Perfusion heterogeneity in rat lungs assessed from the distribution of 4-microm-diameter latex particles.

Pulmonary vascular perfusion has been shown to follow a fractal distribution down to a resolution of 0.5 cm(3) (5E11 microm(3)). We wanted to know whether this distribution continued down to tissue volumes equivalent to that of an alveolus (2E5 microm(3)). To investigate this, we used confocal microscopy to analyze the spatial distribution of 4-microm-diameter fluorescent latex particles trapped within rat lung microvessels. Particle distributions were analyzed in tissue volumes that ranged from 1.7E2 to 2.8E8 microm(3). The analysis resulted in fractal plots that consisted of two slopes. The left slope, encompassing tissue volumes less than 7E5 microm(3), had a fractal dimension of 1.50 +/- 0.03 (random distribution). The right slope, encompassing tissue volumes greater than 7E5 microm(3), had a fractal dimension of 1.29 +/- 0.04 (nonrandom distribution). The break point at 7E5 microm(3) corresponds closely to a tissue volume equivalent to that of one alveolus. We conclude that perfusion distribution is random at tissue volumes less than that of an alveolus and nonrandom at tissue volumes greater than that of an alveolus.     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Reversible and repeatable linear local cell force response under large stretches

Large stretching and un-stretching force response of adherent fibroblasts is measured by micromachined mechanical force sensors. The force sensors are composed of a probe and flexible beams. The probe, functionalized by fibronectin, is used to contact the cells. The flexible beams are the sensing element. The sensors are made of single crystal silicon and fabricated by the SCREAM process. The maximum cell stretch reached is approximately 50 microm, which is about twice of the cell initial size, and the time delay between two consecutive stretching/un-stretching steps is 75 s unless otherwise stated. We find that the force response of the cells is strongly linear, reversible, and repeatable, with a small stiffening at the initial deformation stage. Force response of single cells measured before and after cytochalasin D treatment suggests that actin filaments take almost all the cell internal forces due to stretch. These findings may shed light on the increasing understanding on the mechanical behavior of cells and provide clues for making new classes of biological materials having uncommon properties.     

How does radiation damage in protein crystals depend on X-ray dose?

Is radiation damage to cryopreserved protein crystals strictly proportional to accumulated dose at the high-flux density of beams from undulators at third-generation synchrotron sources? The answer is "yes," for overall damage to several different kinds of protein crystals at flux densities up to 10(15) ph/sec/mm(2) (APS beamline 19-ID). We find that, at 12 keV (1 A wavelength), about ten absorbed photons are sufficient to "kill" a unit cell. As this corresponds to about one elastically scattered photon, each unit cell can contribute only about one photon to total Bragg diffraction. The smallest crystal that can yield a full data set to 3.5 A resolution has a diameter of about 20 microm (100 A unit cell).     

Thin-film IrOx pH microelectrode for microfluidic-based microsystems

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.     

A two-photon FRAP analysis of the cytoskeleton dynamics in the microvilli of intestinal cells

The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.     

Microdosimetric measurements and estimation of human cell survival for heavy-ion beams

The microdosimetric spectra for high-energy beams of photons and proton, helium, carbon, neon, silicon and iron ions (LET = 0.5-880 keV/microm) were measured with a spherical-walled tissue-equivalent proportional counter at various depths in a plastic phantom. Survival curves for human tumor cells were also obtained under the same conditions. Then the survival curves were compared with those estimated by a microdosimetric model based on the spectra and the biological parameters for each cell line. The estimated alpha terms of the liner-quadratic model with a fixed beta value reproduced the experimental results for cell irradiation for ion beams with LETs of less than 450 keV/microm, except in the region near the distal peak.     

Optimizing the crystal size and habit of beta-sitosterol in suspension

The aim of this work was to survey how processing parameters affect the crystal growth of beta-sitosterol in suspension. The process variables studied were the cooling temperature, stirring time and stirring rate during recrystallization. In addition, we investigated the effect a commonly used surfactant, polysorbate 80, has on crystal size distribution and the polymorphic form. This study describes the optimization of the crystallization process, with the object of preparing crystals as small as possible. Particle size distribution and habit were analyzed using optical microscopy, and the crystal structure was analyzed using X-ray diffractometry. The cooling temperature had a remarkable influence on the crystal size. Crystals with a median crystal length of approximately 23 microm were achieved with a low cooling temperature (<10 degrees C); however, a fairly large number of crystals over 50 microm appeared. Higher cooling temperatures (>30 degrees C) caused notable crystal growth both in length and width. Rapid (250 rpm), continuous stirring until the suspensions had cooled to room temperature created small, less than 50 micro m long (median <20 microm), needle-shaped crystals. The addition of surfactant slightly reduced the size of the initially large crystals. Both hemihydrate and monohydrate crystal forms occurred throughout, regardless of the processing parameters. By using an optimized process, it was possible to obtain a microcrystalline suspension, with a smooth texture.     

Variation in microfilariae and infective stages of two types of Wuchereria bancrofti from the Thai-Myanmar border

Comparative morphometric and morphological studies of microfilariae and infective stages were undertaken in nocturnally periodic and subperiodic Wuchereria bancrofti. For microfilariae, the body dimensions of nocturnally periodic (NP) were significantly smaller than nocturnally subperiodic (NSP), i.e., body length 268.03+/-14.75 microm (NP), 307.61+/-11.52 microm (NSP); cephalic space length 4.21+/-0.62 microm (NP), 5.32+/-0.79 microm (NSP); head to nerve ring 49.39+/-5.43 microm (NP), 57.40+/-4.46 microm (NSP); innenk?rper length 33.05+/-5.89 microm (NP), 44.02+/-8.71 microm (NSP); cephalic space width 4.28+/-0.59 microm (NP), 6.04+/-0.68 microm (NSP); body width at nerve ring 5.01+/-0.57 microm (NP), 7.45+/-0.75 microm (NSP). The number of nuclei between the cephalic space and nerve ring of NP (66.67+/-5.19) was also significantly less than in NSP (94.74+/-6.95). For infective stages, the body dimensions of NP were significantly smaller than NSP, i.e., body length 1632.50+/-131.48 microm (NP), 2002.63+/-222.60 microm (NSP); head to nerve ring 103.09+/-7.47 microm (NP), 122.44+/-9.62 microm (NSP); head to oesophago-intestinal junction 567.69+/-94.84 microm (NP), 666.75+/-110.08 microm (NSP); body width at oesophago-intestinal junction 23.15+/-1.55 microm (NP), 26.78+/-1.62 microm (NSP). It is too early to infer the NP type as an additional sibling species of W. bancrofti but it is reasonable to treat it as a new variety and additional work is needed to clarify its status.     

Slow light engineering for high Q high sensitivity photonic crystal microcavity biosensors in silicon

Current trends in photonic crystal microcavity biosensors in silicon-on-insulator (SOI), that focus on small and smaller sensors have faced a bottleneck trying to balance two contradictory requirements of resonance quality factor and sensitivity. By simultaneous control of the radiation loss and optical mode volumes, we show that both requirements can be satisfied simultaneously. Microcavity sensors are designed in which resonances show highest Q～9300 in the bio-ambient phosphate buffered saline (PBS) as well as highest sensitivity among photonic crystal biosensors. We experimentally demonstrated mass sensitivity 8.8atto-grams with sensitivity per unit area of 0.8pg/mm(2). Highest sensitivity, irrespective of the dissociation constant K(d), is demonstrated among all existing label-free optical biosensors in silicon at the concentration of 0.1μg/ml.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 +/- 28 nerve fibers consisting of 63 +/- 24 unmyelinated and 12 +/- 6 myelinated fibers. The PAN was composed of 195 +/- 50 nerve fibers, namely 129 +/- 28 unmyelinated and 66 +/- 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 microm with a maximum between 0.3 and 0.6 microm. The myelinated nerve fibers in the MAN ranged from 1 to 8 microm with a peak between 2 and 5 microm. In the PAN, their diameters ranged from 1 to 12 microm with a clearly visible peak at 4-5 microm and a plateau at 8-9 microm that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Software simulation of tumour motion dose effects during flattened and unflattened ITV-based VMAT lung SBRT

PurposeRestricted studies comparing different dose rate parameters are available while ITV-based VMAT lung SBRT planning leads to perform the analysis of the most suitable parameters of the external beams used. The special emphasis was placed on the impact of dose rate on dose distribution variations in target volumes due to interplay effects.MethodsFour VMAT plans were calculated for 15 lung tumours using 6 MV photon beam quality (flattening filter FF vs. flattening filter free FFF beams) and maximum dose rate of 600 MU/min, 1000 MU/min and 1400 MU/min. Three kinds of motion simulations were performed finally giving 180 plans with perturbed dose distributions.Results6FFF-1400 MUs/min plans were characterized by the shortest beam on time (1.8 ± 0.2 min). Analysing the performed motion simulation results, the mean dose (Dmean) is not a sensitive parameter to related interplay effects. Looking for local maximum and local minimum doses, some discrepancies were found, but their significance was presented for individual patients, not for the whole cohort. The same was observed for other verified dose metrics.ConclusionsGenerally, the evaluation of VMAT robustness between FF and FFF concepts against interplay effect showed a negligible effect of simulated motion influence on tumour coverage among different photon beam quality parameters. Due to the lack of FFF beams, smaller radiotherapy centres are able to perform ITV-based VMAT lung SBRT treatment in a safe way. Radiotherapy department having FFF beams could perform safe, fast and efficient ITV-based VMAT lung SBRT without a concern about significance of interplay effects.     

Myxobolus desaequalis n. sp. (Myxozoa,Myxosporea), parasite of the Amazonian freshwater fish,Apteronotus albifrons (Teleostei,Apteronotidae)

Myxobolus desaequalis n. sp. is described from the gill lamellae of the freshwater fish Apteronotus albifrons, collected in the Amazon River, near the city of Salvaterra, Brazil. Large spherical plasmodia filled with disporic pansporoblasts and spores were observed. Ellipsoidal to pyriform spores are 18.3 microm length x 11.2 microm width x 4.4 microm thickness. The anterior end of the spores contain two extremely unequal pyriform polar capsules measuring: (larger): 11.2 microm length, 4.9 microm width, and an isofilar polar filament with 11 to 12 turns obliquely to the longitudinal axis; (smaller): 4.6 microm length, 2.8 microm width, and an isofilar polar filament with 4 to 5 turns, obliquely to the longitudinal axis.     

Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.     

Postembryonic eye growth in the seashore isopod Ligia exotica (Crustacea,Isopoda)

The eye of Ligia exotica is of the apposition type and has open rhabdoms. The facets are hexagonal, and the dioptric apparatus consists of a flat cornea and a spherical crystalline cone placed in the center of two large cone cells. Each ommatidium has seven regular retinula cells and one eccentric cell; a basement membrane forms the proximal boundary of the retina. With increases in body size from 0.6 to almost 4.0 cm, facet numbers and ommatidial diameters increased from 800 to 1500 and 35 microm to 100 microm, respectively; eye length and width grew from 1.2 to 3.2 and 0.9 to 2.5 mm, respectively; and length of dioptric apparatus and width of retinal layer changed from 70 microm to 180 microm and about 70 microm to 120 microm. Visual angles and interommatidial angles of centrally located ommatidia remained constant at about 30 and 6.9 degrees, respectively. An almost perfect linear relationship was found when eye length was plotted against the product between the square root of the total number of ommatidia and the ommatidial diameter. No difference between males and females was observed in any of the relationships, but the results suggest that, compared with smaller specimens, larger ones possess increased absolute sensitivity in single ommatidia, increased sensitivity to point sources, and overall larger angular visual fields for the eye in its totality. This means that larger individuals of L. exotica (which are also faster) have an advantage over smaller individuals at night, but that smaller individuals may cope better with bright lights. Vision in L. exotica seems useful not only in detecting potential danger, but also in locating and approaching cliffs from a distance of 2-4 m when swimming in seawater.     

Anisotropic mechanical properties of lamellar bone using miniature cantilever bending specimens.

The flexural modulus and work-to-fracture properties of circumferential lamellar bone from baboon tibia are presented, based on experiments with miniature cantilever bending specimens. Data is provided for specimens in three orthogonal directions that show marked anisotropy. The advantages of such miniature specimens (about 150 microm in diameter and 2 mm long) include the possibility of sampling very small volumes within a heterogeneous structure such as osteonal bone, or studying biological materials that are not available in large enough volumes for conventional mechanical analysis.     

Biological effects of ion beams in Nicotiana tabacum L.

The biological effects of ion beams on Nicotiana tabacum L., particularly the induction of chromosome aberrations, were investigated. Dry seeds were exposed to ¹²C⁵⁺, ⁴He²⁺ and ¹H⁺ beams with linear energy transfer (LET) ranging from 1 to 111 keV/μm and irradiated with gamma-rays. Ion beams were more effective in reducing germination and survival of the seeds than gamma-rays. The LD₅₀ for ¹²C⁵⁺ beams, ⁴He²⁺ beams and gamma-rays were 35, 60 and 500 Gy, respectively. The frequencies of mitotic cells with chromosome aberrations, such as chromosome bridges, acentric fragments and lagging chromosomes in the root tip cells of the exposed seeds, increased linearly with increasing doses. Relative biological effectiveness (RBE) values, based on the doses that induced a survival inhibition of 50% and a 10% frequency of aberrant cells, were 14.3–17.5 for the ¹²C⁵⁺ beams, 7.0–8.3 for the ⁴He²⁺ beams and 7.8 for the ¹H⁺ beams. Furthermore, the relative ratios of the chromosome aberration types were significantly different between the ion beam and the gamma-ray regimes: chromosome fragments were more frequent in the former, and chromosome bridges in the latter. Based on these results, we concluded that the repair process of initial lesions induced by ion beams may be different from that induced by low- LET radiation. Received: 29 October 1998 / Accepted in revised form: 25 March 1999     

Response of rat skin to high-dose unidirectional x-ray microbeams: a histological study

There is growing interest in evaluating microbeam radiation therapy as a potential clinical modality. Microbeam radiation therapy uses arrays of parallel, microscopically thin (<100 microm) planes of synchrotron-generated X rays (microplanar beams, or microbeams). Due to the relatively low beam energies involved in microbeam radiation therapy (a median beam energy of 120 keV was used in the present study), the dose penetration of microbeams in tissue is lower than that used in conventional radiotherapy. This lower energy necessitates using a significantly elevated dose to the skin's surface during clinical microbeam therapy to ensure an adequate dose distribution in the target tumor. The findings of the present study, using a rat skin model, indicated that the skin had an extremely high tolerance to microbeam radiation at doses considerably in excess of those that were therapeutically effective in preclinical studies. A histological study was undertaken to evaluate the biological mechanisms underlying this high tolerance. The irradiation configuration employed single-exposure, unidirectional microbeams 90 microm wide, with 300 microm beam spacing on-center. The in-beam skin-surface absorbed doses were in the range 835-1335 Gy. Monte Carlo simulations of the dose distribution indicated that the "valley" dose, i.e. the radiation leakage between adjacent microbeams, was about 2.5% of the in-beam dose. The high tolerance of the rats' skin to microbeams and the rapid regeneration of the damaged segments of skin were attributed to the surviving clonogenic cells situated between the adjacent microplanar beams. In the epidermis, clonogenic cells in the hair follicular epithelium appeared to play a key role in the regeneration process.     

Evaluation of epididymal semen quality using the Hamilton-Thorne analyser indicates variation between the two caudae epididymides of the same bull

Epididymal semen is being more often considered as a potential source of valuable genes for genome resource banks. To utilize this resource as efficiently as possible, storage and freezing fertility and preservation characteristics of epididymal semen have to be examined. Because semen quality should be assessed as objectively as possible, we introduced computer assisted sperm analysis (CASA) of epididymal bull semen. The aims of this study were: to determine the quality of fresh cauda epididymal bull sperm, conventionally and by CASA (Hamilton-Thorne Ceros 12.1); to compare epididymal sperm movement with the motion characteristics of ejaculated semen; and to investigate whether equality of semen characteristics exists between both caudae epididymides of the same bull. In experiment 1, it is shown that epididymal sperm has a lower motility (total: 48.7% versus 79.9%, p < 0.0001 and progressive: 34.4% versus 58.4%, p < 0.0001) and moves less straight (80.5% versus 84.5%, p < 0.0009) with a higher amplitude (6.1 microm versus 5.0 microm, p < 0.0001) than ejaculated semen. The epididymal straight line velocity (85.2 microm/s versus 98.3 microm/s, p < 0.0001) is lower, but the curvilinear velocity (173.5 microm/s versus 156.4 microm/s, p < 0.0001) is higher than those of ejaculated semen. The data in experiment 2 are analysed to determine equality, rather than to find a difference. They illustrate that mean differences, for most semen parameters, between the semen from paired caudae epididymides, deviated more than 20% from the average values of these parameters from all bulls; the exceptions (those parameters within 20% of the average for all bulls) were the percentage of live spermatozoa, the linearity of sperm movement, the weights of testis and epididymis, the weights of the cauda epididymis alone, the volumes, and the amplitudes of movement of the semen (p < 0.05). The mean differences between the percentage of live spermatozoa and the amplitude of movement of the epididymal semen of both epididymides of one bull, were the only values smaller than 10% of the average value of this parameter (p < 0.05). This implies that sperm from one cauda epididymis should not be used as a control for the other because, for most of the semen parameters (concentration, morphology, motility, and beat cross frequency), equality between caudae epididymides of the same bull could not be established.