreases with
increasing
magnification
• Resolving capability
depends on
wavelength of
optical light (400 –
700 nm), objective
lens, and sample
itself.
Confocal Microscope
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Improvement of
optical microscope
• Shallow depth of
field
• Elimination of outof-focus glare
• Collection of optical
sections from thick
specimens
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Patented by Marvin
Minsky in 1957
Confocal Microscope
Confocal Microscope: Components
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Eyepiece, objective
lens, and
stagepiece like
optical
Point illumination
source, or laser, is
used
Pinhole detector
aperture to
eliminate out of
focus light
Confocal Microscope Components
Confocal Microscope: Optics
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Light from laser
floods specimen.
Light then is
focused through
objective lens.
Pinhole aperture
then eliminates
out-of-focus light.
Requires scanning
over specimen.
Optics in Confocal Microscopy
Confocal Microscopy: Advantages
and Disadvantages
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Advantages:
• Improved 3D
imaging.
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Disadvantages:
• Does not really
improve resolution
of optical
microscope (180
nm in focal plane
and 500 nm along
optic axis)
Laser Scanning Confocal
Microscope Configuration
4Pi Microscopy
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Stimulated
Emission Depletion
Microscopy (STEM)
provided resolution
of up to 28 nm.
Two microscope
objectives with a
common focus are
used to increase
microscope
aperture.
Aperture Angle in Microscopy
4Pi vs. Confocal Microscopy
a) Standard Confocal, b) 4Pi,
Profiles in c) Confocal and d) 4Pi
Questions?