Posted by
James Jonkman on
Jun 20, 2013; 12:18pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Signal-bleaching-and-toxicity-vs-exposure-time-for-constant-total-light-dosage-tp7580528p7580538.html
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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.phpI'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