We have developed a compact optical endoscopic probe for in vivo fluorescence optical imaging device. We obtained fluorescence image for the colon tissue of a mouse using a compacted optical endoscopic probe with a designed endoscopic optical lens. In order to demonstrate endoscopic fluorescence imaging for the colon cancer, we have manufactured a compacted optical endoscopic probe to pass through the biopsy channel of electric flexible endoscope. The compacted optical endoscopic probe with maximum outside diameter of 2.8mm consists of fiber-optic illumination part and imaging part. The imaging part consists of a fiber-optic imaging bundle linked to an endoscopic optical lens and focus assembly. We considered a compact structure, sensitivity, and FOV for the design of the endoscopic optical lens. We have suggested an endoscopic optical lens with an FOV of 90 ° and DOF of 3 – 80mm. The optical system consists of glass-based aspheric lenses. The total track is less than 2.5 mm, and the diameter is limited to less than 1.5 mm to obtain a compact system. We have presented the compacted optical endoscopic probe for cancer imaging of a mouse. The proposed endoscopic optical lens showed sufficient sensitivity and a wide field of view for obtaining fluorescence imaging. We demonstrated endoscopic ex vivo fluorescence imaging for the colon cancer of a mouse using the compacted optical endoscopic probe.
We have designed a fiber probe based optical diagnose system for detection of interspecies transmissibility. We have showed the optical performance to measure the optical signal of the target sample by using the manufactured fiber probe. We have confirmed the capability of our system to be utilized to biomedical diagnose applications.
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.
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%.
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.
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.
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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