Download presentation
Presentation is loading. Please wait.
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
Similar presentations
© 2024 SlidePlayer.com Inc.
All rights reserved.