Frequency Domain Analysis for Fluorescence Recovery after Photobleaching

Fourier transformation is evaluated as a means of improving precision in the analysis of fluorescence-recovery-after-photobleaching (FRAP) data. Simulations of FRAP data of 2 ^m points, where m is an integer, are Fourier transformed to obtain the frequency domain data. Analogous to frequency domain techniques in nanosecond spectroscopy, frequency domain analysis of FRAP data is shown to provide more precise results. For a single exponential decay acquired over a time window of five decay constants, frequency domain analysis increases the precision by six fold without requiring that any more data be acquired. For a double exponential decay with decay constants that differ by a factor of two and noise of 5% relative standard deviation, time domain analysis is unable to distinguish this from a single exponential decay (χ² = 1.1), whereas frequency domain analysis reveals that it does not fit to a single exponential decay (χ² = 2.5). For a double exponential decay with five-fold differing decay constants, improved precision is obtained in the frequency domain for both of the decay constants, as well as the fractional amount of each. In contrast to nanosecond spectroscopy, the FRAP analysis described here combines the higher precision of the frequency domain with the direct observation in the time domain to facilitate the assessment of artifacts.