Normally, a molecule can be driven into the excited state by interacting with a photon that falls within the energy of its absorption transition frequency range. For doing fluorescence measurements, one can then monitor emitted light at a longer wavelength (see Stokes shift). However, one can also achieve excitation using photons of longer wavelengths through multiple photon excitation phenomena. If a fluorophore (molecule that fluoresces) normally absorbs light at 300 nm, then one can excite it using a high intensity laser pulse at 600 nm. The power density must be high enough that two photons can interact with the fluorophore cooperatively. The energy of the two photons at the longer (600 nm) wavelength is approximately equal to the energy of a single photon at the shorter wavelength. Similarly, three photons of 900 nm would also have the same effect. If two lasers are used to generate the power density, that excitation will only occur at the focus where the two beams intersect.

This is what makes multiple photon excitation a useful technique in microscopy. A common procedure in looking at microscope images is to tag specific items with fluorescent dyes. For example, if one attaches a rhodamine group specifically to DNA, then one could see where the DNA is located in the cell, as it would light up red when the rhodamine fluoresces. If you wanted to look at a very specific region of the cell, and wanted to eliminate background fluorescence from surrounding regions, two photon excitation gives an extra dimension of focusing, allowing for sharper pinpointing of tagged items. This is also a good way to eliminate inner filter problems, where your sample absorbs too much light in the single photon range to ever see any fluorscence. By exciting at longer wavelengths, you do not get background absorbance attenuating the light reaching the fluorophore.

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