W3 Fluorescence-based Applications in Cell Biology Flashcards
what is fluorescence
absorption of photons turns fluorophore into excited state
excited fluorophore will emit photons
definition of stokes shift
the difference in wavelength between the absorption and emission maxima
what is fluorescence activated cell sorting (FACS)
method to sort and separate cell populations or single cells based on fluorescence
live and fixed cells can be sorted
typically done using labeled antibodies against surface-exposed antigens
how does immunofluorescence work
cells are fixed with PFA > permeabilised using detergents to allow detection of proteins inside cell > sample blocked with BSA to prevent unspecific binding > antigen detected using primary antibodies > primary detected with secondary antibodies
difference between wide field and confocal microscopy
pinhole in confocal microscope eliminates out of focus light > produce a diffraction limited spot/ point spread function
what is Z stacking in confocal imaging
each image overlaps with a certain degree with previous one > continuous volume > reconstruct to get 3 dimensional structure of object > can zoom though step by step to obtain sharp image
properties of spinning disk confocal compared to scanning confocal microscopy
spinning disk is faster and more sensitive
less photobleaching
reduced cytotoxicity
ideal for live cell imaging and imaging thick specimen
pinhole size is fixed
what are line scans
simple way to illustrate whether two or more proteins reside in the same place
measures pixel intensities along a drawn line
can be performed on fixed or live cells
potential artifacts in fixed cell imaging
overfixation or use of inappropriate fixatives can affect protein localisation or deform organelles
gluraraldehyde used for fixing can cross link epitopes on antigens > interfere with antibody recognition and immonolabelling
insufficient labelling can cause wash-out of proteins during permeabilisation step
potential artefacts in live cell imaging
cells are sensitive to light
flzuorophores lose their fluorescence if exposed to too much light (photobleaching)
tagging of proteins can interfere with protein function
tagging protein at wrong place > interfere with protein’s interactions with binding partners or disrupt protein dynamics
what is total internal reflection fluorescence (TIRF) microscopy
imaging is restricted to a small volume close to the ventral membrane
faster and more sensitive than laser scanning confocal
less photobleaching
reduced cytotoxicity
ideal for live cell imaging of membrane and cortical proteins
resolution of wide field, TIRF and confocal
wide field: 1000nm
TIRF: 100nm
confocal: 600nm
what is fluorescence resonance energy transfer (FRET)
used to measure proximity of two fluorophores for analysis of protein-protein interactions in live cells
for FRET to work, emission wavelength of donor must overlap with excitation wavelength of acceptor
applications: interaction, proteolysis and conformational change
what is fluorescence complementation
split fluorescence protein into two halves, each is non fluorescent on its own > fuse each to protein A and B > if proteins interact > fluorophore is reconstituted
what is proximity ligation assay (PLA)
primary antibodies attached to two proteins > secondary antibodies with oligonucleotides bound to it attached to primary antibodies > if proteins close > oligos come close enough to be ligated tgt
but close together does not mean interaction
