Resolving Molecules with Light Flashcards
How does fluorescence of a molecule occur?
An electron drops to the ground state by emitting a photon of light after being excited by some type of energy.
How does the Stokes shift occur?
A photon is supplied by an external source which is absorbed by the fluoroform, creating an excited state. Some energy is dissipated and the electron drops to a lower energy state. After 1-10ns the electron drops to the ground state emitting a photon with a lower energy than the supplied one. This energy difference is the Stokes shift.
How is the photon emitted from the molecule detected?
Using a wavelength-selective filter as the external and emitted photons have different wavelengths.
How does the diffraction limit effect detection of fluoroforms?
If two molecules are close together the two point spread functions (PSF) cannot be resolved separately.
How can fluorescing molecules overcome the diffraction limit? Give 3 reasons.
Any three from:
Not all molecules fluoresce. A molecule can be labelled with a fluorescing molecule.
Stokes shift means light from other sources can be blocked.
Light is emitted from a point. Find the source of light and you have seen the molecule.
Molecules can be switched on and off.
What is blinking?
The spontaneous on/off behaviour of fluoroforms.
What is resolution controlled by in resolving molecules with light? (3 points)
The signal to noise ration of the detector.
The amount of light collected from the molecule.
The mechanical stability of the system.
Why does increasing the numerical aperture increase the resolution?
The amount of collected light affects the signal to noise ratio.
What 2 factors do not affect the resolution of this method?
Wavelength.
Diffraction limit.
What is the point spread function of a molecule?
The image of a point as an Airy disk due to the light passing through the aperture of the microscope.
How can we use a PSF to locate a molecule?
The most intense point of the PSF is where the molecule is.
How does the STORM method work?
By switching molecules on and off and mapping there individual PSFs to recreate their location on a computer.
