Abstract

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 <i>2</i>^<i>m</i> points, where <i>m</i> 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.

Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments

Davide Mazza, Francesca Cella, Giuseppe Vicidomini, Silke Krol, and Alberto Diaspro
Appl. Opt. 46(30) 7401-7411 (2007)

Experimental and theoretical investigation of fluorescence photobleaching and recovery in human breast tissue and tissue phantoms

Sharad Gupta, Bhawna, Pallab Goswami, Asha Agarwal, and Asima Pradhan
Appl. Opt. 43(5) 1044-1052 (2004)

Frequency domain analysis for laser-locked cavity ringdown spectroscopy

T. K. Boyson, T. G. Spence, M. E. Calzada, and C. C. Harb
Opt. Express 19(9) 8092-8101 (2011)

Comparison of frequency-domain and time-domain fluorescence lifetime tomography

Anand T. N. Kumar, Scott B. Raymond, Brian J. Bacskai, and David A. Boas
Opt. Lett. 33(5) 470-472 (2008)

Statistical analysis and optimization of frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection

Yuxiang Lin and Arthur F. Gmitro
J. Opt. Soc. Am. A 27(5) 1145-1155 (2010)