Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy

Abstract

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.

FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging

“…In the case of single exponential decay, the average lifetime obtained through the R-method, should match the real decay constant. Its lifetime accuracy, quantified by the F-Number [26,27], is similar to that of iterative single-exponential decay fitting under identical fluorescent brightness (Fig. 3(b)).…”

Single-laser shot fluorescence lifetime imaging on the nanosecond timescale using a Dual Image and Modeling Evaluation algorithm

“…6). Using such a scheme would also yield F-values around 2.5, which is comparable to the lowest values obtained in the simulations by Edler et al [26].…”

“…Therefore single-shot measurements in the frequency domain can simply not be performed on the same time scale. However, Edler et al [26] showed analytical expressions along with Monte Carlo simulations for several FLI schemes in the frequency domain. For lifetimes between 0.5 and 1.5 ns, the minima of calculated F-values for sinusoidal gain modulations and excitations were in the range 6-9, whereas Dirac excitation in combination with sinusoidal gain modulation resulted in minimum F-values as low as 2.…”

“…In order to present data that could be compared to previously reported simulated results [26], technical details regarding the specific detectors were not included. The noise propagation of an FLI method is commonly illustrated by calculating the figure of merit (F), which is formed as [27]:…”

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

“…[18][19][20] The current state-of-the-art FD-FLIM system requires an image intensifier, the use of which is necessary for low light levels and MHz demodulation frequencies. 11,17,21 The demodulation is done by controlling the voltage of the photo cathode in the image intensifier.…”