What is the frequency of these oscillations?

I will have to double check the data, but I think the frequency is in the kHz.

Can you measure the frequency and let us know? It will help us in providing a solution that matches your requirement.

Frequency of such oscillations gives a good clue as to the source of these oscillations.

Just capturing these oscillations onto a oscilloscope and performing fourier transformation will give you the frequency directly. Some of the advanced oscilloscopes have fourier transformation as in-built feature.

Those often come from computer monitor light leaking into detectors.

Whenever I do experiments, I always dim the computer monitor.  But I could try to dim the monitor even more.

We also see such oscillations in one of our setups. there the problem seems to be an impedence mismatch: Our Perkin-Elmer APDs have a 50Ohm output and are connected to two cards in the computer: An ALV-5000 correlator and a NI counter card. The ALV-card has a 50Ohm input, but the counter has a MegaOhm input ... this mismatch causes reflections of the signals back into the cable, which lead to oscillations on the curve.

Could you post an example ACF? BTW: You can simply read the frequency from the ACF: The first minimum of the pscillatory signal should give you the period of the oscillations (I think up to a factor of 2).

Some other reasons, that are possible for this:

Hello Jan.

Thank you for your suggestions.

1)  We do have an anti-vibration table from Thor Labs.  I am not sure how to inspect the bumpers.  I will look into that immediately.

2)  We do not use ALEX.

3)  We have a Fianium WhiteLase SC480, and we use a Fianium AOTF to select an excitation wavelength from the white laser.  Our laser is pulsed.  The repetition frequency is supposed to be 40 MHz.  However, according to the TimeHarp program, I sometimes see a repetition frequency of 41.5 MHz.

4)  I quickly inspected our setup.  It looks like all of the cables and connectors to our TimeHarp 200 card have an impedance of 50 ohms.  However, I am using connectors from different manufacturers to connect to the Start and SYNC ports of the TimeHarp card.  I do not know if that could make a difference.

5)  I will post a screenshot of a ACF as quickly as possible. 

Hi!

quickly ad 3: The laser shouldn't be the problem then, as at 40MHz you will always average over many pulses in FCS (at least above 1µs).

Best, JAN

Have you checked the settings for you TCSPC Setup, like TAC/CFD/ADC? Misconfiguration of these can also lead to an 'echo' which can look like oszilations. Other than this, i agree with Jan that reflections are a likely source of oszillations.

Check if it is due to background? Do you get these oscillations even when the laser is exciting the sample?

Check if it is due to pickup from the mains? Check if the frequency of these oscillations matches the frequency of the mains.

Check if it is due to some lose connection or contact?

See if you can cut it off using a RC filter.

Thank you for all of your suggestions!  I will try to troubleshoot them later today.  I just wanted to mention one last thing.  I am overfilling my objective lens by quite a bit.  The diameter of the back aperture of my objective lens is 4.3 mm, but the laser beam diameter is 5 mm.  I am still reading literature on how overfilling affects the quality of your signal.

If you overfill your back aperture you achieve the smallest possible (diffraction limited) illuminated volume. For optimal signal your illuminated volume should correlate to the size of your pinhole on the detection side and also the size of your detection area.

Also check to see if you get the same oscillations in the absence of any fluorophore? All other parameters being the same.

Hello.  

Here is a jpeg picture of an "autocorrelation" curve that I measured last week.  

The sample was 14 nM TAMRA, the pinhole aperture was 30 microns, the excitation wavelength was 550 nm, and the Fianium WhiteLase was set to a laser power of 60%.  

My TimeHarp 200 settings were as follows:  acquisition time of 10 minutes, SYNC of -20 mV, CFD of 20 mV, and zero cross of 20 mV.  The photon count rate was around 15 kHz.

As you can see, the correlation is really poor.  I also have these strange oscillations to which I have been referring.

For the future, I am going to try to underfill the objective lens, optimize the settings on TimeHarp 200  card, and inspect whether my signal is being reflected back into the fiber optic cable. 

Hello Jan.

What kind of fiber optic cable do you use in your setup?  Single mode vs multi mode?  Stepped index vs graded index?  What is the NA and the fiber core diameter?

The reason why I am asking is that I am not sure that I am using the optimal fiber optic cable for my setup. 

Dear Niloy

If the instrumental factors are in line, try fitting the autocorrelation/ residual of the data, the oscillations may be due to ultrafast motion which would show up in the fitting of the residual, you might have to compare the fitting with a vibrational Spectra of the compound in the concerned region. The oscillations may not be an artefacts, provided instrumental artefacts are none.

Hi, Bijan Kumar Paul . I reached out to Bryan Donaphon a few months ago as I was seeing the same problem with massive oscillations in FCS acquisitions. He told me that he never solved the problem, and instead moved his FCS measurements to another instrument. In my case, I have a Leica SP8 with Supercontinuum White Light Laser, and a Picoquant FLIM/FCS (Picoharp 300). Turns out for us when we disconnected the "router" (Picoquant PHR800), which takes the 2 detector channels and transfers them to the Picoharp, the oscillations were gone! We sent the router back to Picoquant for repair. When reconnecting it after repair, we still get a bit of fluctuation (though not as bad as before) but this improved by replacing the cable with one of a different length. We will do some further testing in the coming weeks and I will post an update. I hope this helps you to troubleshoot!

Cheers,

James

Hi James

Thanks for sharing, will wait to have the update of your tests.

Cheers