[FLIMfit-users] FLIMfit-users Digest, Vol 21, Issue 2

[R.F. Laine]

Hi Marko,
Just some small further comments on your problem.
I also strongly recommend you perform your analysis using a measured IRF 
rather than estimated one.
Like most people, I use either scatter from the cover slip, Erythrosin B 
(http://www.sigmaaldrich.com/catalog/product/aldrich/200964?lang=en&region=GB) 
or DASPI 
(http://www.radiant-dyes.com/index.php/products/laser-dyes/list-of-laser-dyes).

Constraining your fit as much as possible with as much a priori 
knowledge as possible is usually a good thing for fitting approaches 
like here. So fixing your donor lifetime and performing a global 
analysis (FLIMfit) across all your dataset (with varying a1 and a2) is 
the way to go in my opinion.

Also, on a side note, I think it is important that you use reference 
dyes for Reference reconvolution with single exponential lifetime and it 
is preferable that their lifetimes are shorter than those you are trying 
to measure. I cannot remember the reason why though. Maybe Ian or Sean 
would have some insights on this.

Cheers,

Romain


On 2017-03-10 16:57, Ewan McGhee wrote:
> Hi Marko,
> 
> In response to Ian¹s comments about dyes for IRFs which may be suitable
> for a confocal TCSPC system we use FITC which has a known lifetime of 4 
> ns
> at a pH of 10 (you make up a 0.5 mM stock solution in 0.1M TRIS and pH 
> it
> to be above 10. Then you make your reference standards from that 10uM, 
> 1uM
> etc.(again pH to above 10 if necessary).
> 
> Another dye we use is erythrosin which has a very short (about 100 ps
> lifetime). Which you use will depend upon the emission wavelength of 
> your
> FLIM signal.
> 
> Hopt this helps.
> 
> Cheers,
> 
> Ewan
> 
> On 10/03/2017, 14:58, "FLIMfit-users on behalf of
> flimfit-users-request at lists.openmicroscopy.org.uk"
> <flimfit-users-bounces at lists.openmicroscopy.org.uk on behalf of
> flimfit-users-request at lists.openmicroscopy.org.uk> wrote:
> 
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>> 
>> Today's Topics:
>> 
>>   1. Re: SymphoTime vs. FLIMfit (Munro, Ian)
>>   2. Re: SymphoTime vs. FLIMfit (Dunsby, Christopher W)
>> 
>> 
>> ----------------------------------------------------------------------
>> 
>> Message: 1
>> Date: Fri, 10 Mar 2017 14:52:52 +0000
>> From: "Munro, Ian" <i.munro at imperial.ac.uk>
>> Cc: "flimfit-users at lists.openmicroscopy.org.uk"
>> 	<flimfit-users at lists.openmicroscopy.org.uk>
>> Subject: Re: [FLIMfit-users] SymphoTime vs. FLIMfit
>> Message-ID: <4FD4141D-C7CF-45EF-936F-2C32AF1FF049 at imperial.ac.uk>
>> Content-Type: text/plain; charset="utf-8"
>> 
>> Hi Marko
>> 
>> There is a general problem, in minimisation, of false minima or 
>> multiple
>> minima.
>> Minimisation software simply searches a ( in your case 4D) surface
>> looking for the lowest value of some figure-of-merit (e.g. 
>> chi-squared).
>> A 2D analogy would be trying to find the lowest point in a landscape.
>> If there are 2 equally deep valleys  (2 minima) either side of a ridge
>> then your result can depend on which side of the ridge you start your
>> search.
>> From what you say, you are taking an approach, referred to in FLIMfit 
>> as
>> ?Global binning?.
>> The FLIMfit software offers you the alternative of ?Global fitting?
>> where, rather than fixing one lifetime in advance, you fit both but 
>> tell
>> the software to assume that the 2 lifetimes are constant across the 
>> image.
>> Maybe this approach would shed some light on your problem.
>> 
>> For IRF?s you need something with a known response & ideally emitting 
>> at
>> the same wavelength as your sample.
>> The gold-nanorods are great but IIRC only appropriate for two-photon
>> excitation.
>> Alternatively you can use a dye with either a  very short lifetime or 
>> a
>> mono-exponential decay and a known lifetime (the 'Reference
>> deconvolution? option in ?IRF type?. You can also use a scattering 
>> sample
>> and the scattered excitation light although you need to be careful as
>> the different wavelength may introduce a time-shift and this will then
>> require you to shift the IRF data to compensate.
>> 
>> Best Wishes
>> 
>> Ian
>> 
>> 
>>> On 8 Mar 2017, at 13:16, Marko ?o?tar <markosostar at gmail.com> wrote:
>>> 
>>> Hi,
>>> my name is Marko and I am a phd student on the Institute Ru?er 
>>> Bo?kovi?
>>> in Zagreb, Croatia.
>>> I recently did some FLIM-FRET measurements where I tried to find out
>>> FRET-ing population in a sample by fitting on the double-exponential
>>> model N(t) = A1*exp(?t/t FRET) + A2*exp(?t/t 0). Ideally, I would
>>> measure donor lifetime (t0) from donor only sample (mono-exponential
>>> decay), and then use this as a fixed parameter in subsequent fitting
>>> procedures (with acceptor present). And, that worked fine until
>>> recently, when I came across a sample where very small change in 
>>> donor
>>> lifetime (fixed parameter-I was playing with manually changing it) 
>>> would
>>> yield substantial difference in fitted A1 and A2, with equally good 
>>> fit
>>> quality (chi-squared test). So, now I can't decide which amplitudes 
>>> to
>>> use. Does anybody have some advice how to resolve this issue?
>>> We use Leica confocal microscope paired with PicoQuant TCSPC system 
>>> and
>>> SyphoTime software for analysis. I should mention that I never 
>>> measured
>>> IRF (software offers to approximate it), so I'm not sure how this
>>> affects fitting results. Also, I recently became aware of the FLIM 
>>> fit
>>> software, and I was wondering is there anyone experienced with both
>>> SymphoTime and FLIM fit. What would be the better choice? Could this
>>> fitting issue be resolved by using FLIM Fit (maybe it has some extra
>>> features for assessing goodness of fit?)? Also, could you tell me 
>>> what
>>> would be the best sample (way) for measuring the IRF function (gold
>>> nanorods?- where to get them?).
>>> I would greatly appreciate any advice.
>>> 
>>> Thank you and kind regards,
>>> Marko ?o?tar
>>> _______________________________________________
>>> FLIMfit-users mailing list
>>> FLIMfit-users at lists.openmicroscopy.org.uk
>>> http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users
>> 
>> 
>> ------------------------------
>> 
>> Message: 2
>> Date: Fri, 10 Mar 2017 14:53:31 +0000
>> From: "Dunsby, Christopher W" <christopher.dunsby at imperial.ac.uk>
>> To: "flimfit-users at lists.openmicroscopy.org.uk"
>> 	<flimfit-users at lists.openmicroscopy.org.uk>
>> Subject: Re: [FLIMfit-users] SymphoTime vs. FLIMfit
>> Message-ID:
>> 	<DB5PR06MB1269FAEDD783C018F18426B2AF200 at DB5PR06MB1269.eurprd06.prod.outlo
>> ok.com>
>> 
>> Content-Type: text/plain; charset="utf-8"
>> 
>> Dear Marko,
>> 
>> If you?re using multiphoton excitation, suitable gold nanorods can be
>> purchased from Sigma and just dried on a coverglass, e.g.:
>> 
>> 
>> 
>> http://www.sigmaaldrich.com/catalog/product/aldrich/716820?lang=en&region=
>> GB
>> 
>> This paper provides more information: doi: 10.1364/OE.19.013848.
>> 
>> RE the double exponential decay fitting, the decay parameters will be
>> correlated so the interdependence that you see is normal. Using a high
>> quality IRF and establishing a specific fitting protocol, e.g. by
>> measuring the donor only lifetime from a separate sample and fixing 
>> it,
>> should help you get reproducible and consistent results.
>> 
>> FLIMfit will let you do global analysis, e.g. if you have images then 
>> you
>> can determine the lifetimes globally but the amplitudes for each 
>> pixel.
>> 
>> best regards,
>> 
>> Chris
>> 
>> 
>> From: FLIMfit-users
>> [mailto:flimfit-users-bounces at lists.openmicroscopy.org.uk] On Behalf 
>> Of
>> Marko ?o?tar
>> Sent: 08 March 2017 13:16
>> To: flimfit-users at lists.openmicroscopy.org.uk
>> Subject: [FLIMfit-users] SymphoTime vs. FLIMfit
>> 
>> Hi,
>> my name is Marko and I am a phd student on the Institute Ru?er 
>> Bo?kovi?
>> in Zagreb, Croatia.
>> I recently did some FLIM-FRET measurements where I tried to find out
>> FRET-ing population in a sample by fitting on the double-exponential
>> model N(t) = A1*exp(?t/t FRET) + A2*exp(?t/t 0). Ideally, I would 
>> measure
>> donor lifetime (t0) from donor only sample (mono-exponential decay), 
>> and
>> then use this as a fixed parameter in subsequent fitting procedures 
>> (with
>> acceptor present). And, that worked fine until recently, when I came
>> across a sample where very small change in donor lifetime (fixed
>> parameter-I was playing with manually changing it) would yield
>> substantial difference in fitted A1 and A2, with equally good fit 
>> quality
>> (chi-squared test). So, now I can't decide which amplitudes to use. 
>> Does
>> anybody have some advice how to resolve this issue?
>> We use Leica confocal microscope paired with PicoQuant TCSPC system 
>> and
>> SyphoTime software for analysis. I should mention that I never 
>> measured
>> IRF (software offers to approximate it), so I'm not sure how this 
>> affects
>> fitting results. Also, I recently became aware of the FLIM fit 
>> software,
>> and I was wondering is there anyone experienced with both SymphoTime 
>> and
>> FLIM fit. What would be the better choice? Could this fitting issue be
>> resolved by using FLIM Fit (maybe it has some extra features for
>> assessing goodness of fit?)? Also, could you tell me what would be the
>> best sample (way) for measuring the IRF function (gold nanorods?- 
>> where
>> to get them?).
>> I would greatly appreciate any advice.
>> Thank you and kind regards,
>> Marko ?o?tar
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>> End of FLIMfit-users Digest, Vol 21, Issue 2
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-- 
Dr. Romain Laine, PhD in Biophotonics
Laser Analytics Group
Department of Chemical Engineering and Biotechnology
University of Cambridge
West Cambridge Site
Philippa Fawcett Drive
Cambridge
CB3 0AS