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
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
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; l, 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.
Principle of Phase Contrast Microscopy • Zernicke: Greatest advance in Microscopy (1953) • Phase microscopy requires phase objectives and a phase condensor.
Differential Interference Contrast • 3-D like appearance • DIC polarizer and prisms required; Individual prisms required for each objective. (Relatively expensive)
Fluorescence Microscope Dichroic Mirror Barrier Filter Exciter Filter Exploded View of a Filter Cube Mercury Light Source Objective/ Condensor Specimen
Principle of Confocal Optical Microscopy focus illumination & detection apertures lens above below