Signal, bleaching and toxicity vs. exposure time for constant total light dosage

[Andrew York]

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Simple question:
I take two 3D volumetric images of a cell, (widefield, 488nm laser
excitation, GFP-tagged).
In both cases, I take 10 slices over 1 second.
In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
the laser off for 50 ms between exposures.

Which case bleaches the cell more? Which case is more phototoxic? Which
case gives the most signal? Surely someone has studied this, and the answer
is well known. I assume there's some linear range where total dose is all
that matters, and some non-linear range where this behavior breaks down.

Motivation: my users always want to turn down the laser and crank up the
exposure time, hoping this will be gentler to the cells or give lower
background or higher signal. I usually give my opinion that it won't make
much difference, but I don't have a solid reference to point them to for a
solid answer.

[Julio Vazquez]

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My experience (without much data to support it) is this:

At low illumination intensities, photobleaching seems to depend mostly on the dose of light (illumination intensity x exposure time). I explain this by the fact that, under this regime, a fluorescent molecule has a defined probability of getting destroyed at each excitation/emission cycle, so a population of fluorophores will decay as a function of the amount of exposure received.

At high illumination intensities, several things can happen:

(a) fluorophores can go into a dark state (fluorescence emission goes down, but recovers quickly once exposure stops, as opposed to photobleaching). We can see this on some of our widefield microscopes where illumination intensity is fairly high (e.g Deltavision Elite where we have about 20-30 mW of power focused on the center of the field of view in critical illumination mode). If I do a time series (no delay) at full illumination intensity, image brightness goes quickly down, but recovers to original values if I wait a few seconds and repeat the experiment (i.e I get a saw pattern)

(b) fluorophores can go into an irreversible inactive state forever (photobleaching). This tends to happen on point scanners where local illumination intensity (per area unit) can be significantly higher that on widefield systems. If we do a photobleaching curve at different laser intensities on a point scanner, response is not linear (fluorophores bleach much faster at higher illumination intensities, rather than proportionately). The danger zone (when things start bleaching quite fast) in our experience is around and above 10-30 micro-Watts. Depends on the specimen.

Recently, it has been reported that using resonant scanners in confocal microscopy generates higher signal (i.e integrating ten images at fast speed gives significantly brighter image than collecting 1 image at 10x lower speed). A discussion of the possible mechanisms can be found on the Leica web site:

http://www.leica-microsystems.com/science-lab/brighter-fluorescence-by-resonant-scanning/

(c) other things may be happening I am not aware of. Which fluorophore you use, mounting medium, etc, may have an influence.

A discussion of photobleaching mechanisms can be found in Jim Pawley's Confocal handbook (e.g. Chapter 39 of 3rd Edition, by Alberto Diaspro)

The bottom line is that several mechanisms may be taking place at the same time, the relative importance of which which may depend on the specimen and on the specific microscope used (based on mode of illumination- widefield vs point confocal-, and the intensity of the illumination). When we work with critical samples, we just run a few quick tests to see what works best on any given microscope system.


Julio Vazquez
Fred Hutchinson Cancer Research Center2
Seattle, WA 98109

http://www.fhcrc.org/en.html

==

On Jun 19, 2013, at 11:42 AM, Andrew York wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.

[Jurek Dobrucki]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Andrew,

longer acquisition at a lower intensity of exciting light will often
results in far less bleaching (and less photodamage in live cells).

You are right - this issue was studied. An example is provided by our paper:

Bernas T, Zarebski M, Cook PR, Dobrucki JW .Minimizing photobleaching
during confocal microscopy of fluorescent probes bound to chromatin:
role of anoxia and photon flux.
J Microsc. 2004 Sep;215(Pt 3):281-96

Figure 4I demonstrates that it is possible to (almost) eliminate
photobleaching of eGFP if the photon flux is significanly reduced. The
same dose of light delivered with a laser
beam of much higher intensity results in a rapid loss of signal.

Hope this helps,

Jurek

Jerzy Dobrucki, Ph.D., D.Sc.
Professor of Biophysics
Head, Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
    Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone]; fax. [phone]
[hidden email]
http://www.wbbib.uj.edu.pl/helios




On Wed, Jun 19, 2013 at 8:42 PM, Andrew York
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.

[Claudiu Brumaru]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Andrew,

Regarding the signal intensity, one can double the emitted fluorescence by
doubling the laser intensity (considering that this intensity is low enough
to stay within the linear dependence between the  emission and excitation
light) while doubling the integration time leads to only a square root of 2
increase in S/N.

Claudiu.


On Wed, Jun 19, 2013 at 9:42 PM, Andrew York <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi andrew,

As already mentioned in several replies, the answer is "yes".

Several of the replies discussed confocal imaging (and yes, resonant
scanning helps, although there is an assumption that the manufacturer
does not tweak any settings for different speeds ... the Zeiss LSM510
for example where tweaking definitely was done).

A nice abstract at FOM 2013 from James jonkman and collaborators
compared continuous vs pulsed LED illumination (10 microseconds on, 10
us off ...) and cited earlier papers by others. The abstract is at

http://www.focusonmicroscopy.org/2013/PDF/451_Aswani.pdf

and the cited references are:

[1] T. Nishigaki, C. Wood, K. Shiba, S. Baba, and A Darszon,
"Stroboscopic illumination
using light-emitting diodes reduces phototoxicity in fluorescence cell
imaging,"
BioTechniques, 41, 191-197 (2006)
[2] R. Penjweini, H. Loew, M. Hamblin, and K. Kratky, "Long-term
monitoring of live cell
proliferation in presence of PVP-Hypericin: a new strategy using ms
pulses of LED and the
fluorescent dye CFSE," J. Microscopy, 245, 100-108 (2011)


With respect to GFP (and BFP, CFP, YFP, but apparently not so far RFPs),
the amount of flavin(s) - and a few others, see Bodganov 2012 abstract
below -  has been reported to have a big impact:

Condensed mitotic chromosome structure at nanometer resolution using
PALM and EGFP- histones. <http://www.ncbi.nlm.nih.gov/pubmed/20856676>

*Matsuda* A, Shao L, Boulanger J, Kervrann C, Carlton PM, Kner P, Agard
D, *Sedat*JW.

PLoS One. 2010 Sep 15;5(9):e12768. doi: 10.1371/journal.pone.0012768.

PMID:
    20856676


Anti-fading media for live cell GFP imaging.
<http://www.ncbi.nlm.nih.gov/pubmed/23285248>

*Bogdanov AM*, Kudryavtseva EI, Lukyanov KA.

PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. Epub
2012 Dec 21.

PMID:
    23285248

Photostability is one of the most important characteristic of a dye for
fluorescence microscopy. Recently we demonstrated that vitamins present
in imaging media dramatically accelerate photobleaching of Enhanced
Green Fluorescent Protein (EGFP) and many other green fluorescent and
photoactivatable proteins. Here we tested all vitamins of commonly used
media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and
found that only two vitamins, riboflavin and pyridoxal, decrease
photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can
be used as an imaging medium, which ensures high photostability of GFPs
at the expense of minimal biochemical disturbance. Then, we tested some
antioxidants and found that a plant flavonoid rutin greatly enhances
photostability of EGFP during live cell microscopy. In complete DMEM,
rutin increased EGFP photostability up to the level of vitamin-depleted
DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to
further suppress EGFP photobleaching. Potentially, new medium
formulations can be widely used for fluorescence microscopy of
GFP-expressing cells and model multicellular organisms in a variety of
imaging applications, where photostability represents a challenge.



Cell culture medium affects GFP photostability: a solution.
<http://www.ncbi.nlm.nih.gov/pubmed/19935837>

*Bogdanov AM*, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S,
Lukyanov KA.

Nat Methods. 2009 Dec;6(12):859-60. doi: 10.1038/nmeth1209-859. No
abstract available.

PMID:
    19935837



The Bogdanov group media is available as DMEMgfp from Evrogen. A similar
media is Opti-Klear from Marker Gene Technologies. Researchers can also
arrange for riboflavin free (or at least low) media from their favorite
media companies. Oxygen might also be able to quench GFP (and is needed
for fluorophore maturation).


not mentioned by Bogdanov or Matsuda et al is the proximity of flavin or
pyridoxol (or rutin) to EGFP. With the development of phiLOV, FluBO and
other flavin binding fluorescent proteins, and fusion proteins (FluBO is
YFP-linker-FbFP,
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364895/figure/F1/ ) this
information should become available.


Enjoy,

George
p.s. my thanks to Jonathan Boyd, leica applications specialist, for
showing me fluorescence dimness vs brightness on the leica SP5  standard
vs resonant scanner (wher I don't think Leica was tweaking the numbers
coming out ... HyD detector(s) on SP5 or SP8 could do the test in photon
counting mode).


On 6/19/2013 1:42 PM, Andrew York wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054

[Zdenek Svindrych]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Claudiu,

you are right, but there is one important note to add: in *both* cases
(doubling exposure time vs doubling intensity, linear regime) you collect *
twice* the number of photons which translates to sqrt(2) improvement of S/N,
provided you are limited by photon noise. Anyway, both cases are equivalent,
you get the same result with the same photon dose.

To all:

Generally, of course, there may be other (nonlinear) effects or other
sources of noise (e.g. dark current) that may make a difference. But this is
unlikely in widefield with good camera.

It is strange that (as noted by others) microscopists try to reduce
phototoxicity and photobleaching by decreasing the peak intensity in a point
scanning confocal (faster scanning with averaging) on one hand, while trying
to increase peak intensity in widefield by pulsed illumination... I have not
seen a really convincing paper stating that pulsed illumination reduces
photobleaching.

Best,

zdenek svindrych



---------- Původní zpráva ----------
Od: Claudiu Brumaru <[hidden email]>
Datum: 20. 6. 2013
Předmět: Re: Signal, bleaching and toxicity vs. exposure time for constant
total light dosage

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Andrew,

Regarding the signal intensity, one can double the emitted fluorescence by
doubling the laser intensity (considering that this intensity is low enough
to stay within the linear dependence between the emission and excitation
light) while doubling the integration time leads to only a square root of 2
increase in S/N.

Claudiu.


On Wed, Jun 19, 2013 at 9:42 PM, Andrew York <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I

turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the
answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.
>"

[James Jonkman]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi, all.  The 2 papers cited on our FOM poster (mentioned below by George)
used very different doses for pulsed vs continuous (more than 100 times), so
they're not the least bit convincing.  However, I would welcome feedback on a
webinar I presented on this topic back in May:

http://www.ldgi-xcite.com/news-webinars.php

I'm working on a few follow-up studies now and hope to publish soon.  There
really does seem to be an advantage for pulsing the illumination on the 10 to
100us timescale, thereby allowing the dark state (which is not only dark, but
also less stable, more prone to photobleaching) to relax and freeing those
molecules up for further excitation.

The relevant papers are:
[1] Gerald Donnert, Christian Eggeling & Stefan W Hell, Major signal increase in
fluorescence microscopy through dark-state relaxation", NATURE METHODS,
4:81-86 (2007)

[2] Rolf T. Borlinghaus, "High Speed Scanning Has the Potential to
Increase Fluorescence Yield and to Reduce Photobleaching", MICROSCOPY
RESEARCH AND TECHNIQUE 69:689–692 (2006)

Cheers,
James


On Wed, 19 Jun 2013 20:43:02 -0500, George McNamara
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi andrew,
>
>As already mentioned in several replies, the answer is "yes".
>
>Several of the replies discussed confocal imaging (and yes, resonant
>scanning helps, although there is an assumption that the manufacturer
>does not tweak any settings for different speeds ... the Zeiss LSM510
>for example where tweaking definitely was done).
>
>A nice abstract at FOM 2013 from James jonkman and collaborators
>compared continuous vs pulsed LED illumination (10 microseconds on, 10
>us off ...) and cited earlier papers by others. The abstract is at
>
>http://www.focusonmicroscopy.org/2013/PDF/451_Aswani.pdf
>
>and the cited references are:
>
>[1] T. Nishigaki, C. Wood, K. Shiba, S. Baba, and A Darszon,
>"Stroboscopic illumination
>using light-emitting diodes reduces phototoxicity in fluorescence cell
>imaging,"
>BioTechniques, 41, 191-197 (2006)
>[2] R. Penjweini, H. Loew, M. Hamblin, and K. Kratky, "Long-term
>monitoring of live cell
>proliferation in presence of PVP-Hypericin: a new strategy using ms
>pulses of LED and the
>fluorescent dye CFSE," J. Microscopy, 245, 100-108 (2011)
>
>
>With respect to GFP (and BFP, CFP, YFP, but apparently not so far RFPs),
>the amount of flavin(s) - and a few others, see Bodganov 2012 abstract
>below -  has been reported to have a big impact:
>
>Condensed mitotic chromosome structure at nanometer resolution using
>PALM and EGFP- histones. <http://www.ncbi.nlm.nih.gov/pubmed/20856676>
>
>*Matsuda* A, Shao L, Boulanger J, Kervrann C, Carlton PM, Kner P, Agard
>D, *Sedat*JW.
>
>PLoS One. 2010 Sep 15;5(9):e12768. doi: 10.1371/journal.pone.0012768.
>
>PMID:
>    20856676
>
>
>Anti-fading media for live cell GFP imaging.
><http://www.ncbi.nlm.nih.gov/pubmed/23285248>
>
>*Bogdanov AM*, Kudryavtseva EI, Lukyanov KA.
>
>PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. Epub
>2012 Dec 21.
>
>PMID:
>    23285248
>
>Photostability is one of the most important characteristic of a dye for
>fluorescence microscopy. Recently we demonstrated that vitamins present
>in imaging media dramatically accelerate photobleaching of Enhanced
>Green Fluorescent Protein (EGFP) and many other green fluorescent and
>photoactivatable proteins. Here we tested all vitamins of commonly used
>media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and
>found that only two vitamins, riboflavin and pyridoxal, decrease
>photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can
>be used as an imaging medium, which ensures high photostability of GFPs
>at the expense of minimal biochemical disturbance. Then, we tested some
>antioxidants and found that a plant flavonoid rutin greatly enhances
>photostability of EGFP during live cell microscopy. In complete DMEM,
>rutin increased EGFP photostability up to the level of vitamin-depleted
>DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to
>further suppress EGFP photobleaching. Potentially, new medium
>formulations can be widely used for fluorescence microscopy of
>GFP-expressing cells and model multicellular organisms in a variety of
>imaging applications, where photostability represents a challenge.
>
>
>
>Cell culture medium affects GFP photostability: a solution.
><http://www.ncbi.nlm.nih.gov/pubmed/19935837>
>
>*Bogdanov AM*, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov

S,

>Lukyanov KA.
>
>Nat Methods. 2009 Dec;6(12):859-60. doi: 10.1038/nmeth1209-859. No
>abstract available.
>
>PMID:
>    19935837
>
>
>
>The Bogdanov group media is available as DMEMgfp from Evrogen. A similar
>media is Opti-Klear from Marker Gene Technologies. Researchers can also
>arrange for riboflavin free (or at least low) media from their favorite
>media companies. Oxygen might also be able to quench GFP (and is needed
>for fluorophore maturation).
>
>
>not mentioned by Bogdanov or Matsuda et al is the proximity of flavin or
>pyridoxol (or rutin) to EGFP. With the development of phiLOV, FluBO and
>other flavin binding fluorescent proteins, and fusion proteins (FluBO is
>YFP-linker-FbFP,
>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364895/figure/F1/ ) this
>information should become available.
>
>
>Enjoy,
>
>George
>p.s. my thanks to Jonathan Boyd, leica applications specialist, for
>showing me fluorescence dimness vs brightness on the leica SP5  standard
>vs resonant scanner (wher I don't think Leica was tweaking the numbers
>coming out ... HyD detector(s) on SP5 or SP8 could do the test in photon
>counting mode).
>
>
>On 6/19/2013 1:42 PM, Andrew York wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Simple question:
>> I take two 3D volumetric images of a cell, (widefield, 488nm laser
>> excitation, GFP-tagged).
>> In both cases, I take 10 slices over 1 second.
>> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
>> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
>> the laser off for 50 ms between exposures.
>>
>> Which case bleaches the cell more? Which case is more phototoxic? Which
>> case gives the most signal? Surely someone has studied this, and the

answer

>> is well known. I assume there's some linear range where total dose is all
>> that matters, and some non-linear range where this behavior breaks down.
>>
>> Motivation: my users always want to turn down the laser and crank up the
>> exposure time, hoping this will be gentler to the cells or give lower
>> background or higher signal. I usually give my opinion that it won't make
>> much difference, but I don't have a solid reference to point them to for a
>> solid answer.
>>
>>
>
>
>--
>
>
>
>George McNamara, Ph.D.
>Single Cells Analyst
>L.J.N. Cooper Lab
>University of Texas M.D. Anderson Cancer Center
>Houston, TX 77054