Fluorescence examples

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In the phenomenon of fluorescence, a material absorbs light of one color and emits another. An example of this is a molecule that absorbs blue ($\lambda$=400 nm) light and emits green ($\lambda$= 500 nm) light. An example of the energy-levels of the molecule and one transition that occurs is shown in the figure. 

Do

1. What is the change in the energy between the incoming (blue) photon and the outgoing (green) photon? If the photon gains energy in the transition, give your number as positive and if it loses energy in the transition, give your number as negative. Give your answer in eV. 

2. Where does the energy difference come from/go to?

1. The photon loses energy and that energy goes into changing the shape of the molecule so that it is at a higher potential energy state.
2. The photon gains energy and that energy comes from the molecule, which interacts with ATP and extracts energy from an ATP-ADP reaction.
3. The photon loses energy and that energy goes into the kinetic and vibrational energy of other molecules that interact with the molecule that initially absorbs the photon.
4. The photon gains energy and that energy comes from the thermal (kinetic) energy of other molecules as they collide with the absorbing molecule raising it with some probability into an excited energy state.
5. Something else.

One way that fluorescence is used in biology is in fluorescence microscopy. Instead of viewing a specimen with reflected white light, a specific color is used that causes particular fluorophores to fluoresce. Fluorophores can be attached to particular proteins and thereby show up the locations of those proteins.

A problem with fluorescence microscopy is that the light beam fluoresces fluorophores all along its path through the sample. One way to get a more localized view is to use two-photon microscopy. In this case, the fluorophore absorbs two photons instead of one. This is pretty unlikely so a high intensity of light is needed. Two separate lasers are used and only at the point where the two lasers cross is fluorescence at all likely. So you can map the location of the fluorophores by sweeping your lasers through the sample.

3. Compared with one-photon fluorescence microscopy, what kind of laser would you need to do two-photon microscopy?

1. A laser with twice the frequency of the one-photon one.
2. A laser with the same frequency as the one-photon one but with twice the intensity.
3. A laser with the same frequency as the one-photon one but with half the intensity.
4. A laser with half the frequency of the one-photon one.
5. Something else (explain).

Bill Dorland and Joe Redish 5/15/18