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.