unit 1 - intro to cells + microscopy Flashcards
The method by which cell components are separated by centrifugation at progressively higher speeds is called _______ centrifugation.
differential
Which of the following cell components will be concentrated in the LAST pellet produced by differential centrifugation?
ribosomes
Specific proteins can be localized in the transmission electron microscope with the use of ___
gold-labeled antibodies
What unit of length would you generally use to measure a typical plant or animal cell?
micrometers
Cell biologists employ targeted fluorescent dyes or modified fluorescent proteins in both standard fluorescence microscopy and confocal microscopy to observe specific details in the cell. Even though fluorescence permits better visualization, the resolving power is essentially the same as that of a standard light microscope because the resolving power of a microscope is limited by the __________ of light.
wavelength
What can be used to locat specific proteins in a cell?
Fluorescent-labeled antibodies
Which is a difference between prokaryotes and eukaryotes?
Prokaryotes have one chromosome, eukaryotes have many
“Cell lines” are ___
cancer cells, can be grown in vitro, mostly used in cell biology research, can be purchased.
NOT primary cell cultures
more likely to be different from donor cells than primary cell cultures because mutations arise from continuous divisions
in vitro
tissue culture, grown outside of the organism
in vivo
experiments that are carried out on intact organisms
primary cell culture
obtained from donor and placed in tissue culture
limited number of divisions because the telomeres eventually begin to degrade
secondary cell culture
subculture derived from primary cell culture. limited number of devisions
resolution
ability to tell apart separate points as distinct
clarity and level of detail
brightfield microscope
type of light microscope
stain needed to improve contrast
dead specimen, can’t be used with live cells
magnification
ability of a lens to enlarge the image of an object compared with the real object
combination of eyepiece and objective lenses used
measured as a multiple
resolving power
function of lens design and wavelength of light used to get the image
measured in distance (smaller = clearer)
differential-interference contrast (DIC) microscope
type of light microscope
increases contrast without staining the specimen (can use live cells)
phase-contrast microscope
type of light microscope
increases contrast without staining the specimen (can use live cells)
fluorescent microscope
type of light microscope
uses fluorescent-labeled tags to identify specific molecules, can see location of molecules and can be used in live or dead cells
more expensive than other light microscopes
immunofluorescence
uses a fluorophore/enyme attached to an antibody to target a specific molecule
can be direct (antibody attaches to molecules) or indirect (antibody attaches to a primary antibody that is attached to the molecule)
confocal-laser microscopy
type of light microscope
removes out of focus images
uses laser beams and florescence to produce a bunch of 2D images that can be constructed into a 3D image
expensive but can be used to locate molecules and study live cell interactions
GFP
green florescent protein
technique for protein labelling using fluorescence micropscopy
GFP is fused to protein of interest using recombinant DNA, the tagged protein can be expressed in live cells
photobleaching + FRAP
region of cell expressing GFP labeled protein is bleached with a laser, rate of recovery (or lack of recovery) is a proxy for protein movement within the cell
electron vs. light microscope
electron microscope:
-max magnification: 500,000x
-resolution: 10-0.1 nm
-uses short-wavelength electron beams
-heavy metals like gold used for staining
light microscope:
-max magnification: 1,000x
-resolution: 100 nm
-uses visible light that passes through the specimen and through magnifying lenses
-uses acidic/basic stains or florescent reagents for staining
scanning electron microscopes (SEMs)
focuses beam of electrons on surface of cells coated in a heavy metal, creating images that look 3D
magnification 500,000x, resolution 10 nm
transmission electron microscopes
focus beam of electrons through a very thin slice of specimen, used to study internal structure of cells
magnification 500,000x, resolution 0.1 nm
gold labelling
technique in electron microscopy similar to immunoflorescence– uses gold particles attached to an antibody to target and label specific molecules in the cell
microtome
slices paraffin embedded tissues into thin sheets to be studied (ultra microtome for electron microscopy)
subcellular fractionation
breaking apart the cell and separating the major organelles from one another using centrifugation, allows us to determine function of organelles
differential centrifugation
separates cell components based on size and density. pellet is removed at each speed and the supernatant is centrifuged at sequequentially higher speeds
less precise than density gradient centrifugation
density gradient centrifugation (buoyant density centrifugation)
separates cell components through sedimentation through a dense substance after centrifugation. they settle into bands that are at equilibrium with their density
more precise than differential centrifugation
pellet
heavier organelles that settle to the bottom after centrifugation
supernatant
less dense components that remain at the top after centrifugation
