Analog mean-delay method for high-speed fluorescence lifetime measurement

Abstract

We present a new high-speed lifetime measurement scheme of analog mean-delay (AMD) method which is suitable for studying dynamical time-resolved spectroscopy and high-speed fluorescence lifetime imaging microscopy (FLIM). In our lifetime measurement method, the time-domain intensity signal of a fluorescence decay is acquired as an analog waveform. And the lifetime information is extracted from the mean temporal delay of the acquired signal. Since this method does not rely on the single-photon counting technique, the signals of multiple fluorescence photons can be acquired simultaneously. The measurement speed can be increased easily by raising the fluorescence intensity without a photon-rate limit. We have investigated various characteristics of our method in lifetime accuracy and precision as well as measurement speed. It has been found that our method can provide excellent measurement performances in various aspects. We hav demonstrated a high-speed measurement with a high photon detection rate of approximately 108 photons per second with a nearly shot noise-limited photon economy. A fluorescence lifetime of 3.2 ns was accurately determined with a standard deviation of 3% from the data acquired within 17.8 micros at a rate of 56,300 lifetime determinations per second.

High-speed confocal fluorescence lifetime imaging microscopy by analog mean-delay method

“…The GLPF makes it possible to reconstruct signal with a sampling rate of 100 MS/s. Its impulse response function was controlled to contain no frequency components higher than 50 MHz due to the Nyquist-shannon sampling theorem [11]. The reduced amplitude of the signal after the GLPF was amplified by electric amplifier (AMP, Minicircuits, TB-409).…”

“…As the data acquisition was triggered by the synchronized signal coming from laser diode driver, the initial time position for both signals could be well matched each other. For better precision, the applied integration window size was 30 ns and it was the full width at half maxima (FWHM) of the IRF signal, and four iteration processes were performed [11].…”

“…The evaluated accuracy and photon economy of this method was also compatible with TCSPC method. [11] And the AMD method is also can be applied to confocal system for good spatial resolution and 3D sectioning. By using high-speed resonant scanner and digitizer, the acquisition speed of 7.7 frames per second was achieved for confocal FLIM imaging.…”

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

“…To address this issue, many recent efforts focused on reducing acquisition time by developing high-speed FLIM techniques, using specific detectors and dedicated electronic cards (18)(19)(20). In contrast, the work presented here uses a standard TCSPC FLIM acquisition system.…”

Parametric Optimization for High Speed FLIM Implementation

“…AMD FLIM is an exertion to reconstruct a high frequency signal with a high-bandwidth PMT. Figure 1 shows schematic representation of AMD FLIM [2] and its principle. A photon emitted from pulse laser exhibits timing jitter which makes up Gaussian distribution represented in black in Figure 1.…”