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

  2. 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

  3. Principle of Compound Light Microscopy

  4. 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

  5. Compound Microscopy: Optical Issues

  6. 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.

  7. Bright and Dark Field Contrast

  8. Bright Field Microscopy

  9. Dark Field Microscopy

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

  11. Phase Contrast Microscopy

  12. Differential Interference Contrast • 3-D like appearance • DIC polarizer and prisms required; Individual prisms required for each objective. (Relatively expensive)

  13. Differential Interference Contrast

  14. Fluorescence Microscopy:Principle of Fluorescence

  15. Fluorescence Microscopy

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

  17. Immunofluorescence

  18. Principle of Confocal Optical Microscopy focus illumination & detection apertures lens above below