Intro to Cell Bio Methods Flashcards
what is a flurophore?
molecules that absorb and emit light at specific wavelengths
confocal microscope
scans the sample with narrow excitation beam and collects emitted light through a pinhole so that only light from a single focal plane is collected; creates clear, 3D images by producing a “Z-stack” of planes at increasing distances from the cover glass substrate
deconvolution
uses a Z-stack of images with a standard epifluorescence microscope and uses computer stimulation to subtract the out-of-focus light in each plane and constructs a 3D image
light sheet microscopy
light comes from the side illuminating only 1 focal plane
total internal reflection fluorescence (TIRF)
-excitation beam enters along the edge of the objective barrel and bends sharply when it hits the lens
-evanescent field penetrates cell and excites fluorophores on the basal surface
-method is extremely useful for viewing parts of the cell that are close to the basal surface like the plasma membrane and is extended to visualize the behavior of single molecules in living cells
multiphoton microscopy
produces clear images of thick specimens (10-15 microns) like visualizing neurons deep into the rat cortex
fluorescence resonance energy transfer (FRET)
-allows for detection of intermolecular interactions in cells
-two fluorophores are involved: one donor fluorophore that in an excited state may transfer its excitation energy to a nearby second acceptor fluorophore
-with the donor fluorophore (CFP) on one protein and the acceptor fluorophore (YFP) on another, there will only be fluorescence of YFP if energy from the excited CFP fluorophore is nearby, therefore if you detect YFP fluorescence it would imply interaction of the two proteins with different fluorophores attached
what components make up FRET?
-a donor fluorophore
-an acceptor fluorophore
fluorescence recovery after photobleaching (FRAP)
-measures molecule diffusion in cells
-fluorescent molecules within a cell are imaged then a section of the cell is photobleached with a strong laser
-overtime, the molecules from the surrounding area move into the bleached zone and the fluorescence is recovered
-kinetics of the recovery indicate single or multiple species involved in recovery Ex. determine diffusion of receptor in plane of membrane
electron microscopy
much higher resolution than light/fluorescence microscopy, resolving structures <300 nm
co-immunoprecipitation
-tells you which proteins are interacting with other proteins
-antibody attached to a bead and when you centrigue it down you get binding proteins together
-when you run a gel and separate the proteins in the pellet attached to the bead you get more than your original protein –> considered part of a complex
-carefully choose solubilization method to maintain complexes- typically non-ionic detergent
-non-specific proteins come down whether the protein is there or not
subcellular fractionation
-you could disrupt a cell under general conditions without detergent
-organelles spilled out and they have different sizes and densities
-organelles are separated by centrifugation
fluorescence microscopy
-allows tracking of specific molecules in cells and are configured in different ways for different purposes
1. confocal fluorescence and deconvolution improve images by removing out of focus light
2. two-photon microscopy enables clearer imaging of thick specimens
3. total internal reflection microscopy gives images of cell surface structures with low background
what do biosensors do?
measure biochemical reactions in living cells
what do synthetic biological constructs allow?
allow light and chemical manipulation of cells
how can binding partners be IDed?
co-immunoprecipitation and BioID
