Adjusting a Microscope1 Center components on optic axis2 Focus objective3 Focus condenser4 Adjust 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

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 = 0.4-0.8 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

(50-100 nm thick)• Staining• heavy metals

Variations of Electron Microscopy

• Transmission (TEM)• Scanning (SEM)• Shadow-casting• Freeze-fracture• Freeze-etching• CryoEM• Negative Staining