Marked differences in local bone remodelling in response to different marrow stimulation techniques in a large animal.

Marrow stimulation, including subchondral drilling and microfracture, is the most commonly performed cartilage repair strategy, whereby the subchondral bone plate is perforated to release marrow-derived cells into a cartilage defect to initiate repair. Novel scaffolds and therapeutics are being designed to enhance and extend the positive short-term outcomes of this marrow stimulation. However, the translation of these newer treatments is hindered by bony abnormalities, including bone resorption, intralesional osteophytes, and bone cysts, that can arise after marrow stimulation. In this study, three different marrow stimulation approaches - microfracture, subchondral drilling and needle-puncture - were evaluated in a translationally relevant large-animal model, the Yucatan minipig. The objective of the study was to determine which method of marrow access (malleted awl, drilled Kirschner wire or spring-loaded needle) best preserved the underlying subchondral bone. Fluorochrome labels were injected at the time of surgery and 2 weeks post-surgery to capture bone remodelling over the first 4 weeks. Comprehensive outcome measures included cartilage indentation testing, histological grading, microcomputed tomography and fluorochrome imaging. Findings indicated that needle-puncture devices best preserved the underlying subchondral bone relative to other marrow access approaches. This may relate to the degree of bony compaction occurring with marrow access, as the Kirschner wire approach, which consolidated bone the most, induced the most significant bone damage with marrow stimulation. This study provided basic scientific evidence in support of updated marrow stimulation techniques for preclinical and clinical practice.

Effects of advanced maternal age and race/ethnicity on placental weight and placental weight/birthweight ratio in very low birthweight infants.

To study the association of advanced maternal age (AMA) and race/ethnicity on placental pathology in very low birthweight (VLBW) infants. Retrospective analysis of placental pathology of inborn singleton VLBW infants from a regional level 3 NICU between July, 2002 and June, 2009. Subjects were stratified by age and race/ethnicity. Statistical analysis included One-way ANOVA, Chi Square and multivariable analyses. A total of 739 mother/infant dyads were included. AMA was associated with a decrease in placental weight and placental weight/birthweight ratio. Black/Non-Hispanic mothers ≥35 had a lower placental weight (p = 0.01) and lower placental weight/birth weight ratio (z-score, -0.45 ± 0.71 vs -0.04 ± 1.1, p = 0.01) compared to Black/Non-Hispanic mothers <35 years of age. After controlling for gestational age, race/ethnicity, maternal diabetes, maternal smoking, maternal hypertension and clinical chorioamnionitis, AMA, but not race/ethnicity, remained independently associated with placental weight/birthweight ratio z score (full model r(2) = 0.22, p < 0.01). In our study sample of VLBW infants, placental weight and placental weight/birthweight ratio were lower in mothers of advanced maternal age compared to mothers <35 years of age. Our data suggest that maternal age affects placentation in VLBW infants, which could influence maternal and neonatal outcomes.

Effects of pre-pregnancy obesity, race/ethnicity and prematurity.

To investigate the association between maternal pre-pregnancy obesity, race/ethnicity and prematurity. Retrospective cohort study of maternal deliveries at a single regional center from 2009 to 2010 time period (n = 11,711). Generalized linear models were used for the analysis to estimate an adjusted odds ratio with 95% confidence interval of the association between maternal pre-pregnancy obesity, race/ethnicity and prematurity. Analysis controlled for diabetes, chronic hypertension, previous preterm birth, smoking and insurance status. The demographics of the study population were as follows, race/ethnicity had predominance in the White/Non-Hispanic population with 60.1%, followed by the Black/Non-Hispanic population 24.2%, the Hispanic population with 10.3% and the Asian population with 5.4%. Maternal pre-pregnancy weight showed that the population with a normal body mass index (BMI) was 49.4%, followed by the population being overweight with 26.2%, and last, the population which was obese with 24.4%. Maternal obesity increased the odds of prematurity in the White/Non-Hispanic, Hispanic and Asian population (aOR 1.40, CI 1.12-1.75; aOR 2.20, CI 1.23-3.95; aOR 3.07, CI 1.16-8.13, respectively). Although the Black/Non-Hispanic population prematurity rate remains higher than the other race/ethnicity populations, the Black/Non-Hispanic population did not have an increased odds of prematurity in obese mothers (OR 0.87; CI 0.68-1.19). Unlike White/Non-Hispanic, Asian and Hispanic mothers, normal pre-pregnancy BMI in Black/Non-Hispanic mothers was not associated with lower odds for prematurity. The odds for mothers of the White/Non-Hispanic, Hispanic and Asian populations, for delivering a premature infant, were significantly increased when obese. Analysis controlled for chronic hypertension, diabetes, insurance status, prior preterm birth and smoking. Obesity is a risk factor for prematurity in the White/Non-Hispanic, Asian and Hispanic population, but not for the Black/Non-Hispanic population. The design and evaluation of weight-based maternal health programs that aggregate race/ethnicity may not be sufficient. The optimal method to address maternal pre-pregnancy and intra-pregnancy weight-related health disorders may need to be stratified along race/ethnicity adjusted strategies and goals. However, a more global preventative strategy that encompasses the social determinants of health may be needed to reduce the higher rates of prematurity among the Black/Non-Hispanic population.

The impact of kangaroo mother care on work of breathing and oxygen saturation in very low birth weight infants with respiratory insufficiency.

BACKGROUND: Kangaroo mother care (KMC) is defined as prolonged skin to skin care between a mother and infant with the infant lying in prone position on mom's chest. KMC decreases morbidity and mortality and promotes physiologic stability. The aim of this study is to measure work of breathing (WOB) during KMC in very low birth weight (VLBW) infants on non-invasive respiratory support. METHODS: A prospective observational pilot study was conducted comparing WOB indices during standard care (SC) and KMC. Respiratory inductive plethysmography (RIP) measured WOB indices non-invasively: phase angle and labored breathing index. VLBW infants who were stable on non-invasive respiratory support were randomized to receive RIP measurements during KMC or during SC first. Summary statistics and mixed linear models were used to compare WOB and vital signs. RESULTS: A total of 32 infants were consented for the study, data collection and analysis was completed on 28 infants. There were no significant differences in mean phase angle during KMC or SC (73.5±4.6 SE deg vs 66.8±3.9 SE deg, p = 0.25). No differences in WOB and vital signs were detected. Controlling for respiratory support or randomization/first location did not change the results. CONCLUSION: In this pilot cohort, infants demonstrated no differences in work of breathing indices or oxygen saturation during KMC or SC while receiving non-invasive respiratory support. KMC appears to be safe and well tolerated with no worsened WOB. Larger studies should be performed to confirm our findings.

How many growing vestibular schwannomas tend to stop growing without any treatment?

OBJECTIVE: To determine the long-term, spontaneous growth arrest rates in a large cohort of vestibular schwannoma patients. METHODS: This paper describes a retrospective case series of 735 vestibular schwannoma patients organised into four groups: group A patients showed tumour growth which then stopped without any treatment; group B patients showed tumour growth which continued, but were managed conservatively; group C patients had a growing vestibular schwannoma and received active treatment; and group D patients had a stable, non-growing vestibular schwannoma. Demographics, tumour size and vestibular schwannoma growth rate (mm/month) were recorded. RESULTS: A total of 288 patients (39.2 per cent) had growing vestibular schwannomas. Of the patients, 103 (35.8 per cent) were managed conservatively, with 52 patients (50.5 per cent of the conservative management group, 18 per cent of the total growing vestibular schwannoma group) showing growth arrest, which occurred on average at four years following the diagnosis. Eighty-two per cent of vestibular schwannomas stopped growing within five years. Only differences between age (p = 0.016) and vestibular schwannoma size (p = 0.0008) were significant. CONCLUSION: Approximately 20 per cent of growing vestibular schwannomas spontaneously stop growing, predominantly within the first five years; this is important for long-term management.

Cochlear patency following translabyrinthine vestibular schwannoma resection: implications for hearing rehabilitation.

OBJECTIVE: To examine when cochlear fibrosis occurs following a translabyrinthine approach for vestibular schwannoma resection, and to determine the safest time window for potential cochlear implantation in cases with a preserved cochlear nerve. METHODS: This study retrospectively reviewed the post-operative magnetic resonance imaging scans of patients undergoing a translabyrinthine approach for vestibular schwannoma resection, assessing the fluid signal within the cochlea. Cochleae were graded based on the Isaacson et al. system (from grade 0 - no obstruction, to grade 4 - complete obliteration). RESULTS: Thirty-nine patients fulfilled the inclusion criteria. The cochleae showed no evidence of obliteration in: 75 per cent of patients at six months, 38.5 per cent at one year and 27 per cent beyond one year. Most changes happened between 6 and 12 months after vestibular schwannoma resection, with cases of an unobstructed cochlear decreasing dramatically, from 75 per cent to 38.5 per cent, within this time. CONCLUSION: The progress of cochlear obliteration that occurred between 6 and 12 months following vestibular schwannoma resection indicates that the first 6 months provides a safer time window for cochlear patency.

Benefits of pre-labyrinthectomy intratympanic gentamicin: contralateral vestibular responses.

OBJECTIVE: To determine the impact of pre-operative intratympanic gentamicin injection on the recovery of patients undergoing translabyrinthine resection of vestibular schwannomas. METHODS: This prospective, case-control pilot study included eight patients undergoing surgical labyrinthectomy, divided into two groups: four patients who received pre-operative intratympanic gentamicin and four patients who did not. The post-operative six-canal video head impulse test responses and length of in-patient stay were assessed. RESULTS: The average length of stay was shorter for patients who received intratympanic gentamicin (6.75 days; range, 6-7 days) than for those who did not (9.5 days; range, 8-11 days) (p = 0.0073). Additionally, the gentamicin group had normal post-operative video head impulse test responses in the contralateral ear, while the non-gentamicin group did not. CONCLUSION: Pre-operative intratympanic gentamicin improves the recovery following vestibular schwannoma resection, eliminating, as per the video head impulse test, the impact of labyrinthectomy on the contralateral labyrinth.

Racial differences in prenatal care of mothers delivering very low birth weight infants.

OBJECTIVES: To determine whether there are any racial differences in the prenatal care of mothers delivering very low birth weight infants (VLBW). STUDY DESIGN: Retrospective cohort study of infants cared for at a single regional level III neonatal intensive care unit over a 9-year period, July 1993-June 2002, N = 1234. The main outcome variables investigated included antenatal administration of steroids, delivery by cesarean section, and use of tocolytic medications. Both univariate and multivariate analyses were performed. RESULTS: After controlling for potential confounding variables, white mothers delivering VLBWs had an increased odds of cesarean delivery (odds ratio 1.5, 95% confidence intervals (CI) 1.1-2.0), receiving antenatal steroids (1.3, CI 1.01-1.8), and tocolysis (1.4, CI 1.1-2.0) compared to black mothers. The models controlled for gestational age, multiple gestation, premature labor, clinical chorioamnionitis, maternal age, income, year of birth, and presentation. CONCLUSIONS: In our population of VLBWs, white mothers are more likely to receive antenatal steroids, tocolytic medications, and deliver by cesarean section when compared to black mothers. From our data we cannot determine the reasons behind these racial differences in care of mothers delivering VLBWs.

Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues.

The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.

Increased monocytes and bands following a red blood cell transfusion.

OBJECTIVE: The objective of this study is to analyze the white blood cell changes that occur after a transfusion of red blood cells in order to identify a subclinical inflammatory response in neonates. STUDY DESIGN: Retrospective analysis of infants who received a red blood cell transfusion in an intensive care nursery. White blood cell results within 24 h pre- to 48 h post-transfusion were collected and analyzed. Statistical analysis included ANOVA, T-test, Mann-Whitney U test, Pearson's correlation and multivariable linear regression. RESULT: Monocytes (P=0.02) and bands (P=0.035) were increased post-transfusion. There were no differences in monocytes (P=0.46) or bands (P=0.56) between groups who did or did not have blood cultures obtained. There was no difference in monocytes between groups who did or did not have sepsis (P=0.88). CONCLUSION: We identified an elevation in monocytes and bands in the 48 h following a transfusion in premature infants. Our findings support a possible pro-inflammatory response related to transfusion of red blood cells.

Use of neonatal simulation models to assess competency in bag-mask ventilation.

OBJECTIVE: Providing adequate bag-mask ventilation (BMV) is an essential skill for neonatal resuscitation. Often this skill is learned using simulation manikins. Currently, there is no means of measuring the adequacy of ventilation in simulated scenarios. Thus, it is not possible to ascertain proficiency. The first aim of this study was to measure the pressure generated during BMV as performed by providers with different skill levels and measure the impact of different feedback mechanisms. The second aim was to measure the pressure volume characteristics of two neonatal manikins to see how closely they reflect newborn lung mechanics. STUDY DESIGN: In Phase I to achieve the first aim, we evaluated BMV skills in different level providers including residents (n=5), fellows (n=5), neonatal nurse practitioners (n=5) and neonatologists (n=5). Each provider was required to provide BMV for 2-min epochs on the SimNewB (Laerdal), which had been instrumented to measure pressure-volume characteristics. In sequential 2-min epochs, providers were given different feedback including chest-wall movement alone compared to manometer plus chest-wall movement or chest-wall movement plus manometer plus laptop lung volume depiction. In Phase II of the study we measured pressure-volume characteristics in instrumented versions of the SimNewB (Laerdal) and NeoNatalie (Laerdal). RESULTS: In Phase I, all providers are compared with the neonatologists. All measurements of tidal volume (Vt) are below the desired 5 ml kg(-1). The greatest difference in Vt between the neonatologists and other providers occurs when only chest-wall movement is provided. A linear relationship is noted between Vt and PIP for both SimNewB and NeoNatalie. The compliance curves are not 'S-shaped' and are different between the two models (P<0.001). CONCLUSION: Phase I of this study demonstrates that the SimNewB with the feedback of chest-wall movement alone was the best method of distinguishing experienced from inexperienced providers during simulated BMV. Therefore this is likely to be the best method to ascertain proficiency. Phase II of the study shows that the currently available neonatal simulation manikins do not have pressure-volume characteristics that are reflective of newborn lung mechanics, which can result in suboptimal training.

Inadvertent administration of positive end-distending pressure during nasal cannula flow.

In the clinical setting, nasal cannulas are frequently used to deliver supplemental oxygen to neonates and are not believed to affect the general respiratory status. In contrast, it was hypothesized that clinical changes associated with nasal cannula gas flow may be related in part to the generation of positive end-distending pressure. To test this hypothesis, alterations in esophageal pressure were quantified as an indication of end-distending pressure and thoracoabdominal motion was quantified as an indication of breathing patterns in 13 preterm infants at gas flow levels of 0.5, 1, and 2 L/min delivered by nasal cannula with an outer diameter of either 0.2 or 0.3 cm. Changes in esophageal pressure were assessed by esophageal balloon manometry. Ventilatory patterns were assessed from thoracoabdominal motion by using respiratory inductive plethysmography. Thoracoabdominal motion was quantitated as a phase angle (theta); larger values represent greater asynchrony. The 0.2-cm nasal cannula did not deliver pressure or alter thoracoabdominal motion at any flow. In contrast, the 0.3-cm nasal cannula delivered positive end-distending pressure as a function of increasing levels of gas flow (r = .92) and reduced thoracoabdominal motion asynchrony. The mean pressure generated at 2 L/min was 9.8 cm H2O. These data demonstrate that nasal cannula gas flow can deliver positive end-distending pressure to infants and significantly alter their breathing strategy. This finding raises important concerns about the indiscriminate therapeutic use, size selection, and safety of nasal cannulas for the routine delivery of oxygen in preterm infants.

Risk assessment in a federal regulatory agency: an assessment of risk associated with the human consumption of some species of fish contaminated with polychlorinated biphenyls (PCBs).

The problem of polychlorinated biphenyls (PCBs) became a national concern in 1971 when several accidental contaminations of foods were reported. Extensive efforts were undertaken by FDA to reduce the residues of PCBs in food. However, the PCB levels in several species of fresh-water fish have raised concern about the PCB residues from environmental contamination, and it is this concern which has prompted a reassessment of the human risk involved from consumption of such fish. The human epidemiology and animal toxicity of PCB exposure are reviewed, as well as risk assessment in general. Specific examples to risk assessment involving extrapolation of animal data to humans, based on several levels of human exposure to PCBs in fish, are presented.

Miniature EPSPs and sensory encoding in the primary afferents of the vestibular lagena of the toadfish, Opsanus tau.

The synaptic activity transmitted from vestibular hair cells of the lagena to primary afferent neurons was recorded in vitro using sharp, intracellular microelectrodes. At rest, the activity was composed of miniature excitatory postsynaptic potentials (mEPSPs) at frequencies from 5 to 20/s and action potentials (APs) at frequencies betwen 0 and 10/s. mEPSPs recorded from a single fiber displayed a large variability. For mEPSPs not triggering APs, amplitudes exhibited an average coefficient of variance (CV) of 0.323 and rise times an average CV of 0.516. APs were only triggered by mEPSPs with larger amplitudes (estimated 4-6 mV) and/or steeper maximum rate of rise (10.9 mV/ms, +/- 3.7 SD, n=4 experiments) compared to (3.50 mV/ms, +/-0.07 SD, n=6 experiments) for nontriggering mEPSPs. The smallest mEPSPs showed a fast rise time (0.99 ms between 10% and 90% of peak amplitude) and limited variability across fibers (CV:0.18) confirming that they were not attenuated signals, but rather represented single-transmitter discharges (TDs). The mEPSP amplitude and rise-time relationship suggests that many mEPSPs represented several, rather than a single pulse of secretion of TDs. According to the estimated overall TD frequency, the coincidence of TDs contributing to the same mEPSP were not statistically independent, indicating a positive interaction between TDs that is reminiscent of the way subminiature signals group to form miniature signals at the neuromuscular junction. Depending on the duration and intensity of efferent stimulation, a complete block of AP initiation occurred either immediately or after a delay of a few seconds. Efferent stimulation did not significantly change AP threshold level, but abruptly decreased mEPSP frequency to a near-complete block that followed the block of APs. Maximum mEPSP rate of rise decreased during, and recovered progressively after, efferent stimulation. After termination of efferent stimulation, mEPSP amplitude did not recover instantly and for a few seconds the amplitude distribution of synaptic events showed fewer large-amplitude events than during the control period. This confirms that mEPSP amplitude and rate of rise properties, which are critical for triggering afferent APs, are modified by efferent activity. The depression of afferent AP firing during efferent stimulation corresponded to a decrease in mEPSP frequency and, to a lesser extent, a decrease in mEPSP amplitude and rate of rise, suggesting, a decrease in the level of interaction among TDs contibuting to a mEPSP.

The use of surface oximetry to assess bowel viability.

Tissue depends on perfusion, but tissue oxygenation measurements have been impractical. To develop a method for the clinical evaluation of bowel viability, we studied PO2 on the bowel surface (PSO2). The PSO2 was assessed on the stomach and intestine of 11 dogs. Normal small-bowel PSO2 was 44 +/- 11 mm Hg (mean +/- SD). When bowel vasculature was interrupted PSO2 rapidly decreased. Reductions in PSO2 were related to the degree of ischemia produced. Anastomoses were performed on partially devascularized bowel to determine anastomotic viability at various levels of PSO2. The PSO2 measured just prior to anastomosis predicted viability of the anastomoses at 48 hours. Anastomoses below 30% of predevascularization PSO2 necrosed. Between 30% and 50%, one third of anastomoses leaked. At PSO2 values above 50% of initial normal value all anastomoses healed. Surface oximetry allows accurate intraoperative assessment of bowel perfusion.

Effect of nasal CPAP on thoracoabdominal motion in neonates with respiratory insufficiency.

Thoracoabdominal motion (TAM) profiles were determined in ten infants requiring nasal continuous positive airway pressure (CPAP) and supplemental oxygen, in order to assess the influence of CPAP on chest wall function in infants with respiratory insufficiency. (TAM) was quantitated by respiratory inductive plethysmography, measuring relative motion of the rib cage and abdomen. Baseline pulmonary function (without CPAP support) was assessed from simultaneous measurements of transpulmonary pressure, air flow, and tidal volume. Measurements of (TAM) were acquired at baseline and at nasal CPAP levels of 0, 3, 5, and 8 cm H2O, in randomized order. Without CPAP, relative paradoxical motion occurred, i.e., the rib cage collapsed inward instead of expanding outward early in inspiration. With CPAP, TAM resembled the pattern in preterm infants, without lung disease. We found that nasal CPAP lowers the phase angle in infants with respiratory insufficiency (P less than 0.003), indicating improved synchrony of TAM. In addition, the improvement with nasal CPAP was related to the severity of pulmonary compromise at baseline. We speculate that changes in TAM associated with nasal CPAP arise from an interaction between pulmonary mechanics and an enhanced stability of the chest wall. In this context, the greater synchrony of TAM is suggestive of an improved breathing strategy. This may be a noninvasively obtainable marker of an effective nasal CPAP level in infants with altered pulmonary and chest wall mechanics.

Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumour vascular targeting activity in vivo.

BACKGROUND: Combretastatin A4 Phosphate (CA4P) is a tubulin binding agent which causes rapid tumour vascular shutdown. It has anti-proliferative and apoptotic effects on dividing endothelial cells after prolonged exposure, but these effects occur on a much longer time scale than the reduction in tumour blood flow. This study compared the time course of CA4P effects on endothelial cell shape and reduction in red cell velocity. METHODS: Endothelial cell area and form factor (1-4 pi x area x perimeter-2) were measured for proliferating and confluent HUVECs after CA4P treatment. Recovery of shape after CA4P and colchicine was compared. Window chamber studies of tumours were used to measure red cell velocity. Results 70% reduction in red cell velocity and 44% reduction in HUVEC form factor occurred by 10 minutes. Proliferating HUVECs underwent greater cell shape change after CA4P, which occurred at lower doses than for confluent cells. Cell shape recovered 24 hours after 30 minutes exposure to CA4P, but not after colchicine. CONCLUSIONS: The similar time course of cell shape change and red cell velocity reduction suggests endothelial cell shape change may be involved early in the in vivo events leading to vascular shutdown. Differences in the recovery from the shape changes induced by CA4P and colchicine could underlie the different toxicity profiles of these drugs.

A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles.

Charged-particle microbeams provide a unique opportunity to control precisely, the dose to individual cells and the localization of dose within the cell. The Gray Laboratory is now routinely operating a charged-particle microbeam capable of delivering targeted and counted particles to individual cells, at a dose-rate sufficient to permit a number of single-cell assays of radiation damage to be implemented. By this means, it is possible to study a number of important radiobiological processes in ways that cannot be achieved using conventional methods. This report describes the rationale, development and current capabilities of the Gray Laboratory microbeam.