Global analysis of time correlated single photon counting FRET-FLIM data

Abstract

Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

“…The most reliable and informative method to quantify FRET in a heterogeneous system, such as cells, is fluorescence lifetime imaging microscopy (FLIM). [1][2][3][4][5][6][7][8][9] The theory of time-domain and frequency-domain (FD) time-resolved spectroscopy and a recent review on FLIM are described elsewhere. 10,11 In addition to a careful selection of the donor and acceptor molecules based on their spectral and physico-chemical properties, the geometry of the resulting donor/acceptor complex is very important for designing an efficient FRET system.…”

Effect of noise on modulation amplitude and phase in frequency-domain diffusive imaging

“…(12) and (13) indicate that these random variables are independent each other. Equations (12)(13)(14) imply that γ plays a critical role for the PDFs of andφ, which will be investigated in simulation in the next section. Interestingly, γ is not affected by σ 2 AC and a DC in Eq.…”

“…Equation (15) shows thatĀ, the mean of estimated from noisy sinusoidal outputs is biased and the amount of bias depends on σ 2 and γ in Eqs. (11) and (14). There are no analytic solutions for the moments ofφ, but they can be numerically calculated from Eq.…”

“…Furthermore, for very thick tissues, where it is hard in a frequency-domain measurement to extract meaningful data due to very small number of output photons, an impulse response function can be measured by the time correlated single photon counting (TCSPC) method. 5,14 However, the instrumentation for time-domain systems is usually more complicated and costly than that of frequency-domain systems. More discussion for the comparison between frequency-and time-domain measurements can be found in Refs.…”

“…7,9,14,[19][20][21] In this paper, we derive expressions for means and variances of modulation amplitude and phase that are estimated by Fourier transforming noisy sinusoidal measurements from heterodyne or homodyne methods. Other methods, such as data fitting or maximum-likelihood estimation can be used to extract the frequency-response information, but they generally require more computation time than Fourier analysis.…”