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 (FLIM). Even though the photon economy and the
lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon
counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. 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 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 also proposed dual channel waveform measurement scheme 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 minutes after
electronics components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as
large as 51 ps without a proposed scheme. We have shown that this error can be reduced to 9 ps with our dual-channel
waveform measurement method.
We have demonstrated the high-speed confocal fluorescence lifetime imaging microscopy (FLIM) by analog mean-delay
(AMD) method. The AMD method is a new signal processing technique for calculation of fluorescence lifetime and it is
very suitable for the high-speed confocal FLIM with good accuracy and photon economy. We achieved the acquisition
speed of 7.7 frames per second for confocal FLIM imaging. Here, the highest photon detection rate for one pixel was
larger than 125 MHz and averaged photon detection rate was more than 62.5 MHz. Based on our system, we
successfully obtained a sequence of confocal fluorescence lifetime images of RBL-2H3 cell labeled with Fluo-3/AM and
excited by 4αPDD (TRPV channel agonist) within one second.
Proc. SPIE. 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII
KEYWORDS: Photodetectors, Luminescence, Photons, Interference (communication), Data acquisition, Signal processing, Fluorescence lifetime imaging, Picosecond phenomena, Analog electronics, Signal detection
We present a novel method for high-speed measurements of fluorescence lifetime, in which fluorescence signal for
precise lifetime determination is acquired in a short time on the order of microseconds. Our method is based on analog
signal that contains a number of fluorescence photons in a pulse, on the contrary to the conventional time-correlated
single-photon counting in which only a single photon is permitted for a fluorescence pulse. Because this method does not
have any problem of photon counting pile-up, the measurement speed is not limited by the single-photon constraint and
can increase up to the excitation repetition rate. In order to extract the accurate lifetime information from the analog
signal contaminated by the slow instrumental response function (IRF), we have developed a new signal processing
method, in which the lifetime is determined by difference between mean arrival time of the analog photo-electronic pulse
of fluorescence signal and one of IRF signal. By both experimental and theoretical studies, we have verified that the
measurement accuracy and precision are nearly independent of the width of the IRF so that inexpensive narrowbandwidth
photo-detectors and low-speed electronics can be used for this method. Excellent accuracy and precision have
been obtained experimentally for high-speed measurements completed in a few microseconds. These results suggest that
our method can be well applied to measurement of fast dynamic phenomena and real time fluorescence lifetime imaging
microscope with low cost.
We present a fluorescence lifetime imaging microscope (FLIM) based on a real-time waveform acquisition method. The
fluorophores were excited by a 635-nm gain-switched laser diode, which produced short pulses with duration ~50 ps in a
20-MHz repetition rate. The fluorescence signals were detected by a silicon avalanche photo-diode (APD) in addition to
a wide-band electric amplifier. The converted electric pulses were sampled by a high-speed digitizer of which sampling
rate was 2 GS/s. In order to reduce the sampling interval for analyzing sub-nanosecond lifetimes, an interleaved data
acquisition technique was used. The effective sampling rate was increased to 10 GS/s. In addition, the impulse response
was measured simultaneously with the lifetime signals by an interleaving manner and was used in calibration of the
system. By using these methods, accurate lifetime information was acquired in a short time less than 8 μs.
We describe optical trap lattices, their manipulation, and optical trapping using the digital micromirror device (DMD)-accessory light modulator package (ALP). The proposed device flexibly controls the trap profile, array dimension, hopping over trap lattice, and steering therewithin. In order to generate optical trap lattice with Gaussian intensity profile, desirable input electronic images with LP<sub>01</sub>mode of single mode fiber to the DMD-ALP was loaded, which formed 2-dimensional optical trap lattice with Gaussian intensity profile. We generated 2-dimensional multiple optical trap lattice, where the individual intensity profile took LP<sub>01</sub>mode. This technique flexibly controlled the intensity profile, array dimension, and the hopping over trap lattice. We reported a new 2-dimensional optical trapping by means of the proposed system providing superior benefits in flexible digital control. In order to identify the possibility of optical trapping using the proposed device, single optical trapping was proposed. We demonstrated a polystyrene bead was attracted to a focused beam spot when the focused beam was near by the polystyrene bead and trapped bead was fixed by moving the sample stage of microscope up and down or right and left.
A new method to evaluate the image quality of the fiber bundle using digital micro-mirror device (DMD) along with accessory light modulator package (ALP) is proposed. We have demonstrated that some characteristics of fiber bundle, such as an extent of blurring, gray-level degradation and color aberration can be evaluated by using a single device DMD-ALP, and we have also proved capability of micron-order analysis. Moreover we measured the transmitted image contrast of the fiber bundle using some line pairs made by DMD-ALP, which can be more convenient than previous methods.