Microscopy Flashcards
what is a stereo microscope?
observation of intact species, relatively low magnification
what is a compound light microscope?
histological section and cells
what is an electron microscope transmission and scanning?
sub-cellular details
rank from highest to lowest revolution: stereo microscope, compound light microscope, electron microscope,
highest- electron microscope
middle- compound light microscope
lowest- stereomicroscope
the condensor does what?
collects and focuses a cone of light that illuminates the tissue slide on the stage
what does the objective lens do?
Objective lenses enlarge and project the illuminated image of the object toward the eyepiece
what is light?
electromagnetic radiation, visible radiation is between 400 and 700nm
what is a photon?
elementary particle that defines light
what are the 3 basic dimensions of light?
- intensity (amplitude, brightness)
- frequency (wavelength, perceived as colour)
- polarization (angle of vibration)
what is resolution?
minimal distance of two points that can be distinguished
what is numerical aperture?
an estimate of how much light from the sample is collected by the objective
what is refraction?
light ray bends when entering water
max magnification means max resolution (T or F)?
False
what is the most common form of illumination?
Kohler illumination
what are the different contrasting techniques?
- brightfield
- darkfield
- phase contrast
- polarization contrast
- differential interference contrast
- fluorescence
describe brightfield microscopy
light is transmitted through sample and absorbed, useful for specimens that can be contrasted through dyes, little contrast in unstained specimens
* absorption
describe darkfield microscopy
illuminating rays of light are directed through the sample from the side by putting a dark disk into the condensor that hinders the main light beam to enter the objective, only light that is scattered by structure in the sample enters the objective, used a-lot to view diatoms and other colourless specimens
*scattering
what is phase contrast microscopy?
Incident light is out of phase with transmitted light as it was slowed down while passing through different parts of the sample and when the phases of the light are synchronized by an interference lens, a new image with greater contrast is seen.
*phase interference
what is polarization contrast?
Polarized light is used for illumination. Only when the vibration direction of the polarized light is altered by a sample placed into the light path, light can pass through the analyzer. The sample appears light against a black background. A lambda plate can be used to convert this contrast into colours
*polarization
what is polarization contrast used for?
Polarization contrast is used to look at materials with birefringent properties, in which the refractive index depends on the vibration direction of the incident light, e.g. crystals or polymers.
what is bifringence?
the optical property of a material having a refractive index that depends on the polarization and propagation direction of light
what is differential interference contrast?
Also known as Nomarski microscopy. Uses polarized light for illumination. Synchronizing of the different phases of incident and transmitted light is done by a set of prisms and filters introduced into the light path.
* polarization and phase interference
why is DIC better than phase contrast?
makes better use of numerical aperture of the system and allows the microscope to achieve excellent resolution
what are the steps to tissue preparation?
- fixation
- dehydration
- clearing
- infiltration
- embedding
- trimming
what is a microtome and what is it used for?
sectioning paraffin-embedded tissues for light microscopy
describe fixation
Small pieces of tissue are placed in solutions of chemicals that preserve by cross-linking proteins and inactivating degradative enzymes.
describe dehydration
The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water.
describe clearing
Alcohol is removed in toluene or other agents in which both alcohol and paraffin are miscible.
describe infiltration
The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance.
describe embedding
The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden
describe trimming
The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
what is formaldehyde?
good penetration and fixes by chemical crosslinking of -proteins
a good fixation method should do what?
preserve the cell structure with minimum change from the living state (volume, morphology, localization of macromolecules and organelles, etc.).
what is cytosectioning?
skip processing, faster turnover time, embedded tissue into media after fixation, set by freezing, section at low temperature
what is the oct?
optimal cutting temperature
how is an EM sample prepared?
fixation with glutaraldehyde, better crosslinking but poor penetration, post fix with osmium tetroxide.
what is the difference between a transmission electron microscope and a scanning electron microscope?
TEM- ultramicrotome, resin embedded tissue, diamond or glass knives, ultrathin sections, stained with heavy metals, area very small
SEM- samples critically pointed dried, and coated with conductive material
