W7 Basic Principles and Limitations of Fluorescence Microscopy Flashcards
potential pitfalls in PLA, FRET and bimolecular fluorescence complementation
they are methods in which readout is based on proximity
may be instances in which the two POI interact yet no signal is obtained due to spatial orientation of the fluorescence proteins with respect to each other
what must happen in FRET for energy transfer to occur
donor emission must overlap with acceptor excitation
how to calculate resolution
d = wavelength/2 x NA
ways to increase resolution
use shorter wavelength
use optical components with high numerical aperture (NA)
specimen viewed through imaging medium with relatively high refractive index (n)
combination of all 3 factors
(good resolution: diffraction limit not exceeded)
definition of resolution
term to describe to what extent an optical system can resolve two objects
definition of numerical aperture (NA)
dimensionless number that defines the resolving power of the objective lens
NA of most 40x-100x objective lenses is 1.0-1.4/1.5
relationship between resolution limit and wavelength
resolution limit in optical microscopy is half the wavelength of the light used to image
definition of airy disk
light passes through any size aperture > diffraction > pattern of bright region in center surrounded by series of concentric rings of decreasing intensity around it called airy disk
definition of point spread function (PSF)
the three dimensional image of the airy disk pattern > describes the response of an imaging system to a point source or point object
how does NA effect resolution
low NA > small angle > narrow light cone > low resolution
high NA > large angle > broad light cone > high resolution
formula of NA
NA = n x sin(deter)
n = refractive index of imaging medium
deter = angular aperture of the objective lens
why does adding immersion oil give higher resolution
refractive index of immersion oil matches refractive index of glass coverslips with defined index > higher NA > higher resolution
common colocalisation algorithms
Pearson’s correlation coefficient, PCC (insensitive to pixel intensity)
Manders overlap coefficient, MOC (insensitive to pixel distribution)
