I have used a PicoQuant MicroTime 200 system to do FCS measurements of free Alexa Fluor 488 dye and its conjugates with proteins in simple phosphate buffers. The samples are quite simple. However, I have had a serious problem with the data acquisition: I get a lot of noise in all of my experiments, and the autocorrelation curves can be barely seen, if you can consider it a curve. The same thing seems to happen with the samples provided with the equipment for calibration. I have already tried to do cross-correlation measurements, but the problem persists. I’m new in this field, so I would be grateful for some help.
Hi, Vinnie. Thanks for your answer.
I think I'm already using FLCS. At least I have been applying the FLCS filters. I have already used different dye concentrations, like those that came with the equipment for calibration (Atto488 + 655 at 10^-6, 10^-8, 10^-9 and
Hi, Peter. Thank you for your help.
In this measurement that I showed, the number of photons were really low, but I fixed it by increasing the measurement time (10 min) and doing some other adjustments. The routing channel boxes were correctly marked for both channels A and B, but the program simply marks all the boxes after the FCS calculation. So I think that the autocorrelation is correct.
After doing the correction that you suggested, I managed to improve my results, but it is still bad. It is possible to see a noisy curve at longer times, but at short times the noise is absurd.
Hi, Vinnie.
It's true, I don't get a curve within a short period of time, just a lot of noise. Unfortunately, I won't be able to tell you the counts at this moment, but I'll do it as soon as possible. I'll try to seek advice at the PicoQuant forum too.
Hi, Peter. Thank you for showing me this mistake. I'll try to do it right the next time.
I have tried to do the same measurements with the same calibration sample (which has Atto655, if I'm not wrong) using the 640 nm laser and its appropriate emission filter, dichroic mirror, frequency, intensity percentage etc, just as I did for the 470 nm laser. I have obtained perfect FCS curves, with excellent fits, mainly at 10-9 and
I have a stupid question: did you optimized the pinhole after changing the laser?
Hello Gustavo.
I am having similar issues with my FLCS setup as well. I think my TCSPC histograms are okay, but my FCS curves display strange oscillations. I am not sure whether radio frequencies from the AOTF are interfering with the APD or whether my signal is being reflected back into my cable.
I have a Finaium white laser, and I use an AOTF to select the excitation wavelength. I have a fiber-coupled Perkin Elmer SPCM-AQRH-14. My TCSPC card is TimeHarp 200. I am currently overfilling my objective lens.
I think we should try to stay in contact as we work on our respective setups! It is nice to know that I am not the only one struggling with this issue.
Hi, Vinnie.
As you requested, in blue (the problematic system) I got 2030 counts per second and 368 counts per second per molecule. On the other hand, in red I got 10513 counts per second and 7108 counts per second per molecule (both configurations at
Hi, Peter. Thanks for your help and sorry for delaying my answer.
Since Atto488 is a recommended standard for calibrating the system using the 470 nm laser, I changed to this dye to reduce the possibility of the problem be my sample and then reducing cumulative errors, when trying to find out the source of this strange behavior.
As you already wrote, the problem was really that the laser excitation power reaching the sample was too low. We figured out that the source of this problem was the bad alignment of multiple components that we usually do not disturb. We managed to greatly improve the obtained FCS curves by optimizing the alignment at the Laser Combining Unit, at the Main Optical Unit, and of the dichroic mirror.
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