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%.
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.
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.
Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.
We present the real-time quantitative analysis of Vibrio vulnificus-infected host cells using quantitative phase microscopy (QPM) based on interferometric techniques. This provides the ability to retrieve the phase or optical path-length distribution over the cell with nanometer path-length sensitivity from a single interferogram image. We have used QPM to study dynamic cell morphologic changes and to noninvasively quantify the cell volumes of rat basophilic leukemia RBL-2H3 cells infected with V. vulnificus strains: wild type (MO6-24/O) and RtxA1 toxin mutant (CMM770). During the process of V. vulnificus infection in RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes, and areas were observed in real time using QPM. In contrast, dramatic changes were not detected in RBL-2H3 cells infected with the noncytotoxic RtxA1 toxin mutant. The results showed good correlation between QPM analysis and biochemical assays, such as lactate dehydrogenase assay or β-hexosaminidase release assay. We suggest that QPM is a powerful quantitative method to study the dynamic process of host cells infected with pathogens in a noninvasive manner.
We present the study of the correlation properties of RBC flickering using double-path interferometric quantitative
phase microscopy (QPM) using detrended fluctuation analysis (DFA). For DFA of RBC membrane fluctuations, we
have measured time series thickness variations of a normal RBC for 20 seconds. The amplitude of membrane
fluctuations in RBC have showed significantly larger than the background noise level without a RBC. We have
demonstrated a practical DFA application for QPM by studying the correlation property of RBC membrane fluctuations
in a noninvasive manner. By measuring the fractal scaling exponents of the time series RBC thickness variations
obtained from QPM, we have analyzed the correlation properties of RBC membrane fluctuations and the background
noise without a sample. The exponents for a normal RBC revealed the long-range correlation property in time series
during 20 seconds. However, the averaged exponent for background noise outside a cell was close to the exponent of
white noise.
Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 µg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.
We present the real time quantitative analysis of Vibriovulnificus-infected host cells using high stability quantitative
phase microscopy (HSQPM). It provides the ability to retrieve the phase or optical path length distribution over the cell
from a single interferogram image, which has been measured with nanometer path length sensitivity for long periods of
time. We have applied HSQPM to study dynamic cell morphologic changes and to quantify noninvasively cell volumes
of rat basophilic leukemia RBL-2H3 cells infected with pathogenic bacteria V. vulnificus strains, wild type (MO6-24/O)
and RTX toxin mutant (CMM770). During the process of V. vulnificus wild type infection to RBL-2H3 cells, the
dynamic changes of quantitative phase images, cell volumes and areas were observed in real time using HSQPM. In
contrast, the dramatic changes were not detected in RBL-2H3 cells infected with RTX toxin mutant. The results showed
the good correlation between HSQPM analysis and biochemical assays such as lactate dehydrogenase (LDH) assay and
β-hexosaminidase release assay. We suggest that HSQPM is useful real time quantitative method to study the dynamic
process of host cells infected with pathogen in a noninvasive manner.
The use of AgNP is becoming more and more widespread in biomedical field. But compared with the promising
bactericidal function, other physiological effects of AgNP on cells are relatively scant. In this research, we propose
quantitative phase microscopy (QPM) as a new method to study the degranulation, and AgNP-induced RBL-2H3 cell
degranulation is studied as well. Firstly, HeLa cells as the cell control and PBS as the solvent control, we measured the
cell volume and cross section profile (x-z plane) with QPM. The results showed that the volume and cross section profile
changed only the RBL-2H3 cells exposed to calcium ionophore A23187, which demonstrates the validity of QPM in
degranulation research. Secondly, 50μg/mL of AgNP was used instead of A23187, and the measurement of cell volume
and cross section profile was carried out again. RBL-2H3 cell volume increased immediately after AgNP was added, and
cross section profile showed that the cell surface became granulated, but HeLa cell was lack of that effect. Phase images
obviously indicated the RBL-2H3 cell deformation. Thirdly, stained with Fluo-3/AM, intracellular calcium Ca2+]i of
single RBL-2H3 cell treated with AgNP was observed with fluorescent microscopy; incubated with AgNP for 20min, the
supernatant of RBL-2H3 cells was collected and reacted with o-phthalaldehyde (OPA), then the fluorescent intensity of
histamine-OPA complex was assayed with spectrofluorometer. The results of Ca2+]i and histamine increase showed that
degranulation of AgNP-induced RBL-2H3 cell occurred. So, the cell volume was used as a parameter of degranulation in
our study and AgNP-induced RBL-2H3 cells degranulation was confirmed by the cell volume increment, cross section
profile change, and [Ca2+]i and histamine in supernatant increase.
Three dimensional particle tracking is useful technology to characterize live cell or surrounding environment by tracing
small particles such as fluorescence beads or polystyrene beads which adhered to objective samples. In microscopy
imaging system, the longitudinal(z axis) tracking of the particle is essential for implementation of three-dimensional
particle tracking, however it's been still a challenging topic to find the exact position of the particle in z axis with high
precision.
In this study, we present that a novel technique to find the longitudinal position of the particle, as well as the transverse
position(x,y axis) by applying the numerical reconstruction and focusing with digital holographic microscope.
Transmission type off-axis digital holographic microscope is implemented for this experiment, based on Mach-Zehnder
interferometer and 632.8nm HeNe laser is used as a coherent light source of the microscope and high-speed CMOS
camera is utilized for acquiring the hologram. Digital holographic microscope makes it possible to record and reconstruct
the phase and amplitude image of the sample. In order to find the position of the particle in z axis, we apply the
numerical focusing algorithm, which enables the translation of the imaging focus without actual longitudinal movement
of the sample. To demonstrate the presented method, Brownian movement of 3μm polystyrene sphere suspended in
water is investigated in this experiment.
We present a novel method to determine the effective elastic constant (EEC) and effective restoring
force (ERF) by using volumetric analysis of Red blood cell (RBC)s with Full field quantitative phase
microscopy (FFQPM). We use the simple harmonic oscillator model to determine EEC and ERF. We
investigate the EECs and ERFs of different shape of RBCs (discocyte, acanocyte, stomatocyte, and
spherocyte) and we investigate the effective temporal coherence of RBCs by analyzing temporal
volumetric behavior of the RBCs.
We present the measurement of red blood cell (RBC) volume change induced by Ca2+ for a live cell imaging with
full field quantitative phase microscopy (FFQPM). FFQPM is based on the Mach-Zehnder interferometer combined with
an inverted microscopy system. We present the effective method to obtain a clear image and an accurate volume of the
cells. An edge detection technique is used to accurately resolve the boundary between the cell line and the suspension
medium. The measurement of the polystyrene bead diameter and volume has been demonstrated the validity of our
proposed method. The measured phase profile can be easily converted into thickness profile. The measured polystyrene
bead volume and the simulated result are about 14.74 μm3 and 14.14 μm3, respectively. The experimental results of our proposed method agree well with the simulated results within less than 4 %. We have also measured the volume
variation of a single RBC on a millisecond time scale. Its mean volume is 54.02 μm3 and its standard deviation is 0.52
μm3. With the proposed system, the shape and volume changes of RBC induced by the increased intracellular Ca2+ are measured after adding ionophore A23187. A discocyte RBC is deformed to a spherocyte due to the increased intracellular Ca2+ in RBC. The volume of the spherocyte is 47.88 μm3 and its standard deviation is 0.19 μm3. We have demonstrated that the volume measurement technique is easy, accurate, and robust method with high volume sensitivity (<0.0000452 μm3) and this provides the ability to study a biological phenomenon in Hematology.
We present a novel chromatic dispersion measurement method using a spectral domain interferometer for single mode
optical fiber over a wide spectral range (200 nm). This technique is based on the Mach-Zehnder interferometer using a
white light source and spectrometer. A phase was directly retrieved from a measured spectral interferogram to obtain
relative group velocity, chromatic dispersion and dispersion slope. The measured results with the proposed method were
compared with those obtained using a conventional measurement method. Those results have good agreement with each
other. Our proposed method can simply, accurately, and quickly (< 500 ms) measure chromatic information for a short
length of optical fiber as well as optical devices.
Bio-cells and tissues have intrinsic polarization characteristics, which are changed by external stimulus and internal metamorphosis in cells and tissues and some of the bio-cells and tissues have intrinsic birefringence characteristics, which are also changed by external stimulus and internal metamorphosis in cells and tissues. In this paper, we have developed the polarization microscope for measurement of relative phase which results from birefringence characteristics of materials with improved linear polarizing method and have measured relative phase distribution of onion epidermal cells. From the measurement of the relative phase distribution of onion epidermal cells, decrease of relative phase distribution of onion epidermal cells was investigated as the elapse of time. In decrease of relative phase distribution, relative phase of cell membrane in onion epidermal cells decreased radically as compared with that of cytoplasm because decline of function in cell membrane that takes charge of matter transfer in onion epidermal cells has occurred.
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 LP01mode 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 LP01mode. 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.
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