Posted by
Emmanuel Gustin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Microscopy-used-equipment-tp4594144p4598850.html
Steffen,
Simplified theories of fluorescence of course omit a lot of details. The Jablonski diagrams that are often presented to explain the principle, only have one ground state and one excited state, and perhaps a triplet state. But in reality there is a larger number of electronic states, and therefore more different transitions are possible, as far as quantum mechanical selection rules permit.
We start at the bottom, in the ground state, and by putting in light energy, we move the electrons to a higher state. In first approximation, because of conservation of energy, the excitation wavelength selects a specific excited state -- one out of several possible ones, although the number is limited in practice by the stability of the molecule. Light is then emitted by a radiative transition to a lower state, but that lower state doesn't have to be the original ground state; it can be a state between the excited state and the original ground state.
Excitation and emission spectra can overlap slightly without breaking the law of conservation of energy because the energy that can be converted into light isn't exclusively electronic energy: The transition can include a change in vibrational energy as well. As long as the temperature is above absolute zero, the relaxed excited state is not really the lowest energy level of the excited state; instead there is a spread, given by the Boltzmann distribution, over a number of different vibrational levels associated with the excited state. Therefore it is possible to get out a bit more energy than you put in, by transiting from a high vibrational level in the excited state to a lower vibrational level in the ground state. This is known as anti-Stokes fluorescence.
It converts heat into light, but statistically, of course, that is far less likely to happen than a net conversion of light into heat, unless under very special conditions.
Best Regards,
Emmanuel
--
Emmanuel Gustin, Tel. (+32) 15 46 1586, e-mail:
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-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Steffen Dietzel
Sent: vrijdag 19 februari 2010 14:10
To:
[hidden email]
Subject: Fluorescence theory. was: chlorophyll and associated pigment spectra
At 07:51 19.02.2010, you wrote:
>Hi Christian,
>
>If you use the 633 laser, you'll get the
>expected emission peak at around 695 nm which is
>largely photosystem II emission, then a flat
>tail up to 760 or so which is mostly PSI. There
>are many absorption and emission spectra around,
>though I suspect quite a few of these are for
>isolated pigments in a polar solvent. The
>emission spectra depend very strongly on the
>wavelength(s) of the excitation light, so there
>isn't really a standard emission
How does that fit to the theory of fluorescence?
The theory says that fluorescence occurs when an
electron is falling from the lowest energy level
of the excited state to (any) energy level of the
ground state, emitting a photon during the
process. I thought because of that, the
fluorescent spectrum wavelength is supposed to be
always the same, independant of the mode of excitation.
Did I miss something or is there a problem with the theory?
While I am on it: If the theory is correct, how
can excitation and emission spectra overlap
without breaking the law of conversation of
energy? (Example: If you would excite FITC with
510 nm, how could you obtain the part of the
emission spectrum below 510? I am not sure one
actually would get this part, but if not, this
would seem to argue against the theory of fluorescence.)
Steffen
-- ---------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Marchioninistr. 15, D-81377 München