Phase‐contrast helium‐3 MRI of aerosol deposition in human airways

One of the key challenges in the study of health‐related aerosols is predicting and monitoring sites of particle deposition in the respiratory tract. The potential health risks of ambient exposure to environmental or workplace aerosols and the beneficial effects of medical aerosols are strongly influenced by the site of aerosol deposition along the respiratory tract. Nuclear medicine is the only current modality that combines quantification and regional localization of aerosol deposition, and this technique remains limited by its spatial and temporal resolutions and by patient exposure to radiation. Recent work in MRI has shed light on techniques to quantify micro‐sized magnetic particles in living bodies by the measurement of associated static magnetic field variations. With regard to lung MRI, hyperpolarized helium‐3 may be used as a tracer gas to compensate for the lack of MR signal in the airways, so as to allow assessment of pulmonary function and morphology. The extrathoracic region of the human respiratory system plays a critical role in determining aerosol deposition patterns, as it acts as a filter upstream from the lungs. In the present work, aerosol deposition in a mouth–throat phantom was measured using helium‐3 MRI and compared with single‐photon emission computed tomography. By providing high sensitivity with high spatial and temporal resolutions, phase‐contrast helium‐3 MRI offers new insights for the study of particle transport and deposition. Copyright © 2014 John Wiley & Sons, Ltd.     

A device to control the mass concentration of protein-containing aerosoles in real-time mode

The article is dedicated to the device to measure the mass concentration of protein-containing aerosols, which is now being developed at State Scientific and Research Institute of Biological Instrument Making. The operation of the device is based on the registration of spectral-luminescent and dispersing properties of individual particles of biological nature. Experiments carried out on aerosol static-dynamic stand have demonstrated that the device measures the mass concentration of protein-vitamin concentrate aerosol in real-time mode with 50% accuracy.     

紫外线照射对腺病毒气溶胶活力和粒级分布的影响

目的 研究紫外线照射对腺病毒气溶胶活力和粒级分布的影响.方法 在2000L的腔室中通过TK-3微生物气溶胶发生器将绿色荧光蛋白(GFP)标记的腺病毒形成气溶胶,暴露在预先设定波长的紫外线下,用多级撞击式空气微生物采样器进行采样.对采样样品进行实时荧光定量PCR(FQ-PCR)检测基因组拷贝数.通过在荧光显微镜下计数带绿色荧光的PK15细胞数可直观检测病毒的感染力及活力.结果 带有绿色荧光的PK15细胞数及FQ-PCR检测结果均提示所采集的病毒气溶胶主要分布在第6级.波长为254 nm的紫外线照射5 min时,在显微镜下观察到的荧光数明显减少.波长为254 nm紫外线照射30 min时,6个级别的细胞内均未见绿色荧光.波为365 nm紫外线照射30 min时,绿色荧光数仍较多.波长为254 nm的紫外线照射30 min时没有观察到有绿色荧光的细胞,但是FQ-PCR结果提示病毒基因组拷贝数仍较高.结论 波长为254 nm的紫外线比365 nm的紫外线照射灭活腺病毒气溶胶的效果更显著.两种波长的紫外线照射对腺病毒气溶胶的粒级分布没有显著影响.病毒基因组的存在并不能代表病毒有感染力.     

紫外线照射对腺病毒气溶胶活力和粒级分布的影响

目的研究紫外线照射对腺病毒气溶胶活力和粒级分布的影响。方法在2000L的腔室中通过TK-3微生物气溶胶发生器将绿色荧光蛋白(GFP)标记的腺病毒形成气溶胶,暴露在预先设定波长的紫外线下,用多级撞击式空气微生物采样器进行采样。对采样样品进行实时荧光定量PCR(FQ-PCR)检测基因组拷贝数。通过在荧光显微镜下计数带绿色荧光的PK15细胞数可直观检测病毒的感染力及活力。结果带有绿色荧光的PK15细胞数及FQ-PCR检测结果均提示所采集的病毒气溶胶主要分布在第6级。波长为254nm的紫外线照射5min时,在显微镜下观察到的荧光数明显减少。波长为254nm紫外线照射30min时,6个级别的细胞内均未见绿色荧光。波为365nm紫外线照射30min时,绿色荧光数仍较多。波长为254nm的紫外线照射30min时没有观察到有绿色荧光的细胞,但是FQ-PCR结果提示病毒基因组拷贝数仍较高。结论波长为254nm的紫外线比365nm的紫外线照射灭活腺病毒气溶胶的效果更显著。两种波长的紫外线照射对腺病毒气溶胶的粒级分布没有显著影响。病毒基因组的存在并不能代表病毒有感染力。     

Quantitative assays of pharmacologic aerosols used in studying asthma

To more nearly accurately quantitate the dose of pharmacologic agents delivered to human and animal airways via aerosols, we have developed a monodisperse aerosol containing either methacholine or histamine that permits a light scattering device (tyndallometry) to measure accurately the quantity of inspired and expired particles. These aerosols (described in previous studies) are simultaneously tagged with a radioactive label (technetium 99m) to permit the use of external gamma camera imaging. Present work focuses on the development of assay techniques to measure the quantity of methacholine delivered in these aerosols. The lack of specific radioimmune or radioenzyme assays coupled with the cross-reaction of organic contaminants with conventional chemical reagents for measuring methacholine required the development of separative techniques to isolate the methacholine from the organic aerosol contaminants. With aqueous extraction and column separation we have been able to completely isolate the methacholine from these contaminants. This allows the application of standard spectrophotometric assays for methacholine to quantitate the methacholine in the resulting solution. These separative techniques will permit the use of these aerosols in quantitative studies of airway reactivity.     

Dealing with Cerenkov radiation generated in organic scintillator dosimeters by bremsstrahlung beams

An organic scintillator detector system has been developed for radiotherapy bremsstrahlung dosimetry. The scintillators are connected to photodiodes by light pipes as the photodiodes must be removed and shielded from the incident radiation. The photodiodes see visible and near-visible light emissions from the scintillator as well as Cerenkov and fluorescence radiation that has been generated and trapped in the scintillator and light pipe. The Cerenkov and fluorescence radiation limits the accuracy of the dosimeter. This work examines a range of methods for diminishing the signal contribution of Cerenkov and fluorescence radiation while optimizing the scintillator signal. Three methods of achieving these goals have been used. They are: reflective coatings on the scintillator, long-wavelength-emitting scintillators used in conjunction with the photodiode, and absorptive filters placed between the light guide and photodiode. The contribution of the Cerenkov radiation to the light seen by the photodiode has been modelled and the model predictions have been tested using bremsstrahlung beams of peak energy between 13 and 20 MV, showing agreement with measurement.     

Evaluation of quantitative aerosol techniques for use in bronchoprovocation studies

To investigate airway physiology by use of inhaled aerosols, it is frequently necessary to measure the actual amount of material deposited on the airway wall as well as the site of particle deposition. To satisfy these needs, radiolabeled aerosols and gamma camera techniques have been used to measure regional deposition of inhaled particles. To make quantitative measurements of the amount deposited, previous investigators have used a "phantom" technique to indirectly calibrate the gamma camera for the attenuation of gamma rays through the lungs and chest wall. For this calibration, the phantom is a simulated lung containing a known amount of radioactivity. Radioactive counts emitted from the phantom are assumed to be attenuated in the same manner as the intact human lung. The present article describes a technique to determine directly the amount of inhaled aerosol deposited in the lung and simultaneously to calibrate the gamma camera for each individual subject. We used right angle light scattering and a gamma camera to measure individual values of the deposition fraction (DF) of inhaled aerosol deposited in the lung and the coefficient of attenuation (AC) of gamma rays in normal and obstructed lungs of human subjects. Radiolabeled monodisperse aerosols 1 and 2 microns in diameter were used. Knowledge of the activity of the inhaled aerosol (microcurie per liter), the volume inhaled, and the measured DF determined each subject's AC (counts per minute per microcurie). DF varied by an order of magnitude in normal (0.04 to 0.48) and obstructed (0.16 to 0.75) of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Numerical simulation of light propagation and scattering in turbid biological media.

This article considers the processes of light propagation and scattering in biological tissues. The results obtained made it possible to estimate basic signal parameters and their dependence on various optical parameters with respect to the laser Doppler flowmeter and laser Doppler microscope. We also developed a new method to determine the indicatrix asymmetry of single and multiple light scattering by a suspension of oriented spheroidal particles that simulated erythrocytes in a shear flow. It was found that the angular dependence of the asymmetry index provides infonnation on the shape and orientation of particles. In addition, we obtained single scattering indicatrices, which may improve the accuracy of computer simulation of light scattering by blood.     

用安德森空气生物采样器采集病毒气溶胶的研究

目的以表达绿色荧光蛋白(GFP)报告基因的重组腺病毒(rAdvGFP)为模式病毒,进行气溶胶的主动发生实验和模拟剧烈吹打、振荡混匀等操作,利用安德森采样器进行气溶胶的采集,以建立简单易行的病毒气溶胶采集检测体系。方法用气溶胶发生器模拟模式病毒的气溶胶主动发生,分别在距离发生器管口0、10和20cm处放置采样器及培养皿,在气溶胶发生30s时开始采集,共采集5min。在模拟吹打操作中,将10ml病毒液置于50ml离心管中,用微量移液器反复吸取吹打病毒液;在模拟振荡操作中,将10ml病毒液置于50ml离心管中盖紧,以振荡器最大速度涡旋振荡1min,立即打开管盖采集2min,然后盖紧再重复振荡30s后开盖继续采集。两种模拟操作均在距离管口0和10cm处放置采样器及培养皿,各采集5min。收集各采样器及培养皿中的液体,利用阳离子聚合物聚凝胺浓缩后用PCR方法检测病毒核酸,并接种人胚肾293细胞进行病毒的培养,在荧光显微镜下观察GFP蛋白的表达。结果模拟气溶胶主动发生时,将置于距发生器管口0、10和20cm的采样器和培养皿中的采集液接种细胞均可观察到绿色荧光,并可检测到病毒核酸。模拟剧烈吹打时,置于0cm处的培养皿内可检测到病毒,而置于0cm、10cm处采样器和10cm处培养皿中均未检测到病毒。在剧烈振荡模拟试验中,在距离管口0cm的采样器和培养皿中均可检测到病毒,在置于10cm处的采样器中未检测到病毒,在置于10cm处的培养皿中可检测到病毒。结论利用rAdvGFP模式病毒和安德森采样器初步建立了病毒学实验气溶胶收集检测体系,对模拟危险操作感染性材料所产生的气溶胶做出危险评估,为制定病毒学实验操作规范提供了依据。     

Quantification of particle deposition in asymmetrical tracheobronchial model geometry

The primary objective of this study was to quantify the local inspiratory and expiratory aerosol deposition in a highly asymmetric five-generation tracheobronchial tree. User-enhanced commercial codes and self-developed software was used to compute the air velocity field as well as particle deposition distributions for a large size range of inhalable particles. The numerical model was validated by comparison of our results with experimental flow measurements and particle deposition data available in the open literature. Our simulations show highly localised deposition patterns for all particle sizes, but mainly for the larger particles. As expected, deposition efficiencies and deposition fractions proved to be very sensitive to the particle size. The deposition density in the hot spots can be hundreds and even thousand times higher than the mean deposition density. Present results can be of interest to researchers involved in the assessment of adverse health effects of inhaled aerosols or optimising the drug aerosol delivery into the lung.     

Self-mixing in a diode laser as a method for cardiovascular diagnostics

Our aim is the development of a simple optical method for pulse wave profile, pulse wave delay time, and blood flow measurement. The method is based on recording the Doppler frequency shift related to a moving target--blood vessel walls or small particles. The Doppler signal is detected using the self-mixing that occurs in the diode laser cavity when radiation scatters back from the moving target into the laser and interferes with the field inside. Two different ways can be simultaneously used for the self-mixing signal extraction: a photodiode accommodated in the rear facet of the diode laser package and a resistor from the laser pump current. An experimental device with a pigtail laser diode is developed that is able to detect the pulsation of major arteries with potentially useful information, including the pulse wave profile and the pulse wave delay time. The pulse wave delay time in different regions of the human body is measured relative to the electrocardiogram (ECG) signal. Also the flow velocity of a liquid suspension containing particles the size of erythrocytes (equivalent to blood flow) is measured. Registered signals are stored after digitalization and preprocessed using LabView for a Windows environment. The described device has the application of the self-mixing method and highlights significant advantages of simplicity, compactness, and robustness as well as the self-aligning and self-detecting abilities of such method, compared with the use of conventional interferometric method.     

Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of the exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.     

Development of an aerosol model of murine respiratory mycoplasmosis in mice.

Animal models of murine respiratory mycoplasmosis due to Mycoplasma pulmonis provide excellent opportunities to study respiratory disease due to an infectious agent. The purpose of the present study was to develop and characterize an aerosol model for the production of murine respiratory mycoplasmosis in mice. The exposure of mice for 30 min to aerosols generated with a DeVilbiss 45 nebulizer in a nose-only inhalation chamber consistently reproduced typical lesions. The chamber was operated with a nebulizer air flow of 5.3 liters/min at 5.0 lb/in and a diluting air flow of 20 liters/min, with the nebulizer containing 5 ml of a suspension of viable M. pulmonis organisms (a concentration between 6 X 10(5) to 6 X 10(10) CFU/ml). Infective aerosol particles of less than a 4.0-micron median aerodynamic diameter with a geometric standard deviation of approximately 2.0 reached the lungs and were evenly distributed among the different lung lobes. A minimum 1.5-log loss of viability in the M. pulmonis suspension was demonstrated. With the exception of the 50% lethal dose, all of the parameters previously established by intranasal inoculation could be examined with the aerosol model. The major advantages of the aerosol model were excellent reproducibility of exposure (both between different experiments and between animals in a given experiment), the avoidance of anesthetization, and the ability to immediately deposit the majority of the organisms in the lung. The only disadvantage was the requirement for large volumes of mycoplasmal cultures.     

Two-photon fabrication of hydrogel microstructures for excitation and immobilization of cells

We investigate in vitro fabrication of hydrogel microstructures by two photon laser lithography for single cell immobilization and excitation. Fluorescent yeast cells are embedded in water containing the hydrogel precursor mixtures and cross-linking is used to selectively immobilize a particular cell. Cell viability within the hydrogel precursor is estimated using a life/dead assay and elastic and stiff hydrogel structures are fabricated, immobilizing cells in a microfluidic environment. Additionally, we demonstrate the illumination of cells by on-the-fly fabricated hydrogel waveguide networks connected to an external light source, thereby exciting a fluorescence signal in a single immobilized cell.     

Modelling the effect of charge on selective deposition of particles in a diseased lung using aerosol boli

The technique of using a charged bolus of aerosol to deliver a drug or other agent is advantageous since sites of interest within the lung can be selectively targeted. Ideally, the volume of the bolus should match that of the targeted region allowing the aerosol bolus particles to be confined to the selected area during the pause period after inhalation. Our existing computer model for predicting the deposition of charged aerosol particles has been developed to encompass aerosol boli, some diseased lung morphologies and drug dose administered per breathing cycle. Aerosol deposition in the targeted region is found to be enhanced by increasing particle charge, pause period and particle size. For particles in the size range 1-2.5 microm, aerosol deposition in the region affected by bronchoconstriction does not alter significantly with flow rate variation (range 250-1000 ml s(-1)) for a targeted charged bolus of matched volume. The technique may enable the optimal delivery of therapeutic or other agents to diseased or normal lungs.     

Analytical models for time resolved fluorescence spectroscopy in tissues.

Laser induced fluorescence is a rapidly growing technique for diagnostics and imaging in scattering material, most notably in in vivo biomedical testing. Most previous applications have relied on the measurements of the steady-state emission spectrum, with subsequent analysis of the spectrum for relative concentrations of potential fluorophores. Only recently a few investigators have explored the use of the fluorescence lifetimes as a diagnostic tool by taking advantage of the perturbation of the lifetime by multiple scattering of the excitation and emission light in the tissue. We have developed a model to study the fluorescence signal generated by fluorophores distributed in a scattering medium. This model is based on two coupled time-dependent photon migration phenomena: the transport of the pulsed source laser light and the transport of the induced fluorescent light excited by the source. The coupling of these two is through the source for the induced fluorescence where the strength of the local fluorescence emission depends on the absorption of the laser intensity at that location. Whereas previous research focused mainly on the fluorescence properties of various dyes, compounds and materials, transport phenomena have only recently been addressed by researchers. We have presented general analytical and numerical solutions for finite, infinite, cylindrical and spherical geometries.     

The nasal air sampler: a device for sampling inhaled aeroallergens.

OBJECTIVE: The object is to design, develop, and test a personal aerosol sampling device consisting of impaction samplers worn just inside the nostrils, driven by the wearer's respiration. The device provides a novel and unique measure of individual exposure to aeroallergens. It was conceived as an integral part of an allergen diagnostic system, in which collected aerosols are immunostained with monoclonal antibodies or the patient's IgE and associated particles positively identified using techniques of image analysis. METHODS: Each sampler comprises a slot impactor with a detachable impaction plate covered with either a specially developed medical adhesive or a protein-binding membrane. Sampler performance has been validated by rig tests of aerodynamic resistance and collection efficiency of different sized particles at various flow rates. There have also been field trials with human subjects which show that the sampler can be comfortably worn for periods of up to 4 hours. This is sufficient to gather a representative sample of inhaled allergens in most environments. RESULTS: The sampler collects an increasing proportion of particles in the inhalable range at and above 5 microm. This includes most bioaerosols of interest to allergists. Sampler prototypes have been built by CNC mill and stereolithography. Batches of samplers have been molded in biocompatible materials for field and clinical trials. CONCLUSIONS: The device successfully collects aeroallergens from a patient's own respiration. While developed specifically as a vehicle for the allergen diagnostic system, it can be adapted for studies of other aspects of air quality or for prophylactic use.     

Aerosol particle impaction in the conducting airways

Modelling of inhaled particle deposition in the lungs potentially offers data relevant to assessing hazards from toxic inhaled particles, to studying mucus clearance or lung permeability by aerosol techniques, and to achieving better utilisation of drugs administered as aerosols. Analysis of published modelling studies is complicated by differing approaches to the quantitative estimation of physical factors determining deposition and by differing choices of anatomical data. Published formulae for predicting aerosol particle impaction are compared by applying them to the deposition of 5 micron particles in the human conducting airways using the morphological data of Weibel together with information from other sources about airways branching angles. The results indicate the range of deposition estimates that may be obtained from currently available impaction formulae. All but one of the formulae considered agree in indicating maximum deposition by impaction in, or close to, the segmental or subsegmental bronchi. Data are presented to indicate how the principal physical determinants of deposition depend on particle size, inhalation flow rate and lung volume during inhalation.     

Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness

BACKGROUND: House dust mites (HDMs) are the major source of perennial allergens causing human allergic asthma. Animal models mimicking as closely as possible the allergic features observed in human asthma are therefore interesting tools for studying the immunological and pathophysiological mechanisms involved. Especially the role of eosinophils and allergen-specific immunoglobulin (Ig) E in the pathophysiology of airway hyperresponsiveness (AHR) remains a subject of intense debate. OBJECTIVE: To develop a mouse model of allergic airway inflammation and hyperresponsiveness based on the use of purified house dust mite allergen (Der p 1) as clinical relevant allergen. Furthermore, we studied the effects of low dose allergen exposure on the airway eosinophilia and AHR. METHODS: On day 0, C57Bl/6 mice were immunized with purified Der p 1 intraperitoneally. From day 14-20, the mice were exposed daily to a 30-min aerosol of different concentrations of house dust mite extract. RESULTS: Mice, actively immunized with Der p 1 and subsequently exposed to HDM aerosols, developed AHR, eosinophil infiltration of the airways and allergen-specific IgE. Moreover, lowering the concentration of the HDM aerosol also induced AHR and IgE without apparent eosinophil influx into the airways. Der p 1-sensitized mice exposed to PBS produced IgE, but did not show AHR or eosinophil influx. CONCLUSION: This in vivo model of HDM-induced allergic airway changes suggests that AHR is not related to either eosinophil influx or allergen-specific serum IgE, thereby reducing the importance of these factors as essential elements for allergic AHR.     

Characterization of Respiratory Drug Delivery with Enhanced Condensational Growth using an Individual Path Model of the Entire Tracheobronchial Airways

The objective of this study was to evaluate the delivery of inhaled pharmaceutical aerosols using an enhanced condensational growth (ECG) approach in an airway model extending from the oral cavity to the end of the tracheobronchial (TB) region. The geometry consisted of an elliptical mouth-throat (MT) model, the upper TB airways extending to bifurcation B3, and a subsequent individual path model entering the right lower lobe of the lung. Submicrometer monodisperse aerosols with diameters of 560 and 900 nm were delivered to the mouth inlet under control (25 °C with subsaturated air) or ECG (39 or 42 °C with saturated air) conditions. Flow fields and droplet characteristics were simulated using a computational fluid dynamics model that was previously demonstrated to accurately predict aerosol size growth and deposition. Results indicated that both the control and ECG delivery cases produced very little deposition in the MT and upper TB model (approximately 1%). Under ECG delivery conditions, large size increases of the aerosol droplets were observed resulting in mass median aerodynamic diameters of 2.4–3.3 μm exiting B5. This increase in aerosol size produced an order of magnitude increase in aerosol deposition within the TB airways compared with the controls, with TB deposition efficiencies of approximately 32–46% for ECG conditions. Estimates of downstream pulmonary deposition indicted near full lung retention of the aerosol during ECG delivery. Furthermore, targeting the region of TB deposition by controlling the inlet temperature conditions and initial aerosol size also appeared possible.