Microscopy used equipment

[Collin White]

Hi all,
Looking for a good source of used microscopes or parts for microscopes.
I need a smaller luminescent base for a nikon stereoscope.
Collin

--
Collin White
Research Scientist
Manager CEEH Cytometry Lab
Building Facilities Manager Roosevelt 1
University of Washington
Dept of Environmental and Occupational Health
4225 Roosevelt Way NE #100
Seattle, WA 98105
Phone [phone]
email [hidden email]

[Armstrong, Brian]

You could try LabX.
http://www.labx.com/v2/category_main.cfm?MainCatID=5&CatID=228


Brian Armstrong PhD
Light Microscopy and Digital Imaging
Beckman Research Institute
Neuroscience
X62872
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Collin White
Sent: Thursday, February 18, 2010 12:26 PM
To: [hidden email]
Subject: Microscopy used equipment

Hi all,
Looking for a good source of used microscopes or parts for microscopes.
I need a smaller luminescent base for a nikon stereoscope.
Collin

--
Collin White
Research Scientist
Manager CEEH Cytometry Lab
Building Facilities Manager Roosevelt 1
University of Washington
Dept of Environmental and Occupational Health
4225 Roosevelt Way NE #100
Seattle, WA 98105
Phone [phone]
email [hidden email]


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[Tobias Baskin]

Its not quite in your neck of the woods but try http://www.scopeshop.com/

>
>
>Hi all,
>Looking for a good source of used microscopes or parts for microscopes.
>I need a smaller luminescent base for a nikon stereoscope.
>Collin
>
>--
>Collin White
>Research Scientist
>Manager CEEH Cytometry Lab
>Building Facilities Manager Roosevelt 1
>University of Washington
>Dept of Environmental and Occupational Health
>4225 Roosevelt Way NE #100
>Seattle, WA 98105
>Phone [phone]
>email [hidden email]
>
>
>---------------------------------------------------------------------
>SECURITY/CONFIDENTIALITY WARNING:
>This message and any attachments are intended solely for the
>individual or entity to which they are addressed. This communication
>may contain information that is privileged, confidential, or exempt
>from disclosure under applicable law (e.g., personal health
>information, research data, financial information). Because this
>e-mail has been sent without encryption, individuals other than the
>intended recipient may be able to view the information, forward it
>to others or tamper with the information without the knowledge or
>consent of the sender. If you are not the intended recipient, or the
>employee or person responsible for delivering the message to the
>intended recipient, any dissemination, distribution or copying of
>the communication is strictly prohibited. If you received the
>communication in error, please notify the sender immediately by
>replying to this message and deleting the message and any
>accompanying files from your system. If, due to the security risks,
>you do not wish to receive further communications via e-mail, please
>reply to this message and inform the sender that you do not wish to
>receive further e-mail from the sender.
>
>---------------------------------------------------------------------

[Christian-103]

Does anyone have the autofluorescent spectra for plants due to the pigments in the chloroplasts?

I know there is a very strong peak beginning in the 600 nm range, but there is also one in the 520 nm range as well when excited with 488 nm.

 

I have not been able to locate it on the usual spectral sites.

 

Thanks

 

Christian

[Rosemary.White]

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 spectrum.

cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
M 61 2 420 972 028



On 19/02/10 5:33 PM, "Christian" <celowsky21@...> wrote:

Does anyone have the autofluorescent spectra for plants due to the pigments in the chloroplasts?

I know there is a very strong peak beginning in the 600 nm range, but there is also one in the 520 nm range as well when excited with 488 nm. I have not been able to locate it on the usual spectral sites. Thanks Christian

[Christian-103]

Rosemary,

Thank you for the rapid reply!

Often when GFP is weak (ex488/em522), such as in mitochondrial targetting, the chloroplasts often in overlay become yellow as the autofluorescence becomes very noticeable.  Furthermore, if there is GFP in the chloroplasts which is not quite bright, it can be very difficult to demonstrate its presence to reviewers.  I always set up against wild type material, and I'm always looking for ways to explain to users what is going on with their "green" chloroplasts which should be red!

Thanks.

Christian

[Rosemary.White]

HI Christian,

Basically, you can see chlorophyll autofluorescence over much of the FP spectrum.  I either collect autofluorescence in a chlorophyll-only region, often above 650 nm (except when using Cy5 or PI then have to split between these and Chl), and add to the total image in a contrasting colour, or subtract out chlorophyll autofluorescence.  Depends on what you need to see and measure and how tricky your fluorescence quantification is if you’re going down that track.
cheers,
Rosemary


On 19/02/10 6:07 PM, "Christian" <celowsky21@...> wrote:

Rosemary,

Thank you for the rapid reply!

Often when GFP is weak (ex488/em522), such as in mitochondrial targetting, the chloroplasts often in overlay become yellow as the autofluorescence becomes very noticeable.  Furthermore, if there is GFP in the chloroplasts which is not quite bright, it can be very difficult to demonstrate its presence to reviewers.  I always set up against wild type material, and I'm always looking for ways to explain to users what is going on with their "green" chloroplasts which should be red!

Thanks.

Christian

[Steffen Dietzel]

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

[Howard Berg]

To address the first part of your discussion, chlorophyll emission  
detected in our Meta system is always the same from different plants  
and with different excitations, with a peak near 680.

Howard



On Feb 19, 2010, at 7:10 AM, Steffen Dietzel wrote:

> 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

[Christian-103]

Howard,

Have you checked the entire spectra from ~500 nm through ~750 nm with only an excitation of ~488 nm?

The reason is said "associated pigments" is due to the fact that you can easily see grana stacks (the location of the chlorophylls) in the far red channel, but you can not in the roughly 520 nm range.  I assume it's one of two things, either it's not bright enough to resolve the stacks or it's different pigments.

I often use only the 488 nm laser line to image GFP and chlorophyll, but there is a point where "chloroplasts" will fill the image in the 522 nm channel.  Again, 35S promoters are not a problem, it's the native promoters or when small organelles are targeted.

I can not be the only person who has seen "orange" chloroplasts or even false yellow ones, can I?

Christian



--- On Fri, 2/19/10, Howard Berg <[hidden email]> wrote:

From: Howard Berg <[hidden email]>
Subject: Re: Fluorescence theory. was: chlorophyll and associated pigment spectra
To: [hidden email]
Date: Friday, February 19, 2010, 8:38 AM

To address the first part of your discussion, chlorophyll emission 
detected in our Meta system is always the same from different plants 
and with different excitations, with a peak near 680.

Howard



On Feb 19, 2010, at 7:10 AM, Steffen Dietzel wrote:

> 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

[Emmanuel Gustin]

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. [phone],    e-mail: [hidden email]


-----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

[Guy Cox-2]

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
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
http://www.guycox.net

---

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

[Christian-103]

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 <[hidden email]> wrote:

From: Guy Cox <[hidden email]>
Subject: Re: Fluorescence theory. was: chlorophyll and associated pigment spectra
To: [hidden email]
Date: Friday, February 19, 2010, 7:46 PM

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
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
http://www.guycox.net

---

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

[Rosemary.White]

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
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
http://www.guycox.net <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net>

---

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