Phasor domain clustering of Fluorescence Lifetime data has gained a lot of attention because of fit-free algorithm and the ability to resolve heterogeneity of the sample. Despite the popularity, the optimal modulation frequency has not been discussed yet. Especially, an optimal condition must be considered where the two clusters are too close to discriminate, or it is challenging to acquire enough photons. In this study, we found optimal modulation frequency to resolve two clusters in the phasor domain. We theoretically derived the Davies-Bouldin index (DBI) of two clusters in the phasor domain and predicted the optimal frequency based on it. We verified the theoretical prediction via two different fluorescence droplets experiments.
KEYWORDS: Single photon, Photons, Time resolved spectroscopy, Time metrology, Temporal resolution, Photomultipliers, Electronics, Digital photography, Digital electronics
Because the axial resolution of an optical microscope is in the order of the wavelength of light, imaging a nano-scale object is very challenging. Many nanometer-sectioning imaging technics such as total internal reflection fluorescence (TIRF) microscopy and metal-induced energy transfer (MIET) imaging have been invented to overcome this limitation. However, the measurement ranges of these methods along the axial direction are too short to cover even a single cell. Here we propose a new long-measurement-range nanometer-sectioning imaging scheme by using MIET, focal plane shifting, and sophisticated signal analysis. We have verified the principle and the feasibility of our proposed method by using an artificial sample; it is shown that the axial measurement range is extended from 100 nm to 500 nm.
Fluorescence anisotropy is a powerful tool for observing molecular rotational speed, which is widely applied for molecular conjugation measurements (quantitative assay, membrane tagging, and protein-protein interaction). Fluorescence anisotropy (r) can be obtained by subtracting horizontal polarized fluorescence (H) from vertical polarized fluorescence (V), and dividing by non-polarized fluorescence (F). Since F equals to V+2H, V and H can be thought of as weighted sum and subtraction of F and r·F, respectively. Using phasor approach, which is graphical plotting technique based on fluorescence lifetime imaging microscopy, V and H are located on internal and external dividing points of F and r·F. Considering those four phasor points (V, H, F, and r·F) lie on a straight line, one can easily guess the value of the r. We first introduced phasor plot to fluorescence anisotropy, and confirmed that this method dramatically simplifies fluorescence anisotropy analysis with graphical intuition.
KEYWORDS: Fluorescence lifetime imaging, Sensors, Luminescence, Photodetectors, Signal detection, Monte Carlo methods, Single photon, Analog electronics, Physics, Temporal resolution
GaAsP hybrid detectors, which is new kind of photodetector, has been known as its excellent performance in time correlated single photon counting technique. We have verified that this detector also shows excellent performance in analog mean-delay method, which is another kind of time-domain FLIM, so one can expect enhancement of performance in time-domain FLIM when using the hybrid detector.
Phasor plot analysis is one of the most powerful analysis technique in fluorescence lifetime imaging microscopy, especially for analysis of heterogeneous mixtures. Compared to frequency domain fluorescence lifetime measurement, time domain measurement offers information in various frequencies at once measurement, but needs high frequency sampling for stable signal acquisition, which requires a lot of memory in hardware and a long time for analysis, furthermore in TCSPC, acquisition time is extremely long due to low photon count rate. We suggest a new system with low pass filter, which leads to about 100 times faster measurement speed while maintaining precision and accuracy in usual modulation frequency.
KEYWORDS: Luminescence, Fluorescence lifetime imaging, Analog electronics, Signal processing, Signal detection, Super resolution, Photodetectors, Convolution, Microscopy, Fluorescence resonance energy transfer
Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging tool widely used in monitoring cells, organelles, and tissues in biosciences. Since fluorescence lifetimes of most probes are a few nanoseconds, 20 ps measurement resolution is normally required. This requirement is quite challenging even with the fastest available optical and electronic devices, and several brilliant time-domain super-resolution techniques have been proposed for FLIM. The analog mean-delay (AMD) method is a recently introduced time-domain super-resolution technique for FLIM. Detailed constraints in the AMD method and their impact on the performance of the AMD super-resolution lifetime measurement are presented with experiments and simulations.
The Analog mean delay (AMD) method is a multiphoton detection fluorescence lifetime measurement method. We have investigated the effect of the linearity of a photodetector response on the performance of the AMD method, which is a multiphoton detected fluorescence lifetime measurement method. A Monte-Carlo simulation scheme was adapted to generate various electronic signals for the AMD method with different linearity conditions of a photodetector. It is found that the photon economy is better if a photodetector with better linearity is used especially when the number of detected photons per pulse is low.
A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.
We have simulated the effects of the number of bits and the sampling rate of a digitizer on the performance of lifetime measurements. We found that the number of bits of a digitizer is important to obtain certain accuracy in lifetime measurement. There exists a certain critical sampling frequency of a digitizer required to separate a certain lifetime differences in a double exponentially decaying intensity profile. We did these simulations by using Monte Carlo simulations with least-square curve fitting algorithms.
Angles of polygon scanners have been measured by using rotary encoders, autocollimators or indexing tables. These methods produce precise angle values but require removal of polygon mirror from its motor. For resolving this inconvenience, we introduce a simple angle measurement method by measuring timing jitters of a scanned beam in the time-domain with a high-speed detector and a digitizer while a polygon scanner is rotating at its full speed. Our setup includes a 635 nm wavelength semiconductor laser, a high-speed photodiode, two lenses, and a high-speed digitizer. A polygon scanner with 12 facets were tested with a rotating frequency of near 350 Hz. To detect the signal of the photodiode, we used a high speed digitizer which has a sampling rate of 2Gs/s with 256MB on-board memory. We obtained repeated pulsed sequential photodiode signals for 12 mirror facets of the scanner. Angle variations and their jitters for 12 scanner mirror facets were successfully calculated from measured data. We have repeated same experiments with a photomultiplier tube and compared results with those measured by a photodiode.
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