Microscopy Techniques for Biomaterial Characterization: A Primer
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Microscopy Techniques for Biomaterial Characterization:
A PrimerPrabhas V. Moghe
Lecture 3September 21, 1999
RU CBE 533 or BME 553; NJIT BME 698
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Outline• Physics of Compound Light Microscopy• Light Microscopy Modes
Bright Field & Dark FieldPhase ContrastDifferential Interference ContrastFluorescenceConfocal Laser Scanning Mode
• Techniques For Biomaterial Topography AnalysisAtomic Force MicroscopyProfilometryConfocal Laser Scanning Microscopy - Case Studies
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Principle of Compound Light Microscopy
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Physics of Optical Microscopy• The ability of a microscope objective to "grasp" the various rays coming from each illuminated part of the specimen is related to the angular aperture of the objective. N.A. = n . sin (u); n= refractive index; u=1/2 subtended angle- Max theoretical N.A. of a dry objective is 1- Max theoretical N.A. of oil immersion objectives is 1.5
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Compound Microscopy: Optical Issues
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Optical Microscopy Issues: Resolution
• Resolution is defined as the ability of an objective to separate clearly two points or details lying close together in the specimen.
R=0.61λN.A.
where R=resolution distance; , the wavelength oflight used; N.A. = the numerical aperture.
- As N.A. increases, resolution gets better (R smaller).- Longer wave lengths yield poorer resolution.
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Bright and Dark Field Contrast
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Bright Field Microscopy
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Dark Field Microscopy
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Principle of Phase Contrast Microscopy
• Zernicke: Greatest advance in Microscopy (1953)• Phase microscopy requires phase objectives and a phase condensor.
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Phase Contrast Microscopy
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Differential Interference Contrast
• 3-D like appearance• DIC polarizer and prismsrequired; Individual prismsrequired for each objective.(Relatively expensive)
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Differential Interference Contrast
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Fluorescence Microscopy:Principle of Fluorescence
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Fluorescence Microscopy
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Fluorescence Microscope
MercuryLight Source
ExciterFilter
Dichroic MirrorBarrierFilter
Objective/Condensor
Specimen
Exploded View of a Filter Cube
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Immunofluorescence
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Principle of Confocal Optical Microscopy
illumination& detectionapertures
focus
above belowlens