Basic Microscopy – An Overview – October 2005 Protistology Course MBL, Woods Hole, MA.
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Transcript of Basic Microscopy – An Overview – October 2005 Protistology Course MBL, Woods Hole, MA.
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Basic Microscopy Basic Microscopy – An Overview –– An Overview –
October 2005October 2005
Protistology CourseProtistology Course
MBL, Woods Hole, MAMBL, Woods Hole, MA
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Brief History of the MicroscopeBrief History of the Microscope
What‘s a microscope?What‘s a microscope?
Definition of MagnificationDefinition of Magnification
Conventional Viewing DistanceConventional Viewing Distance
Leeuwenhoek > Compound > Stereo Leeuwenhoek > Compound > Stereo Microscope Microscope
The “Telescope”, a simple detourThe “Telescope”, a simple detour
How to make the specimen visible – How to make the specimen visible – Contrast!Contrast!
Definition of ContrastDefinition of Contrast
Techniques: Techniques: BrightfieldBrightfield PhasePhase DarkfieldDarkfield PolPol DIC (Differential Interference Contrast)DIC (Differential Interference Contrast) FluorescenceFluorescence Optical Sectioning – an expansion of FluorescenceOptical Sectioning – an expansion of Fluorescence
Setting up the Microscope (Lab) Setting up the Microscope (Lab)
Koehler IlluminationKoehler Illumination
Resolution & Empty MagnificationResolution & Empty Magnification
Age
nda
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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.
MB ~ 2x MA
A B
What is “Magnification”?
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Conventional Viewing Distance
250 mm
1x
?
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“Magnification” 1x
f = 250 mm
1x
1x
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Magnification via Single Lens
f = 250 mm
1x
Example: f=50mm
5x
Magnifying Glass (Loupe)Lensf
mmM
250
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The simple microscope
Leeuwenhoek Microscope
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The -corrected Compound Microscope
Eyepiece
Tube
Objective f250mm
250mm
f
f250mm
M
EyepieceObjectiveMicroscope Compound M MM
Tube lens
(Zeiss: f=164.5mm)
Objective
Eyepiece
EyepieceObjective
Tube
f250mm
ff
M
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Q: What happens if we take the objective away?
Tube lens
(Zeiss: f=164.5mm)
Objective
Eyepiece
Eyepiece
Tube
f
fTelescopeM
Eyepiece
Tube
f250mm
250mm
fM
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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Upright microscope.
Inverted microscope
Mixed Illumination
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Other Ways to Illuminate
Reflectors
Ring Lights
Fiber Optics
LED’s
Etc.
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Which Microscope types are typically illuminated this way?
Reflectors
Ring Lights
Fiber Optics
LED’s
Etc.
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“Couldn’t 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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1896: Drawing by Horatio S. Greenough
1897 – the first Stereo Microscope in the world, built by Zeiss, according to the “Greenough” principle
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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 BreakShort Break