We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 having absorption at UV wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film of an electron-beam excitation assisted (EXA) optical microscope. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal. The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA optical microscope is useful for analysis of growth of a crystal, bioimaging, and so on.
We present high spatial-resolution label-free imaging with an electron-beam excitation-assisted optical microscope (EXA microscope). The EXA microscope improves the spatial resolution down to 100 nm. To realize the high spatial resolution, a nanoscale optical spot is generated by irradiating a fluorescent thin film with a focused electron beam whose spot size is less than 10 nm. The size of the optical spot becomes smaller than the diffraction limited spot size and is reduced to about 100 nm, because the light emission is localized in nanometer-sized region. In this microscopy, it is not necessary to label a specimen for imaging beyond the diffraction limit of the light. The specimen stage is separated from the vacuum chamber of the scanning electron microscope by the fluorescent thin film and a specimen under atmospheric pressure can be imaged.
We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 having absorption at UV wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film we deposited. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal measured with an atomic force microscope (AFM). The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA microscope is useful for analysis of growth of a crystal, bio-imaging, and so on.
We have developed a confocal fluorescence laser scanning microscopy (CFLSM) incorporating a liquid crystal on silicon spatial light modulator (LCOS-SLM). To achieve high-resolution and high-contrast imaging for deeper part of the tissue with CFLSM, high numerical aperture objective lenses are required to tightly focus excitation light to meet Rayleigh limit(criterion) for the specimens. However, mismatch of refractive index at the boundary of interfacing materials, such as atmosphere, glass cover, and biological tissues, causes spherical aberration. Recently, we proposed a numerical method for correcting spherical aberration. In this method a pre-distorted wavefront pattern for aberration correction is calculated by ray tracing from a hypothetical focal point inside a specimen to the pupil plane. The resulting microscope can correct such spherical aberration. We observed 6.0μm fluorescent micro-beads dispersed three-dimensionally in agarose gel to confirm effectiveness of aberration correction. We reconstructed a three-dimensional image by taking 20 images by changing the depth with 1 μm interval and stacking them. It was apparent that the longitudinal/depth resolution was improved and that the intensity of fluorescence image was increased with aberration correction. While this method is applicable to other laser scanning microscopes, it has potential to enhance the signals for various super-resolution microscopic techniques, such as stimulated- emission-depletion (STED) fluorescence microscopy.
To observe molecular transport in a living cell, a high-speed CMOS image sensor for multi-point fluorescence correlation spectroscopy is developed. To achieve low-noise and high-speed simultaneously, a prototype CMOS image sensor is designed based on a complete pixel-parallel architecture and multi-channel pipelined pixel readout. The prototype chip with 10×10 effective pixels is fabricated in 0.18-μm CMOS image sensor technology. The pixel pitch and the photosensitive area are 56μm and 10μm in diameter without a microlens, respectively. In the experiment, the total sampling rate of 606kS/s is achieved. The measured average random noise is 24.9LSB, which is equivalent to about 2.5 electrons in average.
We propose a method for high precision modulation of the pupil function of a microscope objective lens to improve the performance of multifocal multi-photon microscopy (MMM). To modulate the pupil function, we adopt a spatial light modulator (SLM) and place it at the conjugate position of the objective lens. The SLM can generate an arbitrary number of spots to excite the multiple fluorescence spots (MFS) at the desired positions and intensities by applying an appropriate computer-generated hologram (CGH). This flexibility allows us to control the MFS according to the photobleaching level of a fluorescent protein and phototoxicity of a specimen. However, when a large number of excitation spots are generated, the intensity distribution of the MFS is significantly different from the one originally designed due to misalignment of the optical setup and characteristics of the SLM. As a result, the image of a specimen obtained using laser scanning for the MFS has block noise segments because the SLM could not generate a uniform MFS. To improve the intensity distribution of the MFS, we adaptively redesigned the CGH based on the observed MFS. We experimentally demonstrate an improvement in the uniformity of a 10 × 10 MFS grid using a dye solution. The simplicity of the proposed method will allow it to be applied for calibration of MMM before observing living tissue. After the MMM calibration, we performed laser scanning with two-photon excitation to observe a real specimen without detecting block noise segments.
In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not
by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the
cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and
ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence
spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of
tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was
monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence
spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral
analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of
three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in
the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors
showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of
4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring
tumor.
KEYWORDS: Endoscopes, 3D image processing, Distortion, 3D metrology, 3D displays, Endoscopy, Cameras, Distance measurement, Laser systems engineering, Laser therapeutics
Conventional endoscopic images do not provide quantitative 3-D information. We present an endoscope system that can measure the size and position of an object in real time. Our endoscope contains four laser beam sources and a camera. The procedural steps for 3-D measurements are as follows. First, to obtain the function that maps 2-D coordinates of an image point to its 3-D coordinates in 3-D space, we observe a standard chart with the endoscope lens and determine the correspondence between the image and object height. In addition to the mapping, this function can correct barrel-shaped distortion of endoscopic images. The system detects laser spots on an object surface automatically using a template matching method, and maps the 2-D coordinates of the laser spots to the 3-D coordinates by the triangulation method. Then the system calculates the magnification ratio on the object plane, which is perpendicular to the optical axis and passes the laser spot, so that the system can superimpose a ruler whose scale fits the 3-D coordinates of the object. Thus, physicians can measure the size and position of objects in real time on undistorted images similar to placing rulers on the surface of an organ.
Due to very complex structure of nasal area that is covered by facial bones, a tracking of surgical instruments
on the preoperative CT image is very important for obtaining an improved image guidance as well as preventing
surgical accidents in the paranasal sinus surgery. In this contribution, we present our recently developed an efficient
and compact navigation system for paranasal sinus surgery and its first clinical trial.
In our system, we use an optical-based 3D range imaging device intra-operatively, in order to achieve
registration and a tracking of instruments. Before the intervention, the range image of patient's face is acquired by a
3D range scanner and registered to corresponding surface extracted from the preoperative CT images. The surgical
instrument fitted with spherical markers that also can be measured by range scanning device, is tracked during the
procedure. The main advantages of our system are (a) markerless on the patient's body, (b) an easy semiautomatic
registration, (c) frameless during surgery, thus, it is feasible to update a registration and to restart the tracking when
a patient moves. In this paper, we describe a summary of used techniques in our approach including the benefits and
limitations of the system, experimental results using a precise model based on a human paranasal structure and a
first clinical trial in the surgical room.
The photodynamic therapy (PDT) on tumors is quite effective and widely applied but usually carried out without an immediate evaluation of results. We measured the tumor fluorescence in mice with a fiber probe connected to a linear array spectral analyzer (PMA-11, Hamamatsu Photonics). The spectrum showed a transient change in fluorescence color from red to green during Photofrin-mediated PDT. In order to examine the source of green fluorescence, the mitochondria were accessed under a Nipkow disk-scanning confocal microscope in the HeLa cell in culture after labeling them with a red fluorescent protein (DsRed1-mito) and staining the cell with Photofrin (Axcan Scandipharm). Changes in fluorescence color from red to green were observed in the area of mitochondria upon their swelling during irradiation. This finding in vitro provided clear evidence that the change in fluorescence color from red to green observed in vivo was due to the mitochondrial destruction associated with the cell-death by PDT. This technique of spectral monitoring in tumor may be useful for detection of the cell-death signal during PDT in patients.
This paper presents a segmentation method of brain tissues from MR images, invented for our image-guided neurosurgery system under development. Our goal is to segment brain tissues for creating biomechanical model. The proposed segmentation method is based on 3-D region growing and outperforms conventional approaches by stepwise usage of intensity similarities between voxels in conjunction with edge information. Since the intensity and the edge information are complementary to each other in the region-based segmentation, we use them twice by performing a coarse-to-fine extraction. First, the edge information in an appropriate neighborhood of the voxel being considered is examined to constrain the region growing. The expanded region of the first extraction result is then used as the domain for the next processing. The intensity and the edge information of the current voxel only are utilized in the final extraction. Before segmentation, the intensity parameters of the brain tissues as well as partial volume effect are estimated by using expectation-maximization (EM) algorithm in order to provide an accurate data interpretation into the extraction. We tested the proposed method on T1-weighted MR images of brain and evaluated the segmentation effectiveness comparing the results with ground truths. Also, the generated meshes from the segmented brain volume by using mesh generating software are shown in this paper.
Neurosurgical navigation systems using preoperative images have a problem in their accuracy caused by brain deformation during surgery. To address this problem the use of laser range scanner in order to obtain intraoperative cortical surface, is under study in our currently developing neurosurgical navigation system. This paper presents preliminary results of registration of intraoperatively acquired range and color images to preoperative MR images, within the context of image-guided surgery. We register images by performing two procedures: mapping of color image on the range image; and registration between color-mapped range images and preoperative medical images. The color image is mapped on the range image using camera calibration. Point-based rigid registration of preoperative images to the intraoperative images is performed through detection and matching of common fiducials in the images. Experimental results using intraoperatively acquired range images of cortical surface demonstrated the ability to perform registrations for MR images of the brain. In the future, we will focus on incorporating the above registration results into a biomechanical model of the brain to predict brain deformation during surgical procedures.
In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Cα (PKCα) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCα fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.
KEYWORDS: Confocal microscopy, Microscopes, In vivo imaging, Luminescence, Objectives, Signal detection, Real time imaging, Photodynamic therapy, Tumors, Clinical research
To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.
In this paper, we describe the design and implementation of a one-chip camera device for a capsule endoscope. This experimental chip integrates peripheral circuits required for the capsule endoscope and the wireless transmission function based on a data transmission method using human body conduction. The integrated functional blocks include an image array, a timing generator, a clock generator, a voltage regulator, a 10b cyclic A/D converter, and a BPSK modulator. It can be operated autonomously with 3 pins (VDD, GND, and DATAOUT). A prototype chip which has 320x240 effective pixels was fabricated using 0.25μm CMOS image sensor process and the autonomous imaging was demonstrated. The chip size is 4.84mmx4.34mm. With a 2.0 V power supply, the analog part consumes 950µW and the total power consumption at 6 fps (20MHz carrier frequency) is about 3mW.
By employing the total internal reflection fluorescence (TIRF) microscope with an ultra high NA (1.65) objective lens, we demonstrated detailed dynamics of exocytosis in various types of secretory vesicles. However, the TIRF microscopy could be applied to observations only on the plasma membrane and its immediate vicinity. To observe the vesicles in the deeper region of cytoplasm, we modified the TIRF optics to project a slit beam thinner than 1 μm in width to the cell. The slit beam illumination spotted single secretory vesicles inside the cell better and their movement and exocytosis easier. By scanning the slit beam, a fluorescence microscopy was possible at a high signal-to-noise ratio useful for
measurement and analysis of single exocytosis in neurons and endocrine cells.
Optical imaging with a high detecting power is very instrumental to dynamic observations of bio-molecular objects in water. To pioneer this field of imaging researches further, we used novel objective lenses of 1.65 and 1.45 in NA comparatively with 1.35 NA lens. The lenses of ultra high NA have a high resolving power and very useful properties for observation of high contrast images of fluorescently labeled molecules and subcellular organelles including DNA and membrane fusion proteins.
We designed Apo 100X NA 1.65 and Plan Apo 60X NA 1.45 objective lenses. When we use three lenses for water- immersed specimens, the marginal angle available for the total internal reflection becomes larger compared with NA 1.40 objective lens. Therefore, the alignment of the laser beam to the objective lens becomes very easy. We also designed the TIRFM illuminator. A single mode fiber is connected between the laser and illuminator. The laser beam is conducted through the fiber and supplied to the illuminator with a pre-adjusted positioning. Almost no additional alignment of illumination light is necessary.
A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.
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