Re: Fluorescence theory. was: chlorophyll and associated pigment spectra

Posted by Rosemary.White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Microscopy-used-equipment-tp4594144p4601473.html

Yes, exactly.  The different emission spectra (often with different excitation wavlengths) in the literature are usually from experiments on isolated photosystems or pigments under various conditions in which the experimenters are trying to understand the energy dynamics of these isolated components or systems.

Imaging chloroplasts in intact tissues using red light, e.g. 633 nm laser, will give you quite long term steady fluorescence with little bleaching, if that’s what you’re after.  I’ve always thought of chloroplast fluorescence as an energy-shedding mechanism, like heat loss.  As Guy mentions, the loss of far-red emission means that you’ve killed off part of the energy transfer pathway to the photosystem core, so you now see fluorescence from accessory pigments.

cheers,
Rosemary


On 20/02/10 1:08 PM, "Christian" <celowsky21@...> wrote:

Guy,

I made a nod at the facts, only because most of this list works in the much more simple animal systems.  Hemoglobin has nothing compared to the fun in chloroplasts.

I have never noted shifts over time of the signal in my  "~520" channel from imaging.  It all seems to fade equally but only with great effort can I bleach it.  I DO see massive shifts from far red to green in stressed, infected, dying, dehydrated, you name it, chloroplasts, and often refuse to guess if GFP is present or not based on "unhealthy" chloroplasts.

Of course, this all should become much more simple with a spectral system, which is the direction I hope to move in.

Thanks for the reply.

Christian


--- On Fri, 2/19/10, Guy Cox <guy.cox@...> wrote:

From: Guy Cox <guy.cox@...>
Subject: Re: Fluorescence theory. was: chlorophyll and associated pigment spectra
To: CONFOCALMICROSCOPY@...
Date: Friday, February 19, 2010, 7:46 PM

Fluorescence theory. was: chlorophyll and associated pigment spectra People here seem to be talking about imaging "chlorophyll" and imaging "chloroplasts" as if they are one and the same thing.  This is far from the case.

Green plant chloroplasts contain chlorophyll a and chlorophyll b, which have different excitation and emission spectra.  They also contain carotenoids which have the function of capturing energy outside the blue and red absorption bands of chlorophyll and transferring it to the chlorophyll system.  There are also two modified forms of chlorophyll which form the reaction centres for photosystem1 and 2, and these have different spectra again.  

Remember that the aim of all this is that it should not fluoresce - the energy is non-radiatively transferred to the two reactions of photosynthesis (which are spatially separated).  If you get fluorescence it's because this system isn't working.  The carotenoid system can uncouple itself in case of overload, in which case you'll get the fluorescence from these (quite a mix).  Otherwise you'll get fluorescence from one or more of the forms of chlorophyll.  But once this starts happening bleaching is quite quick, and then you'll get chlorophyll breakdown products with different fluorescence again.  Badly bleached chloroplasts fluoresce in the green.  

The moral is that you need to you need to be minimal with your excitation levels for successful chloroplast imaging.

                                                          Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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---

From: Confocal Microscopy List on behalf of Steffen Dietzel
Sent: Sat 20/02/2010 12:10 AM
To: CONFOCALMICROSCOPY@...
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