Why Homoscleromorph Sponges Have Ciliated Epithelia: Evidence for an Ancestral Role in Mucociliary Driven Particle Flux.

Ciliary beating and metachronal waves are fundamental to effective mucociliary transport. The ciliary beat frequencies (CBFs) and metachronal wave directions of multiple cilia beating in culture media were measured simultaneously using digital microscopic images. The degree of synchronisation between ciliary beats was determined by the correlation between ciliary signals at two different locations. The wave propagation directions of cilia were determined from a two-dimensional correlation map by a principal axis method. The standard deviation of measured wave directions in a region of interest was defined as a measure of metachronal wave disorder (MWD). Considerable variation was found in the beat frequencies and metachronal wave directions of cilia beating on epithelium. The pooled mean of MWDs was 23.4 +/- 8.8 degrees, and the pooled mean of CBFs was 10.1 +/- 1.9 Hz on 120 cells from five healthy subjects. The means of the MWD and the CBF from subjects were highly correlated (correlation = -0.83). The higher the CBF, the lower the level of the MWD.

Introduction: In primary ciliary dyskinesia there is an alteration of ciliary beat frequency and/or ciliary beat pattern. Aims and objectives: The aim of this preliminary study was to assess the ciliary beat frequency in patients affected of respiratory illnesses that could be present in primary ciliary dyskinesia. Methods: Nasal nitric oxide (nNO) was measured in 20 patients affected of respiratory illnesses in whom primary ciliary dyskinesia was suspected. Ciliated epithelium was obtained by brushing the inferior nasal turbinate. Beat frequency measurements were made by digital high speed video analysis. Abnormal beat pattern was considered when less than 80% of the measurements were normal beat pattern. Circular, static, rigid and dyskinetic (flickering or others) patterns were considered as abnormal patterns. Results: Twenty patients (2-55 years-old) were studied. Newborn respiratory distress was present in 4 patients, bronchiectasis (10), recurrent otitis media (10), sinusitis (5), middle lobe syndrome (2), recurrent pneumonia (7), gastro-esophageal reflux (3). Nasal nitric oxide ranged from 44 ppb to 1019 ppb. Beat frequency ranged from 7,26 Hz to 14,46 Hz. Static beat pattern was predominant in 8 patients, dyskinetic beat pattern in 2 patients and circular beat pattern in one patient. In one patient there were no cilia. In all patients with beat frequency below 8,5 Hz, beat pattern were abnormal. There was an overlap on beat frequency values in patients with normal and abnormal beat pattern. Conclusions: In this study, similar ciliary beat frequencies were found in patients with normal and abnormal beat patterns. Genetic study or cell culture could help to differentiate between primary and secondary ciliary dyskinesia.

Epidemiological data support a correlation between smoking and increased incidence of ectopic pregnancy, yet the causal mechanism responsible for this relationship is unknown. The purpose of this study was to examine the effect of solutions containing dissolved mainstream (MS) or sidestream (SS) cigarette smoke on ciliary beat frequencies (CBF) in explants of hamster oviducts. MS smoke is the puff inhaled by an active smoker, while SS smoke leaves the burning end of cigarette. SS smoke is inhaled by both active and passive smokers. Experiments were performed in handmade perfusion chambers using infundibula from hamster oviducts. After a short incubation in Earle's balanced salt solution containing HEPES buffer (EBSS-H), chambers were flushed with one of six types of smoke solution prepared in EBSS-H, and incubation continued 19 min. A second perfusion (washout) was then done using EBSS-H alone to determine whether effects induced by the smoke solutions could be reversed. CBF were determined at three times in both the smoke and washout solutions, and means were compared to values obtained in the initial EBSS-H incubation. All smoke solutions except the SS particulate solution inhibited CBF in a dose-dependent manner. Whole MS and whole SS smoke solution at the highest strength tested caused the greatest inhibition and in some cases completely stopped ciliary beating. Both single-strength and 0.1-strength MS gas phase solutions, which contained concentrations of nicotine in the range found in typical human smokers, produced about 50% inhibition of ciliary beating. Inhibition was generally seen within 2-12 min of adding smoke solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by defective ciliary beating. Diagnostic guidelines of the American Thoracic Society and European Respiratory Society recommend measurement of nasal nitric oxide (nNO) for PCD diagnosis. Several studies demonstrated low nNO production rates in PCD individuals, but underlying causes remain elusive. Objectives: To determine nNO production rates in a well-characterized PCD cohort, including subgroup analyses with regard to ultrastructural and ciliary beating phenotypes. Methods: This study included 301 individuals assessed according to European Respiratory Society guidelines. Diagnostic cutoffs for nNO production rates for this study cohort and subgroups with normal and abnormal ultrastructure were determined. Diagnostic accuracy was also tested for the widely used 77 nl/min cutoff in this study cohort. The relationship between nNO production rates and ciliary beat frequencies (CBFs) was evaluated. Results: The study cohort comprised 180 individuals with definite PCD diagnosis, including 160 individuals with genetic diagnosis, 16 individuals with probable PCD diagnosis, and 105 disease controls. The 77 nl/min nNO cutoff showed a test sensitivity of 0.92 and specificity of 0.86. Test sensitivity was lower (0.85) in the subgroup of 47 PCD individuals with normal ultrastructure compared with 133 PCD individuals with abnormal ultrastructure (0.95). The optimal diagnostic cutoff for the nNO production rate for the whole study cohort was 69.8 nl/min (sensitivity, 0.92; specificity, 0.89); however, it was 107.8 nl/min (sensitivity, 0.89; specificity, 0.78) for the subgroup of PCD with normal ultrastructure. PCD individuals with normal ultrastructure compared with abnormal ultrastructure showed higher ciliary motility. Consistently, PCD individuals with higher CBFs showed higher nNO production rates. In addition, laterality defects occurred less frequently in PCD with normal ultrastructure. Conclusions: Measurements of nNO below the widely used 77 nl/min cutoff are less sensitive in detecting PCD individuals with normal ultrastructure. Our findings indicate that higher nNO production in this subgroup with a higher cutoff for the nNO production rate (107.8 nl/min) and higher residual ciliary motility is dependent on the underlying molecular PCD defect. Higher nNO production rates, higher residual CBFs, and the lower prevalence of laterality defects hamper diagnosis of PCD with normal ultrastructure. Adjusting the cutoff of nNO production rate to 107.8 nl/min might promote diagnosing PCD with normal ultrastructure.

In vitro nasal and tracheal ciliary beat frequencies (CBFs) were measured photometrically in brush samples of 15 patients undergoing bronchoscopy. Tracheal and nasal CBF values in these patients were found to be significantly correlated (r = .74, p less than .01), and the latter did not differ significantly from the nasal CBF of 80 subjects serving as controls. Premedication with morphine and atropine and local anesthesia with lidocaine hydrochloride significantly decreased the CBF. Among the control subjects, a significantly higher CBF (13.6 +/- 1.5 Hz) was found in the young age group (10 to 19 years) as compared to other groups (12.2 +/- 1.7 Hz). Gender and ethnic origin did not influence CBF in any age group. These data support the possible use of the in vitro study of nasal epithelium to reflect the CBF in the lower respiratory tract.

Very little information is available on the relationship of ciliary beat frequency (CBF) to mucus transport velocities (MTV) in airways distal to the trachea in intact lungs. Ciliary beat frequencies and mucus transport rates in airways are not uniform at all levels of the lung, both of these increasing from the small airways to the trachea. The purpose of this paper is to report the relationship of CBF to MTV in normal airways and compare these relationships to abnormalities of mucus transport in animals with spontaneous chronic respiratory disease.

A new approach to recover 3-D shape from a Scanning Electron Microscope (SEM) image is described. With an ideal SEM image, 3-D shape can be recovered using the Fast Marching Method (FMM) applied to the Eikonal equation. However, when the light source direction is oblique, the correct shape cannot be obtained by the usual one-pass FMM. The new approach modifies the intensities in the original SEM image using an additional SEM image of a sphere and Neural Network (NN) training. Image modification is a two degree-of-freedom (DOF) rotation. No assumption is made about the specific functional form for intensity in an SEM image. The correct 3-D shape can be obtained using the FMM and NN learning, without iteration. The approach is demonstrated through computer simulation and validated through real experiment.

Polymer gel dosimeters have been proved as a useful tool in the verification of treatment planning for three-dimensional radiation therapy. The degree of radiation-induced polymerization (DP) is a function of absorbed dose, and it can be determined in macro-scale by several imaging modalities, such as magnetic resonance imaging (MRI), optical computed tomography, and computed tomography (CT). In this study, we determined the DP for the polymer gel dosimeter in micro-scale by texture analysis on scanning electron microscope (SEM) images. The irradiated n-NIPAM gels with absorbed doses of 5 to 20 Gy delivered by the linear accelerator were freeze-dried and sliced. The SEM images were acquired at magnification of 50t, 500ntn and 3500t , respectively. Four image texture indices, including the entropy, contrast, energy and homogeneity, were estimated from the grey level co-occurrence matrix of each SEM image. The relationships between the radiation dose and texture indices were determined. The SEM images with 500t and 3500t magnification showed significant changes in morphological features of the irradiated gel. The r2 of the linear fitting from 3500t SEM images was 0.8194 (entropy), 0.6024 (contrast), 0.7681 (energy), and 0.9978 (homogeneity), respectively. We conclude that the texture analysis on SEM images is a feasible method to estimate the dose response of the polymer gel dosimeter.

Ciliated respiratory epithelial cells have to tolerate variations in local pH caused by the respiratory cycle and potential ventilation-perfusion mismatches. We showed previously that peripheral bronchiolar cilia beat at a lower frequency than bronchial cilia, and have now investigated whether they show differences in tolerance to changes in pH. Using the image analysis system applied in the previous study, we compared variations in the ciliary beat frequencies (CBF) of bronchi and bronchioles sampled from human lung resections at various pH in vitro. Application of nonparametric tests (the variance of samples was not similar) indicated that CBF was not significantly modified when pH was varied between 7.5 and 10.5 for bronchi, and between 5.5 and 10.5 for bronchioles. Reversible and significantly lower CBF were observed below pH 7.0 for bronchi and below pH 5.0 for bronchioles. Extreme pH values such as 11.0 or 3.0 were lethal within a few minutes. Thus, respiratory ciliary beating is able to tolerate external pH variations between 3.5 and 10.5 without permanent impairment. In addition we found that alkaline pH values are more favourable than acidic ones and that bronchiolar ciliated cells are more tolerant to acidic pH than bronchial cells.

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Nanomanipulation under scanning electron microscopy (SEM) enables direct interactions of a tool with a sample. We recently developed a nanomanipulation technique for the extraction and identification of DNA contained within sub-nuclear locations of a single cell nucleus. In nanomanipulation of sub-cellular structures, a key step is to identify targets of interest through correlating fluorescence and SEM images. The DNA extraction task must be conducted with low accelerating voltages resulting in low imaging resolutions. This is imposed by the necessity of preserving the biochemical integrity of the sample. Such poor imaging conditions make the identification of nanometer-sized fiducial marks difficult. This paper presents an affine scale-invariant feature transform (ASIFT) based method for correlating SEM images and fluorescence microscopy images. The performance of the image correlation approach under different noise levels and imaging magnifications was quantitatively evaluated. The optimal mean absolute error (MAE) of correlation results is 68±34 nm under standard conditions. Compared with manual correlation by skilled operators, the automated correlation approach demonstrates a speed that is higher by an order of magnitude. With the SEM-fluorescence image correlation approach, targeted DNA was successfully extracted via nanomanipulation under SEM conditions. A fast method for correlating scanning electron microscopy (SEM) and fluorescence images when handling subcellular objects has been acheived. Yu Sun of the University of Toronto, Canada, and co-workers in China and Canada developed the image correlation technique to improve the implementation of a method for extracting DNA from cells. Such correlation is essential, because SEM images lack sufficient contrast to enable structures of interest to be identified and because of the need to use low-energy (and hence low-resolution) SEM imaging to avoid damaging DNA. Their correlation technique employs image processing algorithms to automatically correlate the two microscopy techniques and is an order of magnitude faster than manual image correlation by skilled operators. The researchers have demonstrated the method's usefulness by using it to extract DNA from cells under SEM imaging.

We have tried to estimate the electron beam profile from a scanning electron microscope (SEM) image by using Wavelet multiresolution analysis for in-process SEM inspection. At first, an ideal secondary electron (SE) profile for step edge is calculated by using the Monte Carlo simulator. Then the SE profiles observed by the electron beam with Gaussian profiles are simulated with the electron beam diameter as a parameter. Wavelet analyzed results of SE profiles show that it is possible to estimate the size of electron beam profile from the Wavelet coefficient of SE profile. Next we apply the proposed estimation method to SEM images of test pattern. The procedure is as follows. 1) Noise included in SEM images is reduced by using the denosing method by Wavelet transform. 2) The SE profile for edge is extracted from a SEM image and is normalized. 3) The normalized SE profile is decomposed by Wavelet multiresolution analysis. 4) By comparing the obtained Wavelet coefficient of SE profile with the relation between the electron beam diameter and the Wavelet coefficient, the electron beam profile is estimated. Proposed procedure was applied to the SEM image of test pattern to obtain the beam profile.

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Laser microsurgery on zebrafish larvae combined with Scanning Electron Microscopy (SEM) imaging can in particular provide accelerated insights into the tissue regeneration phenomena. Conventional SEM exposes, however, specimens to high vacuum environments, and often requires laborintensive and time-consuming pretreatments and manual positioning. Moreover, there are virtually no technologies available that can quickly immobilize the zebrafish larvae for high definition SEM imaging. This work describes the proof-of-concept design and validation of a microfluidic chip-based system for immobilizing zebrafish larvae and it’s interfacing with Environmental Scanning Electron Microscope (ESEM) imaging. The Lab-on-a-Chip (LOC) device was fabricated using a high-speed infrared laser micromachining and consists of a reservoir with multiple semispherical microwells, which hold the yolk of zebrafish larvae, and drain channels that allow removing excess of medium during SEM imaging. Paper filter is used to actuate the chip and immobilization of the larvae by gentle suction that occurs during water drainage. The trapping region allows multiple specimens to be positioned on the chip. The device is then inserted directly inside the ESEM and imaged in a near 100% humidity atmosphere. This facilitates ESEM imaging of untreated biological samples.

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The modern high‐performance personal computer (PC) has very recently expanded the range of utilization of digital scanning electron microscopy (SEM) images, and the PC will be used increasingly with SEMs. However, the image quality of digital SEM images may be considerably influenced by scanning and digitization conditions. In particular, the effects of the aliasing error peculiar to digital data are often serious in the low‐magnification acquisition (undersampling) of SEM images, and moreover even a high‐magnification image (oversampling) is disturbed by the undersampled noise (a sort of aliasing error). Furthermore, the signal‐to‐noise ratio of a digitized SEM image is closely related to the performance of the analog‐to‐digital converter. To prevent a flood of low‐quality digital images with artifacts by the aliasing and additional noise, we propose a method using very high‐density sampling (scanning). In addition, we will discuss how to handle digital SEM images from the point of view of the sampling and quantization.