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Choosing FCS Acquisition Options

FCS data acquisition is very simple, but choosing the wrong options can lead to ambiguous or complicated results. Carefully considering the following options will ensure that you acquire meaningful data.

  • Laser excitation intensity: Must be set to a suitable intensity for your fluorescent dyes to avoid photobleaching and intersystem crossing (triplet state excitation), yet achieve sufficient average intensity for FCS measurements.
  • Delay Before Acquistion: The time between sample preparation and measurement depends on the type of assay being performed. Assays that monitor reaction dynamics over time should be measured as soon as possible, but assays that monitor systems at equilibrium should be measured when you are sure equilibrium has been reached. A significant delay between preparation and measurement can lead to aggregation of your sample and can affect some fluorescent dyes.
  • Acquisition Duration: Longer data acquisition leads to cleaner, more robust data. However, very long acquisition times increase the chance that your data will be affected by artifacts such as large fluorescent particulates or drift in intensity over time.