Microscopy Techniques for Biomaterial Characterization:

A PrimerPrabhas V. Moghe

Lecture 3September 21, 1999

RU CBE 533 or BME 553; NJIT BME 698

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

Principle of Compound Light Microscopy

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

Compound Microscopy: Optical Issues

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.

Bright and Dark Field Contrast

Bright Field Microscopy

Dark Field Microscopy

Principle of Phase Contrast Microscopy

• Zernicke: Greatest advance in Microscopy (1953)• Phase microscopy requires phase objectives and a phase condensor.

Phase Contrast Microscopy

Differential Interference Contrast

• 3-D like appearance• DIC polarizer and prismsrequired; Individual prismsrequired for each objective.(Relatively expensive)

Differential Interference Contrast

Fluorescence Microscopy:Principle of Fluorescence

Fluorescence Microscopy

Fluorescence Microscope

MercuryLight Source

ExciterFilter

Dichroic MirrorBarrierFilter

Objective/Condensor

Specimen

Exploded View of a Filter Cube

Immunofluorescence

Principle of Confocal Optical Microscopy

illumination& detectionapertures

focus

above belowlens