FCSXpert™ Solutions: Fluorescence Correlation Spectroscopy Simplified!.
FCSXpert Banner

Customer Support

FCS Troubleshooting Tips

There are a number of things that can be done experimentally to make analysis of FCS data more robust. Some of these include:

  • Minimize Laser Excitation: Increased laser power results in a number of photophysical events that might affect your FCS data collected with the QuantumXpert. It is a good idea to use the minimum laser power possible (greatest attenuation) while still obtaining between 50 and 100 Kcps intensity (See Choosing FCS Acquisition Options).
  • Use the Fluorescence Intensity Monitor: (FCSXpert™ Software) The fluorescence intensity monitor allows you to select the appropriate level of attenuation prior to acquiring correlation data.
  • Work Within Recommended Dye Concentration Range: The range of dye concentrations that can be used with the QuantumXpert FCS Spectrometer is 50 pM - 50 nM. The effective probe concentration can be manipulated to extend this range by controlling the specific activity of the dye-labeled probe.
  • Choose the Minimum Acquisition Time: If your counts are sufficiently high, multiple scans of short duration will result in better quality data than a single scan of long duration. Although increasing the acquisition time improves counting statistics for the correlation functions, it also increases the probability of a counting artifact due to detector noise or simply dust particles in the sample.
  • Select the Right Dyes: The dyes should not interfere with normal interaction of probe and target, and should be selected to minimize photobleaching and triplet state conversion (See Choosing Fluorescent Dyes for FCS Assays).
  • Use High Quality Probes: Probe specificity and purity will have a large impact on the quality of your FCS measurements (See Choosing & Preparing Biological Probes for FCS Assays.
  • Select the Right Buffer Conditions: Buffers should be selected for both compatibility with the interaction being measured, as well as compatibility for FCS measurements (Choosing Buffers for FCS Assays).
  • Do the Control Measurements: The control measurements described in Selecting Control Samples for FCS Assay will provide you with the necessary information to ensure effective analysis of your FCS data.