Analog Mean-delay Research Articles

Analysis of biexponential decay signals in the analog mean-delay fluorescence lifetime measurement method

Abstract Signals obtained in fluorescence lifetime imaging microscopy (FLIM) often contain biexponential decay components, and unmixing of them is a critical issue in FLIM especially for fluorescence resonance-energy-transfer (FRET) applications. Even though the analog mean-delay (AMD) method is the fastest fluorescence lifetime measurement scheme for point-scanning confocal microscopy, its ability to analyze biexponential decay signals has not been demonstrated, yet. In this paper we have demonstrated that biexponential decay signals can be effectively analyzed with the AMD method. Effects of various measurement conditions on the accuracy of biexponential decay signal analysis are presented with numerical simulations and experimental demonstrations.

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

Multispectral analog-mean-delay fluorescence lifetime imaging combined with optical coherence tomography.

The pathophysiological progression of chronic diseases, including atherosclerosis and cancer, is closely related to compositional changes in biological tissues containing endogenous fluorophores such as collagen, elastin, and NADH, which exhibit strong autofluorescence under ultraviolet excitation. Fluorescence lifetime imaging (FLIm) provides robust detection of the compositional changes by measuring fluorescence lifetime, which is an inherent property of a fluorophore. In this paper, we present a dual-modality system combining a multispectral analog-mean-delay (AMD) FLIm and a high-speed swept-source optical coherence tomography (OCT) to simultaneously visualize the cross-sectional morphology and biochemical compositional information of a biological tissue. Experiments using standard fluorescent solutions showed that the fluorescence lifetime could be measured with a precision of less than 40 psec using the multispectral AMD-FLIm without averaging. In addition, we performed ex vivo imaging on rabbit iliac normal-looking and atherosclerotic specimens to demonstrate the feasibility of the combined FLIm-OCT system for atherosclerosis imaging. We expect that the combined FLIm-OCT will be a promising next-generation imaging technique for diagnosing atherosclerosis and cancer due to the advantages of the proposed label-free high-precision multispectral lifetime measurement.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method.

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection.

High-speed time-resolved laser-scanning microscopy using the line-to-pixel referencing method.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to visualize photophysical characteristics of biological targets. However, conventional FLIM methods have some limitations that restrict obtaining high-precision images in real time. Here, we propose a high-speed time-resolved laser-scanning microscopy by incorporating a novel line-to-pixel referencing method into the previously suggested analog mean-delay (AMD) method. The AMD method dramatically enhances the photon accumulation speed for achieving the certain precision compared to the time-correlated single-photon counting (TCSPC) method while maintaining high photon efficiency. However, its imaging pixel rate can still be restricted by the rearm time of the digitizer when it is triggered by laser pulses. With our line-to-pixel referencing method, the pulse train repeats faster than the trigger rearm time can be utilized by generating a line trigger, which is phase-locked with only the first pulse in each horizontal line composing an image. Our proposed method has been tested with a pulsed laser with 40 MHz repetition rate and a commercial digitizer with a 500 ns trigger rearm time, and a frame rate of 3.73 fps with a pixel rate of 3.91 MHz was accomplished while maintaining the measurement precision under 20 ps.

Precision and accuracy of the analog mean-delay method for high-speed fluorescence lifetime measurement

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.

High-speed confocal fluorescence lifetime imaging microscopy (FLIM) with the analog mean delay (AMD) method

We demonstrate a high-speed confocal fluorescence lifetime imaging microscopy (FLIM) whose accuracy and photon economy are as good as that of a time-correlated single photon counting (TCSPC). It is based on a new lifetime determination scheme, the analog mean delay (AMD) method. Due to the technical advantages of multiple fluorescence photon detection capability, accurate lifetime determination scheme and high photon detection efficiency, the AMD method can be the most effective method for high-speed confocal FLIM. The feasibility of real-time confocal FLIM with the AMD method has been demonstrated by observing the dynamic reaction of calcium channels in a RBL-2H3 cell with respect to 4αPDD stimulus. We have achieved the photon detection rate of 125 times faster than a conventional TCSPC based system in this experiment.

Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging

The analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy. Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as those of the state-of-the-art time-correlated single photon counting method, there have been some speculations and concerns about the accuracy of this method with respect to the absolute lifetime value of a fluorescence probe. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of the lifetime to be measured. We have found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. We have proposed electrical and optical referencing techniques that may suppress these errors. It is shown that there may exist more than 2 ns drift in a measured temporal waveform during the period of the first 12 min after electronic components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as large as 51 ps without any referencing technique. We have shown that this error can be reduced to 9 ps with our electronic referencing technique. It is demonstrated that this can be further reduced to 4 ps by the optical referencing technique we have introduced.

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

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.