Adjusting a Microscope 1Center components on optic axis 2Focus objective 3Focus condenser 4Adjust illumination lamp voltage (intensity) iris diaphragm.

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Presentation transcript:

Adjusting a Microscope 1Center components on optic axis 2Focus objective 3Focus condenser 4Adjust illumination lamp voltage (intensity) iris diaphragm brightness resolution contrast

Sample Preparation fixation organic solvents (acetone, alcohols) formaldehyde glutaraldehyde sectioning for tissues or thick samples embed in parrafin or resin cut with microtome staining dyes differentially bind to DNA, RNA and protein provides more contrast

Light Microscopy Modifications Phase Contrast Differential Interference Contrast (Normarski) Confocal Scanning Fluorescence Dark Field (diffracted light) Image Enhancement

Phase Contrast and Differential Interference Contrast requires special objective and condenser lens phase differences are converted into intensity differences distinguish objects that only differ slightly in refractive index or thickness

Light Microscopy Modifications Phase Contrast Differential Interference Contrast (Normarski) Confocal Scanning Fluorescence Dark Field (diffracted light) Image Enhancement

video cameras + computers used to enhance images correct imperfections in optical systems overcome limitations of human eye seeing image in dim light seeing small intensity differences against bright background does not increase actual resolution brightness resolution contrast

Limit of Resolution distance at which two objects can be resolved resolution limit = 0.61 /numerical aperture (NA is a lens property) of visible light = m Electron Microscopy samples are analyzed with electrons particles traveling near the speed of light behave as a wave wavelength with velocity resolutions of 2 nm or less

Sample Preparation Fixation glutaraldehyde osmium tetroxide Dehydration ethanol (step-wise) Embedding plastic resins Sectioning ultramicrotome ( nm thick) Staining heavy metals

Variations of Electron Microscopy Transmission (TEM) Scanning (SEM) Shadow-casting Freeze-fracture Freeze-etching CryoEM Negative Staining

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