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

17. Immunofluorescence

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