Susumu Terakawa

The Activation of Exocytotic Sites by the Formation of Phosphatidylinositol 4,5-Bisphosphate Microdomains at Syntaxin Clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.

Design and implementation of a one-chip wireless camera device for a capsule endoscope

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.

Mechanical fragmentation and transportation of calcium phosphate substrate by filopodia and lamellipodia in a mature osteoclast.

The functions of filopodia and lamellipodia in mature osteoclasts are not well known in the process of bone resorption. We investigated the function of filopodial/lamellipodial movement in mature osteoclasts by video‐enhanced contrast‐differential interference contrast (VEC‐DIC) microscopy. Mature osteoclasts, which were isolated from Japanese white rabbits, were cultured on calcium phosphate (CP)‐coated quartz coverslips to observe filopodial/lamellipodial movement and the formation of CP‐free areas precisely. Filopodia broke the CP substrate into pieces and transported them to the cell body by capturing them at the tip. Lamellipodia destroyed the CP substrate, and transported it to the cell body by capturing small particles in a mass. This study suggests two functions of filopodia and lamellipodia in mature osteoclasts, i.e., the mechanical fragmentation of the CP substrate and the transportation of the CP particles to the cell body.

Fiber-coupled confocal microscope (FCM) for real time imaging of cellular signals in vivo

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.

Intracellular dynamics observed by mode switching of microscope with a light incidence to the interface at alternate angles through the ultra high NA objective

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.

Stroboscopic near-field imaging for the analysis of contraction of muscle cells.

We have developed a stroboscopic near‐field optical microscope for observation of biological specimens and observed glycerinated muscles before and after muscle contraction with the developed system. In the system, the optical field distribution localized near the specimen is recorded as the surface topographic distribution of a photosensitive film surface. Our system is very useful for observing living biological specimens with high resolutions, because it is possible to get stroboscopic image by using a photosensitive film as detecting optical distributions instead of a scanning of probes. We have succeeded in observing inner structures of muscle cells with sub‐wavelength resolution and achieved higher contrast than an ordinary optical microscope.

Segmentation of brain volume based on 3D region growing by integrating intensity and edge for image-guided surgery

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.

Registration of laser range image of cortical surface to preoperative brain MR images for image-guided neurosurgery: preliminary results

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.

Model for monitoring the process of photodynamic therapy in patients

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.

Slit-scanning microscope with a high-NA objective lens for analysis of synaptic function

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.