In situ biochemical characterization of Venus cloud particles using a life-signature detection microscope.

Much of the information about the size and shape of aerosols forming haze and the cloud layer of Venus is obtained from indirect inferences from nephelometers on probes and from the analysis of the variation of polarization with the phase angle and the glory feature from images of Venus. The microscopic imaging of Venus' aerosols has recently been advocated. Direct measurements from a fluorescence microscope can provide information on the morphology, density, and biochemical characteristics of the particles; thus, fluorescence microscopy is attractive for in situ particle characterization of the Venus cloud layer. Fluorescence imaging of Venus cloud particles presents several challenges owing to the sulfuric acid composition and corrosive effects. In this article, we identify the challenges and describe our approach to overcoming them for a fluorescence microscope based on an in situ biochemical and physical characterization instrument for use in the clouds of Venus from a suitable aerial platform. We report that pH adjustment using alkali was effective for obtaining fluorescence images and that fluorescence attenuation was observed after the adjustment, even when the acidophile suspension in concentrated sulfuric acid was used as a sample.

Cell culture on hydrophilicity-controlled silicon nitride surfaces.

Cell culture on silicon nitride membranes is required for atmospheric scanning electron microscopy, electron beam excitation assisted optical microscopy, and various biological sensors. Cell adhesion to silicon nitride membranes is typically weak, and cell proliferation is limited. We increased the adhesion force and proliferation of cultured HeLa cells by controlling the surface hydrophilicity of silicon nitride membranes. We covalently coupled carboxyl groups on silicon nitride membranes, and measured the contact angles of water droplets on the surfaces to evaluate the hydrophilicity. We cultured HeLa cells on the coated membranes and evaluated stretch of the cell. Cell migration and confluence were observed on the coated silicon nitride films. We also demonstrated preliminary observation result with direct electron beam excitation-assisted optical microscope.

Intracellular calcium ion concentration measurement using a phase-modulation fluorescence lifetime method with compensation for phase shift due to the presence of proteins.

The calcium ion concentration in cells was measured by a phase-modulation fluorescence lifetime method with compensation for proteins. A high-accuracy measurement of the calcium ion concentration is best realized by fluorescence lifetime measurements, because the fluorescence lifetime is independent of the fluorescence intensity. The fluorescence intensity is easily varied by the scattering of excitation and emission light in cells, photobleaching, the concentration of fluorochromes, and wavelength dispersion of optical elements. A phase-modulation fluorescence lifetime measurement, however, provides high accuracy and precision, and can measure not only the calcium ion concentration, but also other ion concentrations, such as that of magnesium, sodium, and potassium. We have examined the phase-modulation fluorescence lifetime shift using protein compensation in cells, and have measured the calcium ion concentration in cells stimulated with bradykinin.

Dynamic and high-resolution live cell imaging by direct electron beam excitation.

We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.

Electron beam excitation assisted optical microscope with ultra-high resolution.

We propose electron beam excitation assisted optical microscope, and demonstrated its resolution higher than 50 nm. In the microscope, a light source in a few nanometers size is excited by focused electron beam in a luminescent film. The microscope makes it possible to observe dynamic behavior of living biological specimens in various surroundings, such as air or liquids. Scan speed of the nanometric light source is faster than that in conventional near-field scanning optical microscopes. The microscope enables to observe optical constants such as absorption, refractive index, polarization, and their dynamic behavior on a nanometric scale. The microscope opens new microscopy applications in nano-technology and nano-science.

Intravital oxygen radical imaging in normal and ischemic rat cortex.

OBJECTIVE: We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging. METHODS: In anesthetized adult rats, a fluorescent dye (5 microL), MitoSOX (5 micromol/L) for superoxide radical (.O2-), and hydroxyphenyl fluorescein (20 micromol/L) for hydroxyl radical (.OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-microm diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n=3/mimicking each dye), and based on the results, FI of ROS indicators was corrected. RESULTS: After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n=5/each, P<.01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged .OH, but did not do so in .O2- generation. CONCLUSION: ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.

Three-dimensional measurement endoscope system with virtual rulers.

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.

In vivo calcium imaging of cerebral cortex in hypoxia-ischemia followed by developmental stage-specific injury in rats.

The parietal area is a part of the cortex that is vulnerable in the rat to hypoxia-ischemia (HI) within the early postnatal period. To investigate the localizing mechanism of this cortical injury, we spatiotemporally detected the cortical intracellular calcium changes, as revealed by a calcium-sensitive fluorescence dye, Rhod 2-AM, during 1h of HI on postnatal days 7-21 in vivo. The calcium level rose to different levels at different cortical points in all animals within the first 20 min. Over the whole cortical area in the camera field, the changes in three groups significantly differed across time at 30 and 60 min, and a chronic increase appeared at days 7-8. After 3h of reperfusion, microtubule-associated protein 2 (MAP-2) immunoreactivity confirmed that parietal injury was more serious at day 7, whereas the imaging of calcium distribution did not segregate the injured and uninjured areas. Our in vivo findings in the whole brain structure indicate that the age-specific vulnerability of the parietal cortex injury is affected indirectly by the chronic increase in the late HI phase in the early postnatal period, suggesting that each cortical area differs postnatally with respect to the development of calcium regulation and signal transduction involving neural cell death and/or survival.