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Hello Michael,
The idea, at least for Argolight, is to provide solution to measure the reliability of your systems in the conditions you will use, so that you know there is no bias added by the system. Then of course, if your protocol has inherent fluctuation in the biological part, we cannot correct for it.
Le 23 mars 2015 à 17:14, Cammer, Michael <[log in to unmask]> a écrit :
> Am I missing something?
>
> I don’t understand how you can have a standard independent of the actual experimental condition since each fluorophore behaves differently and the specific biological/chemical environment may make a difference too.
>
> Also, you need to measure the illumination at the sample independently from the intensity on the detection side.
>
> Regards,
> Michael
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Temporary location: SK2-7
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Andrew York
> Sent: Tuesday, March 17, 2015 4:36 PM
> To: [log in to unmask]
> Subject: Re: Photobleaching Standard
>
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>
> A reasonable standardized sample to try:
> http://www.gattaquant.com/products/gatta-brightness.html
> I haven't tried them myself, but I'd like to. If anyone has, please tell me about your experience.
>
> This wouldn't directly tell you photobleaching rates, but since you actually know how many dye molecules you're starting with, it should make bleaching statistics simple to gather, and simple to interpret. It won't get at interesting sample-dependent bleaching questions, but it should help if you want to compare microscopes quantitatively.
>
> On Mon, Mar 16, 2015 at 1:26 PM, Neil Anthony <[log in to unmask]> wrote:
>
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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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>>
>> Cool, thanks Sam. That's exactly the kind of answer I was looking
>> for; it's great to get the very specific details from an expert.
>>
>> Thanks
>> Neil
>>
>>
>>
>>
>> On 3/12/2015 2:40 PM, Sam Lord wrote:
>>
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>>>
>>> Hi Neil,
>>>
>>> Great question.
>>>
>>> On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[log in to unmask]>
>>> wrote:
>>>
>>>> I would presumably
>>>> calculate the intensity decay over a time lapse as a metric, but
>>>> wasn't sure if the sample type was a factor as I don't know much
>>>> about the photochemistry.
>>>>
>>> I have a pet peeve: when a paper reports photobleaching half-lives or
>>> bleaching decay times *without accounting for the rate of absorbing
>>> photons*.
>>> Photobleaching is a statistical process that typically occurs from
>>> the excited state of the molecule (e.g. from intersystem crossing to
>>> a triplet state and then further excitation and photodegratation) or
>>> from an excited producing singlet oxygen that then reacts with that
>>> molecule or a nearby molecule.
>>>
>>> Either way, the rate of photobleaching is going to depend on how
>>> frequently the dye cycles into the excited state. If you compare two
>>> samples without taking into account the excitation rate, you might
>>> think one sample is more susceptible to bleaching when really it is
>>> just brighter.
>>>
>>> I recommend using the photobleaching quantum yield as an absolute
>>> measure. It is the probability of photobleaching with each photon
>>> absorbed, or the ratio of the bleaching rate to the photon absorption
>>> rate. It is easy to calculate if you know the extinction coefficient
>>> of the dye and the irradiance and wavelength of light you're using.
>>> See equation 2.1 of my paper here:
>>>
>>> Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E.
>>> (2010).
>>> Molecules and methods for super-resolution imaging. Methods in
>>> Enzymology, 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3
>>>
>>> http://everydayscientist.com/pdfs/sjl16.pdf
>>>
>>> Alternatively, maybe a more practical metric would be to change the
>>> illumination so that each sample has the same brightness on the
>>> camera or the same signal to noise ratio or some other useful
>>> quantity. Then record the bleaching half-time. That would tell you
>>> exactly what you really want to know: given a certain starting image
>>> quality, how long can I observe my sample? This measure should indeed
>>> scale with the photobleaching quantum yield, but it also takes into
>>> account the "brightness"
>>> of the dye (i.e. the fluorescence quantum yield times the extinction
>>> coefficient), as well as microscope factors like the chromatic
>>> aberrations and camera quantum efficiency at different wavelengths.
>>>
>>> This is a pragmatic measure, but it would be a little hard to
>>> directly compare completely different dyes, samples, microscopes,
>>> etc. That's why I like absolute metrics, like the photobleaching
>>> quantum yield and dye brightness.
>>>
>>> For a great reference on dye brightness, see:
>>>
>>> A guide to choosing fluorescent proteins
>>> http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Me
>>> thod s%20-%20Choosing%20fluorescent%20proteins.pdf
>>>
>>> (Note the measure that Shaner used for photo stability does indeed
>>> take into account absorption rate: "Time for bleaching from an
>>> initial emission rate of 1,000 photons/s down to 500 photons/s (t1/2;
>>> for comparison, fluorescein at pH
>>> 8.4 has
>>> t1/2 of 5.2 s).")
>>>
>>> The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid
>>> the pitfalls of multi-exponential fitting and reporting weighted
>>> decay constants (see Lakowicz or equation 2.2 of my paper above).
>>> Half-life is a super simple and pragmatic measure of sample
>>> photobleaching.
>>>
>>> On that note, can anybody recommend a good reference
>>>> that discusses the subtleties of photobleaching?
>>>>
>>> As far as I know, there is no single review that really encompasses
>>> all the subtleties of photobleaching. A lot of those subtleties are
>>> not well understood.
>>> People often blame singlet oxygen, but it's way more complicated than
>>> that (e.g.
>>> triplet/ground-state oxygen is an excellent triplet quencher, so it
>>> is actually good to have around to reduce dyes in the triplet
>>> bottleneck; see Hubner. J.
>>> Chem. Phys.
>>> 2001, 115, 9619). There are many paths to photobleaching, they are
>>> difficult to observe, they yield a huge range of photoproducts, and
>>> each dye is likely to have a completely different way it prefers to
>>> die.
>>>
>>> There are some great examples of papers where the authors discovered
>>> the photobleaching path of a particular dye or subset of dyes:
>>>
>>> Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
>>> Photoswitching mechanism of cyanine dyes. Journal of the American
>>> Chemical Society, 131, 18192-3.
>>>
>>> Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking
>>> and switching behaviour of single molecules of green fluorescent protein.
>>> Nature, 388,
>>> 355-8.
>>>
>>> Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with
>>> improved photostability. Organic Letters, 9, 2775-7.
>>>
>>> Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways
>>> in
>>> single-
>>> molecule FRET experiments. Journal of the American Chemical Society,
>>> 129,
>>> 4643-
>>> 54.
>>>
>>> Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of
>>> Trolox as antiblinking and antibleaching reagent. Journal of the
>>> American Chemical Society, 131, 5018-9.
>>>
>>> Berglund AJ. (2004). Nonexponential statistics of fluorescence
>>> photobleaching. The Journal of Chemical Physics, 121, 2899-903.
>>>
>>> Best,
>>>
>>> -Sam
>>>
>>
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