Microscopy Techniques for Biomaterial Characterization: A Primer Prabhas V. Moghe Lecture 3 September 21, 1999 RU CBE 533 or BME 553; NJIT BME 698.

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Presentation transcript:

Microscopy Techniques for Biomaterial Characterization: A Primer Prabhas V. Moghe Lecture 3 September 21, 1999 RU CBE 533 or BME 553; NJIT BME 698

Outline Physics of Compound Light Microscopy Light Microscopy Modes Bright Field & Dark Field Phase Contrast Differential Interference Contrast Fluorescence Confocal Laser Scanning Mode Techniques For Biomaterial Topography Analysis Atomic Force Microscopy Profilometry Confocal 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. where R=resolution distance;, the wavelength of light 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 prisms required; Individual prisms required for each objective. (Relatively expensive)

Differential Interference Contrast

Fluorescence Microscopy: Principle of Fluorescence

Fluorescence Microscopy

Fluorescence Microscope Mercury Light Source Exciter Filter Dichroic Mirror Barrier Filter Objective/ Condensor Specimen Exploded View of a Filter Cube

Immunofluorescence

Principle of Confocal Optical Microscopy illumination & detection apertures focus abovebelow lens