MIC 205 1st Edition Lecture 3 Outline of Last Lecture II. Golden Age of MicroIII. ExperimentsIV. Scientific MethodV. FermentationOutline of Current Lecture VI. Microscopy-General Principlesa. Electromagnetic Spectrumb. Refraction & Magnificationc. Resolution & ContrastVII. Limits of ResolutionVIII.Types of Light MicroscopesIX. Electron MicroscopesCurrent LectureMicroscopy-General Principles:1) Electromagnetic Spectrum- shorter wavelength = greater resolving power- Resolving power: ability to see small things2) Refraction & Magnification- can’t see anything unless the light bends around the object - Refraction: bending of light (radiation)- Refraction & magnification depend on the medium (air, water, oil) through which the radiation travels3) Resolution & Contrast- (Inversely Proportional) Increased resolution, decreased contrast- Staining is used to increase contrast- Contrast can be improved by using phased &/or polarized lightLimits of Resolution: -Light Microscope = 200nm to 10nm (hard to see viruses, but can see cells)-Electron Microscope = 10nm to 1mm (proteins, ribosomes, viruses)Light Microscopes: white light is ALL light colors, black is the ABSENCE of all colorsTypes of Light Microscopes:These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Bright Field- single magnifying lens (SIMPLE microscope)- 1 or 2 ocular lenses = Compound microscope- light rays pass through the specimen into a series of objective lenses for magnification-TOTAL mag. = ocular mag. X objective mag.Dark Field- for observing pale objects (only light rays scattered by the specimen enter)- Specimen appears light against a dark background-This increases contrast and enables observation of detailsPhase Contrast- light in phases that produce contrast (used on living organisms)- Appears 3D on grey backgroundFluorescence- use of UV light- Appears colored on a black background- Localizes specific cell structures of interest by inserting the color (immunofluorescence)Confocal Fluorescence- uses lasers to illuminate the sample & removes out of focus light- This increases resolution and contrast- A series of digital images can be stacked to create a 3D image Electron Microscopes:-Uses electrons to illuminate the sample (@ a wavelength from 0.01nm to 0.001nm)-Short wavelength = increased amount of resolution and magnification power-Appear in black and white-There are 2 types of Electron Microscopes:- Transmission- appear in cross sections- Scanning Probe- provides a surface view, extremely sensitive (can view single molecules & large atomic
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