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.