Basic Microscopy An Overview

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Basic Microscopy An Overview

• October 2005
• Protistology Course
• MBL, Woods Hole, MA

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• Brief History of the Microscope
• Whats a microscope?
• Definition of Magnification
• Conventional Viewing Distance
• Leeuwenhoek gt Compound gt Stereo Microscope
• The Telescope, a simple detour
• How to make the specimen visible Contrast!
• Definition of Contrast
• Techniques
• Brightfield
• Phase
• Darkfield
• Pol
• DIC (Differential Interference Contrast)
• Fluorescence
• Optical Sectioning an expansion of Fluorescence
• Setting up the Microscope (Lab)
• Koehler Illumination
• Resolution Empty Magnification

Agenda
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• Other things you may wish to know but there
  wont be enough time today
• Components of the Microscope
• Different Light Sources
• Field / Aperture Diaphragms
• Condensers why
• Objectives what to consider in the selection
  process
• Adapting Cameras
• Understanding Numerical Aperture
• Diffraction Experiment

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Objects appear to the eye at different
magnifications, depending on their distance from
the eye. Accommodation (lens) will make it
possible.
What is Magnification?
MB 2x MA
A
B
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Objects appear to the eye at different
magnifications, depending on their distance from
the eye. Accommodation (lens) will make it
possible.
What is Magnification?
MB 2x MA
A
B
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Conventional Viewing Distance
?
1x
250 mm
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Magnification 1x
1x
f 250 mm
1x
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Magnification via Single Lens
1x
f 250 mm
Magnifying Glass (Loupe)
5x
Example f50mm
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The simple microscope
Leeuwenhoek Microscope
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The ?-corrected Compound Microscope
Eyepiece
Tube lens (Zeiss f164.5mm)
Objective
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Q What happens if we take the objective away?
Eyepiece
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Tube lens (Zeiss f164.5mm)
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Objective
250mm
f


M
Tube


f
250mm
Eyepiece
Answer We have created a Telescope
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AxioImager Upright Research Microscope
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Axiovert 200 Inverted Research
Microscope
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The basic light microscope types
Upright microscope .
Inverted microscope
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Illumination via Transmitted Light
The specimen must be transparent !
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Upright microscope .
Inverted microscope
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Illumination via Reflected (Incident) Light
Eg. Fluorescence, Opaque Samples
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Upright microscope .
Inverted microscope
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Mixed Illumination
Upright microscope .
Inverted microscope
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Other Ways to Illuminate
Reflectors Ring Lights Fiber Optics LEDs Etc.
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Which Microscope types are typically illuminated
this way?
Reflectors Ring Lights Fiber Optics LEDs Etc.
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• Couldnt one build a microscope for both
  eyes, and thereby generate spatial images?
• Question addressed to Ernst Abbe in 1896
• by Horatio S. Greenough

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1897 the first Stereo Microscope in the world,
built by Zeiss, according to the Greenough
principle
1896 Drawing by Horatio S. Greenough
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Greenough Type
Telescope Type
Introduced first by Zeiss - 1946
Introduced first by Zeiss - 1897
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Greenough Stereo Microscopes
SteMi DV4
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Greenough Stereo Microscopes
SteMi 2000 (2000-C, 2000-CS)
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Research Stereo Microscopes
SteREO Discovery V12
SteREO Lumar V12
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• Short Break