Hi,

As John mentioned, it is possible to get lifetime analysis software from Edinburgh Instruments. I think FAST (See; http://www.edinburghphotonics.com/spectrometers/software/fast/) is very good and robust, particularly if you need to do global analysis across multiple wavelengths. PicoQuant also has lifetime analysis software (though I've never used it) and I'm sure there will be others out there too. Of course, there will be a cost to these commercial software packages.

If you don't want to write your own code then perhaps DecayFit (See; http://www.fluortools.com/software/decayfit) would be an option. I think it was originally written for MATLAB but it seems that a stand-alone version is now available. I'm not sure it has the capability to perform the analysis procedure Aleksander suggested (I've not really used it much) but it is free and, if required, it is possible to alter the scripts behind it (in the MATLAB version at least).

I suggest getting a copy of Principles of Fluorescence Spectroscopy by Lakowicz if you don't already have one. Advanced Time-Correlated Single-Photon Counting Techniques by Becker is also good if you want to know more about the technical aspects of TCSPC.

All the best,

Darren

Saurabh,

Non dairy creamer in water will work just fine for generating an instrument response function. Adjust the optical density of the solution to about 0.1.

I do not have the most recent version of Origin. The version I have does not have the immediate ability to deconvolute a decay that is convoluted with an irf. You may be able to buy a software package from Edinburgh instruments.

Best of luck,

John

i used water too during my TCSPC experiments. they were just fine. But people do use milk, silica and tinopol for IRFs.

Personally I never liked the origin 8 fittings. I would rather that you use a proper software package. You can do proper fittings and also get the % contributions which seem to me more accurate. Good luck.

Origin will work fine for deconvolution of TCSPC data and fitting multiple decay curves, but you can't just use the built-in functions, you'll have to write a script that performs the deconvolution. If that's not something you're comfortable with, then a commercial package might be better.

The non dairy creamer in water works well but in my opinion the LUDOX solution is better for the simple fact that sometimes your measuremnt takes some time to aquire. Using the creamer you risk having it settle to the bottom of your cell. The coloid solition in LUDOX remains in suspention and does not settle on you in the middle of a measurment.

As for the deconvolution I eould look for a software package to do the job

People often use colloidal silica/ ludox solutions to detemine the instrument response function, but this method may sometimes lead to erroneous results. In general, the IRF may depend on light wavelength.  Therefore, instead of using a nonfluorescent scatterer , I would advise to use a fluorescence lifetime standard with known lifetime and emission spectrum approximately matching the emission spectrum of the sample under investigation.

Thanks Aleksander! I am using Fluorescein dianion as my lifetime standard. Wouldn't I still need IRF to deconvolute my decay profile?? Also, could you give me few insights on what should a good IRF look like? Thanks!

I recommend to perform the following:

1. record the fluorescence decay of your sample ,

2. record the fluorescence decay of your standard at the same emission wavelength,

3. assuming a single exponential decay for your standard and the same IRF, perform the simultaneous deconvolution of the two decay profiles (1 and 2).

As a result you should obtain decay parameters of your sample and the decay time of your standard (you can check whether it agrees with literature data). in this method, the IRF does not have to be determined directly and in fact knowledge about its profile is not neeed at all. The only question is: does your deconvolution software allow to perform such a procedure. Otherwise you would need to customize the existing software or write your own...

Greetings!

Hi,

As John mentioned, it is possible to get lifetime analysis software from Edinburgh Instruments. I think FAST (See; http://www.edinburghphotonics.com/spectrometers/software/fast/) is very good and robust, particularly if you need to do global analysis across multiple wavelengths. PicoQuant also has lifetime analysis software (though I've never used it) and I'm sure there will be others out there too. Of course, there will be a cost to these commercial software packages.

If you don't want to write your own code then perhaps DecayFit (See; http://www.fluortools.com/software/decayfit) would be an option. I think it was originally written for MATLAB but it seems that a stand-alone version is now available. I'm not sure it has the capability to perform the analysis procedure Aleksander suggested (I've not really used it much) but it is free and, if required, it is possible to alter the scripts behind it (in the MATLAB version at least).

I suggest getting a copy of Principles of Fluorescence Spectroscopy by Lakowicz if you don't already have one. Advanced Time-Correlated Single-Photon Counting Techniques by Becker is also good if you want to know more about the technical aspects of TCSPC.

All the best,

Darren

Hi guys,

Regarding the IRF and in connection with the mentioned software 'DecayFit' by Darren A. Smith, I have a question. Actually, in that program when you analyse your data, a % scatter for the IRF can be selected. Can anyone explain me under which criteria I should set it? Sometimes the fitting gets much better when the % scatter is remarkably higher. It shouldn't be the same for all the measurements, which have been taken over the same sample?

Also, if the fitting is a bi-exponential, for getting the average lifetime, should I take this scatter into account or I just get the parameters associated to the intensity amplitudes?

Many thanks!

Dear Yurema Teijeiro Gonzalez,

As I said previously, I haven't actually used Decay Fit very much so I am perhaps not the best person to answer you.

I assume you have seen the guide here: http://www.fluortools.com/software/decayfit/documentation/fit. If not, it may help you with your fitting. Otherwise you could try and contact Søren Preus (see; http://www.fluortools.com/about) and ask about the issues you are facing (though I have no idea if he is still actively monitoring Decay Fit – I have never been in correspondence with him).

Anyway, it is difficult to give you a precise answer because there are many things that depend on the specific details of your experiment; however, I will outline some thoughts (which may or may not be appropriate for you).

As the name suggests, the scatter parameter is a correction for any scattered excitation light detected during acquisition. Whether you should include it in your analysis will depend on your sample and your system.

Ideally, you should not observe scatter in your sample decay. If you are not doing so already, it is possible that using an emission (long pass) filter may help reduce any scatter detected. Unfortunately, it may not be possible to get rid of scatter completely. Scattered light may be misinterpreted as a short lifetime component in the decay, which is why it sometimes needs to be corrected for.

Including the scatter parameter may cause a problem if you expect to have a lifetime component that is very short (on the order of the IRF – or channel – width), since it will result in an underestimate of the contribution (a-factor amplitude) of a short lifetime component. Of course, conversely, excluding the scatter parameter may result in an overestimate of the contribution of a short lifetime component.

(Rather than fit with the scatter parameter, you could potentially fit the decay with an ‘extra’ lifetime component. It may be useful to see if you get the same fit parameters as when using the scatter parameter. Alternatively, you could see what values you get when you perform a tail fit without deconvolution (starting the fit range at a channel after the peak of the decay).)

If the fluorescence lifetime(s) that you are measuring are long (compared to the IRF width) then things are perhaps a little easier: you should, in all likelihood, include the scatter parameter (especially if you are getting large values and much better quality of fit).

You could perhaps record the lifetime of a standard with a known lifetime and similar photophysical properties (excitation/emission wavelength and lifetime) as your sample. Then you can determine if fitting with the scatter parameter is important for you. If you need the scatter parameter to obtain the expected lifetime of the standard then it probably means you should include it in the analysis of your sample.

Whatever you do, be consistent. Comparing the fits of different samples if often far more enlightening (and meaningful) than a specific decay analysis (which may be prone to error and artefacts, such as scattered light).

If it is present, you should account for scattered light in your fitting; however, you should not include the scattered light contribution in your calculation of average lifetime (it has, in effect, already been accounted for).

I hope that helps.