Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Signal-bleaching-and-toxicity-vs-exposure-time-for-constant-total-light-dosage-tp7580528p7580534.html
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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.pdfand 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:
>
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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.
>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054