broadband excitation vs. narrow band

[Boswell, Carl A - (cboswell)]

Hi all,
I was told once that there are no stupid questions, so let's test that
assumption.

The question has to do with photobleaching vs. excitation energy.  To get X
photons from a fluor, would there be less photobleaching using a single
wavelength excitation source at peak excitation wavelength, or a broadband
(20-30nm) light source centered on the peak excitation of the molecule, or
would there be no difference?  My assumption is that lower "power"
(brightness?) would be needed for the broadband source, but would the
overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from
"inefficient" excitation, i.e. off-peak excitation, to get the same output?
If a fluor is less efficiently excited, is it less efficiently bleached,
even though more power may be needed to get equivalent output?

 Thanks,
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[John Runions]

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Eli Rothenberg-2]

Hi Carl,
I actually think these are all good questions.
To answer dryly I would say these would depend on the specific system you are using, namely the buffer, dye you're and environment (in-vivo/vitro).
To give you a broader answer, and again this would depend on the dye system you're using- two main factors that affect
photobleaching would be:
1) the absorption states of your dye molecule.
different states (different wavelengths) would have
a different probability of pushing your dye into photobleaching.
2) Photon flux, or excitation cycles. exciting your molecule
when it's already excited would cause it to photobleach even
faster, so if you have a molecule with a short excited-state lifetime, and you match your flux accordingly, you'll be able to "squeeze" more excitation cycles.

I would say the combination of these two factors, and their
coupling to other "environmental" factors would determine
the answer to your questions.

Which to say again, depends on your system, for instance,
if you use broad excitation, it might work well for some dyes, assuming that some of the photons may even contribute to photoactivate a dark state. On the other hand,
it might speed up photobleaching with residual energy
in an already excited molecule.

Overall, I think single wavelength excitation may be better.


 Eli

---- Original message ----

>Date: Tue, 18 Nov 2008 09:51:58 -0700
>From: Carl Boswell <[hidden email]>  
>Subject: broadband excitation vs. narrow band  
>To: [hidden email]
>
>Hi all,
>I was told once that there are no stupid questions, so let's test that
>assumption.
>
>The question has to do with photobleaching vs. excitation energy.  To get X
>photons from a fluor, would there be less photobleaching using a single
>wavelength excitation source at peak excitation wavelength, or a broadband
>(20-30nm) light source centered on the peak excitation of the molecule, or
>would there be no difference?  My assumption is that lower "power"
>(brightness?) would be needed for the broadband source, but would the
>overall photon flux be greater to get equivalent output?
>
>To take this one step farther, is there less or more bleaching from
>"inefficient" excitation, i.e. off-peak excitation, to get the same output?
>If a fluor is less efficiently excited, is it less efficiently bleached,
>even though more power may be needed to get equivalent output?
>
> Thanks,
>Carl
>
>Carl A. Boswell, Ph.D.
>Molecular and Cellular Biology
>University of Arizona
>520-954-7053
>FAX [phone]

________________________________
Eli Rothenberg, Ph.D.
Postdoctoral Fellow,
Department of Physics,
NSF Center for the Physics
of Living Cells,
University of Illinois - Urbana,
1110 W. Green St.
Urbana, 61801.
Illinois, USA
Tel: [phone];
Email: [hidden email]

[Andrew Resnick]

My interpretation of the excitation and emission curves is that they
are fundamentally probability curves, or efficiency curves.  That is,
the excitation spectrum is multiplied by the absorption curve to get
x% of power absorbed (averaged in time).  Same with emission curves-
they are a probability that the emitted photon has a certain
frequency.  As for photobleaching, I'm not positive on how that event
occurs- if it's fundamentally a probabilistic event or a deterministic event.

This topic raises the issue of how a continuum "classical" spectrum
arises from quantum-mechanical (discrete) events.  But I suspect that
photobleaching occurs based on the total absorbed power, and not the
detailed frequency of the absorbed photon.

At 11:51 AM 11/18/2008, you wrote:

>Hi all,
>I was told once that there are no stupid questions, so let's test
>that assumption.
>
>The question has to do with photobleaching vs. excitation
>energy.  To get X photons from a fluor, would there be less
>photobleaching using a single wavelength excitation source at peak
>excitation wavelength, or a broadband (20-30nm) light source
>centered on the peak excitation of the molecule, or would there be
>no difference?  My assumption is that lower "power" (brightness?)
>would be needed for the broadband source, but would the overall
>photon flux be greater to get equivalent output?
>
>To take this one step farther, is there less or more bleaching from
>"inefficient" excitation, i.e. off-peak excitation, to get the same
>output? If a fluor is less efficiently excited, is it less
>efficiently bleached, even though more power may be needed to get
>equivalent output?
>
>Thanks,
>Carl
>
>Carl A. Boswell, Ph.D.
>Molecular and Cellular Biology
>University of Arizona
>520-954-7053
>FAX [phone]

Andrew Resnick, Ph. D.
Instructor
Department of Physiology and Biophysics
Case Western Reserve University
216-368-6899 (V)
216-368-4223 (F)

[Boswell, Carl A - (cboswell)]

To John and others:

As usual, I made assumptions that were not valid.  When I mentioned excitation and bleaching in my recent query, I was referring to the unavoidable bleaching of fluors when they are viewed for image capture, not the purposeful bleaching of dyes for FRAP, etc.

 

However, the combination of laser lines for FRAP is a useful techinque to remember.

 

 

thanks for your interest.

C

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Tuesday, November 18, 2008 10:08 AM

Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Kurt Thorn]

My experience is that the bleaching rate of fluorophores at different
wavelengths varies from fluorophore to fluorophore.  Naively you would
expect that the photobleaching spectrum should look the same as the
excitation spectrum but this is not always the case.

I first noticed this when doing some FRET experiments with CFP and YFP
where it turns out the CFP illumination bleaches YFP pretty
efficiently.  When we measured this carefully it turns out the CFP
illumination (centered on 430 nm) bleaches YFP about twice as quickly as
illumination centered on YFP excitation maximum (around 500 nm).  We
similar results for some red fluorescent proteins - for instance,
tdtomato, tdimer2, and tHcRed are all bleached more quickly by GFP
illumination light (490 nm) than by excitation on peak (555 nm).  
However other red proteins like mRFP1 and mCherry are bleached very
inefficiently by GFP illumination light.

Since we were mostly interested in FRET pairs we never did a careful
measurement of the photobleaching spectrum of any of these proteins - we
just used the filters we had for epifluorescence imaging.  But it's
clear that the behavior can be complicated.

Kurt

Carl Boswell wrote:

> Hi all,
> I was told once that there are no stupid questions, so let's test that
> assumption.
>
> The question has to do with photobleaching vs. excitation energy.  To
> get X photons from a fluor, would there be less photobleaching using a
> single wavelength excitation source at peak excitation wavelength, or
> a broadband (20-30nm) light source centered on the peak excitation of
> the molecule, or would there be no difference?  My assumption is that
> lower "power" (brightness?) would be needed for the broadband source,
> but would the overall photon flux be greater to get equivalent output?
>
> To take this one step farther, is there less or more bleaching from
> "inefficient" excitation, i.e. off-peak excitation, to get the same
> output? If a fluor is less efficiently excited, is it less efficiently
> bleached, even though more power may be needed to get equivalent output?
>
> Thanks,
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone [phone]
fax   [phone]

[Mario-2]

As, Kurt pointed it out it will depend on the fluorophore plus the
local environment (meaning local chemistry). Experiments are the only
way to tell for sure.

However, there are some basics that need to be considered. Most
fluorophores that consist of multiply bonded conjugated rings systems
have broad absorption bands that depend on the number of internal
vibrational states. Photon absorption generally takes about 10^-12
sec or less with vary rapid energy redistribution among the various
vibrational states until a photon emission occurs, generally in the
1-10 nsec. range. Just as there is a broad absorption spectra because
of the large number of vibrational states accessible, the same is
true for the emission spectra. Most of us are familiar with the
mirror images common to the absorption vs emission spectra.

Excited molecules can lose all their absorbed energy via vibrational
levels transferring energy as heat, internal bond rearrangement into
a permanently non-radiative molecule, aka photo-bleaching, excitation
of environmental molecules such as oxygen to a reactive singlet
species or superoxide radical each of which can break open a
fluorophore ring system again resulting in photobleaching. Then there
are singlet-triplet transitions (a classic problem with fluorescein).
In any event, there are a lot of complex paths that an excited state
molecule can take to reach the ground state. Some destructive, some
not. If wavelengths are used to excite in the short wavelength part
of the excitation spectrum, more energy has to be dissipated to reach
states that favor emission. Intuitively one might expect that on a
per absorbed photon basis this would have a higher probability of
leading to internal rearrangement and photobleaching than exciting at
lower energies whether at the peak or longer wavelengths. Kurt's
posting would seem to bear this out.

Still, the only way to make meaningful assessments of this issue is
if bleach rates are measured in terms of the the number of absorbed
photons at whatever wavelength.

Sorry for the brevity of this description; I will try to add to this latter,

Mario

>Hi all,
>I was told once that there are no stupid questions, so let's test
>that assumption.
>
>The question has to do with photobleaching vs. excitation energy.
>To get X photons from a fluor, would there be less photobleaching
>using a single wavelength excitation source at peak excitation
>wavelength, or a broadband (20-30nm) light source centered on the
>peak excitation of the molecule, or would there be no difference?
>My assumption is that lower "power" (brightness?) would be needed
>for the broadband source, but would the overall photon flux be
>greater to get equivalent output?
>
>To take this one step farther, is there less or more bleaching from
>"inefficient" excitation, i.e. off-peak excitation, to get the same
>output? If a fluor is less efficiently excited, is it less
>efficiently bleached, even though more power may be needed to get
>equivalent output?
>
>Thanks,
>Carl
>
>Carl A. Boswell, Ph.D.
>Molecular and Cellular Biology
>University of Arizona
>520-954-7053
>FAX [phone]

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]
[hidden email]