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

[Ewan McGhee]

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:
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>   1. Re: SymphoTime vs. FLIMfit (Munro, Ian)
>   2. Re: SymphoTime vs. FLIMfit (Dunsby, Christopher W)
>
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>----------------------------------------------------------------------
>
>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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