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.
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). The major advantage of this method is that the mean delay effect caused by a slow measurement system can be completely removed. The measurement speed can be very fast compared to the conventional TCSPC method because the AMD method can detect multiple photons simultaneously for a single excitation pulse. More accurate fluorescence lifetimes can be determined with more photons such that an accurate fluorescence lifetime image can be acquired quickly by the AMD method. In this study, we demonstrated cancer discrimination based on real-time AMD(Analog Mean-Delay)-FLIM(Fluorescence Lifetime Imaging Microscopy). We subcutaneously injected MDA-MB-231 breast cancer cell lines into nude mice. After subcutaneous (SC) injection of sodium fluorescein, the fluorescence lifetime of sodium fluorescein was measured by real-time AMD-FLIM. The fluorescence lifetime of sodium fluorescein depends on the local pH and pH differs between abnormal and normal tissues, cancer tissue can be discriminated from normal tissue by measuring the fluorescence lifetime of pH-sensitive sodium fluorescein. The measured fluorescence lifetime of sodium fluorescein inside the normal and abnormal tissues were 4.15~4.28 ns and 2.36~3.18 ns. Since the measured fluorescence lifetime for abnormal tissues were well differentiated from those for normal tissues, the fluorescence lifetime of sodium fluorescein could be used as an indicator to increase the accuracy of cancer detection with confocal microscopy or endoscopy.
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 presented the plastic based ultra-compact aspheric lens for disposable epidural spinal endoscope. We have also showed the analysis of the stray light distribution on the image plane using optical illumination system design software (Light Tools). The optical system consists of the aspheric lens with a size of 1.4mm (total track of optical system). The effective length and field of view (FOV) is 0.66mm and 90 degrees. The distortion of the optical system is below 25%. The curves of modulation transfer function (MTF) are higher than 0.3 at 80 line pairs/mm (lps/mm) in image space. For the analysis of stray light, we assumed that the 98 percent of incident light is absorbed inside lens barrel and the rest is scattered on the inner surfaces of the lens barrel. The average value of stray light is 0.16% in the image intensity. The maximum stray light and minimum stray light of the proposed optical system is 0.57% and 0.0005% in the image intensity, respectively. The effective transmission rate of the proposed optical system is 89.6%.
Not only static characteristics but also dynamic characteristics of the red blood cell (RBC) contains useful information for the blood diagnosis. Quantitative phase imaging (QPI) can capture sample images with subnanometer scale depth resolution and millisecond scale temporal resolution. Various researches have been used QPI for the RBC diagnosis, and recently many researches has been developed to decrease the process time of RBC information extraction using QPI by the parallel computing algorithm, however previous studies are interested in the static parameters such as morphology of the cells or simple dynamic parameters such as root mean square (RMS) of the membrane fluctuations. Previously, we presented a practical blood test method using the time series correlation analysis of RBC membrane flickering with QPI. However, this method has shown that there is a limit to the clinical application because of the long computation time. In this study, we present an accelerated time series correlation analysis of RBC membrane flickering using the parallel computing algorithm. This method showed consistent fractal scaling exponent results of the surrounding medium and the normal RBC with our previous research.
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 (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 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. As a referencing technique, we already proposed dual-channel waveform measurement scheme that may suppress these errors. In this study, we have demonstrated real-time confocal AMD-FLIM system with dual-channel waveform measurement technique.
KEYWORDS: Cameras, Endoscopes, Color reproduction, Endoscopy, Sensors, Light sources and illumination, Laparoscopy, In vivo imaging, Image display, Light sources, Xenon, Lamps, Medical imaging, RGB color model
Most of cameras like CCTV cameras or video cameras are shoot in available light or lightings. But the medical cameras like endoscope or laparoscopy are shoot in the situation in vivo. Generally, inside ad body is lightless and moist. So we can shoot the inside with a single light from outside the body. So medical cameras should be set more clearer and vivider in low-light than in high-light. The camera setting of disposable endoscopes is especially very important because those are low price and should be guaranteed against faulty workmanship. In this study, We suggest effective conditions for camera settings of disposable endoscopes, and request value of test measurement item with analysis from the commercial endoscopes.
We have studied the RBC membrane properties between a normal RBC and a RBC in Paroxysrnal nocturnal hemoglobinuria (PNH) patient using common path interferometric quantitative phase microscopy (CPIQPM). CPIQPM system has provided the subnanometer optical path length sensitivity on a millisecond. We have measured the dynamic thickness fluctuations of a normal RBC membrane and a RBC membrane in PNH patient over the whole cell surface with CPIQPM. PNH is a rare and serious disease of blood featured by destruction of red blood cells (RBCs). This destruction happens since RBCs show the defect of protein which protects RBCs from the immune system. We have applied CPIQPM to study the characteristic of RBC membrane in PNH patient. We have shown the morphological shape, volume, and projected surface for both different RBC types. The results have showed both RBCs had the similar shape with donut, but membrane fluctuations in PNH patient was shown to reveal the difference of temporal properties compared with a normal RBC. In order to demonstrate the practical tool of the CPIQPM technique, we have also obtained the time series thickness fluctuation outside a cell.
We have studied the method to improve the illumination efficiency in DFE with optical illumination design software. We have showed the result of illumination efficiency according to the change of geometrical shape in distal tip of plastic optical fiber (POF). We simulated the illumination efficiency in the case of the polished POF distal tip and the unpolished one, respectively using optical illumination system design software. We obtained the illumination efficiency was increased by about 46 percent in the polished distal tip more than in the unpolished distal tip when a light emitting diode (LED) was directly excited to the distal tip of POF. In order to demonstrate the simulated results, we showed the polishing fabrication of the distal tip in POF and have measured the illumination efficiency of the polished POF using LED. The measured results showed that illumination efficiency was increased by about 23 percent in the polished distal tip more than in the unpolished distal tip of POF. We have demonstrated the optimized geometrical shapes of the POF for minimization of the illumination loss. We have suggested the method to improve the illumination efficiency by 69 percent for a single fiber illumination delivery system of DFE.
KEYWORDS: Fluorescence lifetime imaging, Microscopy, Analog electronics, Confocal microscopy, Monte Carlo methods, Calcium, Molecules, Linear filtering, Signal to noise ratio, Signal detection, Data acquisition, Signal generators
We present a study on the characteristics of the AMD method. We have demonstrated that the photon economy of the AMD method is not degraded for longer lifetimes even when the applied integration window size is increased. By an extension of MCS, the photon economy with respect to different designs of the Gaussian low-pass filter (GLPF) used in the AMD setup was also studied. When a GLPF with the highest cutoff frequency of 100 MHz is applied, the most effective photon economy performance is achieved for lifetimes of 1, 3.2, 5, and 8 ns.
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