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Biomedgradstudent's picture

Hi everyone,
 I am brand new in the field of biophysics, let alone fluorescent spectroscopy. I believe that the latter method is used to quantify protein interactions by measuring fluorescent intensities and providing excitation/absorption spectra. I have difficulty in understanding the following related terms/methods “time-resolved”, “polarization”, “lifetime” “anisotropy” and “steady-state.” I appreciate if anyone can shed light into this.


Sami Tuomivaara
Sami Tuomivaara's picture


Fluorescence is a very interesting phenomenon, and can be utilized in various ways in biochemistry and related disciplines.

Time-resolved and steady-state fluorescence spectroscopy are the two reciprocal ways in which fluorescence can be utilized in studying events at molecular level. In time-resolved spectroscopy, one uses short (order of picoseconds) excitation pulse that excites the fluorophore(s) to a higher energy state. After the pulse, one starts detecting the decay of the emission with high time-resolution to get a decay curve. Because one records individual datapoint (measuring the remaining intensity) in various time points every few picoseconds or so, this method is called time-resolved. How fast the curve decays depends on the lifetime(s) of the fluorophores.

Lifetime is the average time that excited fluorophore stays in its excited state, before emitting a photon (that can be detected) and hence decaying into ground state. The lifetimes of many fluorophores are in the regime of 0.1 to 10 nanoseconds. If lifetime is low, the decay curve goes to zero rapidly, if it is high, the decay curve decays slower. Because of many (bio)molecular processes such as molecular rotation occur at this timescale, fluorescence is very valuable tool studuing them.

In steady-state spectroscopy, one uses continuous excitation and continuous detection simultaneously to detect the fluorescence emission. The signal is averaged  for long time (long time compared to the molecular events, perhaps up to seconds to get good signal to noise ratio). In steady-state experiment, information regarding the lifetimes of the fluorophores is lost.

Polarization and anisotropy refer to how polarized excitation light behaves when "hitting" the sample.
Polarized light means a light which is passed through some device that let's through only photons with some particular "orientation". If one excites the sample with polarized light, the polarization of the emitted photons is different from that of the excitation light. This is due to the rotation of the fluorophore between the excitation and emission events. Small fluorophore rotates very fast and the correlation between excitation and emission light polarizations is low. Because the incoming light is of high polarized, the detected light (because low correlation) is of low polarization. Large molecules rotate slowly, and the correlation between excitation and emission light polarizations is high, hence the polarization is high as well. Polarization and anisotropy are basically the same thing, they both describe the correlation between incoming and outcoming light, and hence tell us something about the timescale of the fluorophore rotation. Polarization/anisotropy experiments are especially handy in studying interactions between molecules because molecules alone rotate faster than their complexes (which is of course larger).

You can look up easily the equations describing lifetime, polarization and anisotropy by googling them. There are also some educational articles available that describe fluorescence and its uses.