Automatic detection of adult cardiomyocyte for high throughput measurements of calcium and contractility.

Simultaneous calcium and contractility measurements on isolated adult cardiomyocytes have been the gold standard for the last decades to study cardiac (patho)physiology. However, the throughput of this system is low which limits the number of compounds that can be tested per animal. We developed instrumentation and software that can automatically find adult cardiomyocytes. Cells are detected based on the cell boundary using a Sobel-filter to find the edge information in the field of view. Separately, we detected motion by calculating the variance of intensity for each pixel in the frame through time. Additionally, it detects the best region for calcium and contractility measurements. A sensitivity of 0.66 ± 0.08 and a precision of 0.82 ± 0.03 was reached using our cell finding algorithm. The percentage of cells that were found and had good contractility measurements was 90 ± 10%. In addition, the average time between 2 cardiomyocyte calcium and contractility measurements decreased from 93.5 ± 80.2 to 15.6 ± 8.0 seconds using our software and microscope. This drastically increases throughput and provides a higher statistical reliability when performing adult cardiomyocyte functional experiments.

In Vivo Visualization and Quantification of Mitochondrial Morphology in C. elegans.

Caenorhabditis elegans is a highly versatile model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology.

A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

Proteomics and microscopy tools for the study of antimicrobial resistance and germination mechanisms of bacterial spores.

Bacterial spores are ubiquitous in nature and can withstand both chemical and physical stresses. Spores can survive food preservation processes and upon outgrowth cause food spoilage as well as safety risks. The heterogeneous germination and outgrowth behavior of isogenic spore populations exacerbates this risk. A major unknown factor of spores is likely to be the inherently heterogeneous spore protein composition. The proteomics methods discussed here help in broadening the knowledge about spore structure and identification of putative target proteins from spores of different spore formers. Approaches to synchronize Bacillus subtilis spore formation, and to analyze spore proteins as well as the physiology of spore germination and outgrowth are also discussed. Live-imaging and fluorescence microscopy techniques discussed here allow analysis, at single cell level, of the 'germinosome', the process of spore germination itself, spore outgrowth and the spore intracellular pH dynamics. For the latter, a recently published improved pHluorin (IpHluorin) under control of the ptsG promoter is applicable. While the data obtained from such tools offers novel insight in the mechanisms of bacterial spore awakening, it may also be used to probe candidate antimicrobial compounds for inhibitory effects on spore germination and strengthen microbial risk assessment.

Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications.

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca2+ measurements) and FRAP imaging.

Developmental trajectories of structural and pragmatic language skills in school-aged children with Williams syndrome.

BACKGROUND: This study aimed to compare developmental courses of structural and pragmatic language skills in school-aged children with Williams syndrome (WS) and children with idiopathic intellectual disability (IID). Comparison of these language trajectories could highlight syndrome-specific developmental features. METHOD: Twelve monolingual Dutch-speaking children with WS aged 5.10 to 13.3 years were assessed by means of standardised structural language tests measuring receptive and expressive vocabulary and sentence comprehension and production. Pragmatic language was evaluated by means of an expressive referential communication task and a retelling test. All of these language abilities were re-evaluated with the same measures after a period of 18 to 24 months. Performance was compared to 12 children with IID pairwise matched for chronological age (CA) and non-verbal fluid reasoning (Gf) at Time 1. Non-verbal mental age (NVMA) was taken into account when delineating developmental trajectories. RESULTS: Children with WS outperformed children with IID on expressive vocabulary development. In contrast, sentence comprehension was significantly poorer than in children with IID at the second time point. Increased variability and rather poor performance on pragmatic language tasks were demonstrated in the WS group. Irrelevant and off-topic extraneous information transfer continued to be a syndrome-specific characteristic of children with WS. CONCLUSION: The data provide new insights into diverging developmental trajectories across language domains. Expressive structural language skills tend to progress more rapidly than receptive language skills in children with WS causing more distinctive language profiles over time. Some children with WS seem to benefit from the growth in expressive structural language abilities to enhance their expressive pragmatic language skills, while in some others these abilities remain challenging. This study highlights the need for continued follow-up of language challenges in WS and for a dynamic and individualised interventional approach.

Living with locked-in syndrome: an explorative study on health care situation, communication and quality of life.

PURPOSE: to explore and describe the health care situation, the use of aids, the way of communicating and the quality of life of Locked-in syndrome patients in Flanders (Belgium) and to collect information on their fulfilled and unfulfilled needs. METHOD: in depth interviews with eight LIS-patients by means of an extensive questionnaire consisting of five parts: (i) general information and medical history, (ii) health care, rehabilitation and follow-up, (iii) speech and communication, (iv) quality of life, (v) needs and problems experienced. RESULTS: the patients' condition, mostly caused by ischemic stroke, persisted for a mean period of 6 years 8 months. Their mean age was 41;10 years. At the moment of our study all the patients were living at home. Care was provided by an extensive care team. Some recovery of head and neck movements was mentioned, recovery of upper and lower limb mobility however was very limited. Most patients use an alphabet system to communicate, all of them had access to and made use of a PC with internet connection. Except for the domain of physical functioning, the quality of life scores of our patient group are rather high. As for unfulfilled needs, half of the patients experience a lack of information on their condition and a lack of appropriate information on (communication) aids. CONCLUSION: most results seem to be in line with those of other studies, though larger scale and follow-up studies are needed to confirm these findings.

The effects of deformation, ischemia, and reperfusion on the development of muscle damage during prolonged loading.

Deep tissue injury (DTI) is a severe form of pressure ulcer where tissue damage starts in deep tissues underneath intact skin. In the present study, the contributions of deformation, ischemia, and reperfusion to skeletal muscle damage development were examined in a rat model during a 6-h period. Magnetic resonance imaging (MRI) was used to study perfusion (contrast-enhanced MRI) and tissue integrity (T2-weighted MRI). The levels of tissue deformation were estimated using finite element models. Complete ischemia caused a gradual homogeneous increase in T2 (∼20% during the 6-h period). The effect of reperfusion on T2 was highly variable, depending on the anatomical location. In experiments involving deformation, inevitably associated with partial ischemia, a variable T2 increase (17-66% during the 6-h period) was observed reflecting the significant variation in deformation (with two-dimensional strain energies of 0.60-1.51 J/mm) and ischemia (50.8-99.8% of the leg) between experiments. These results imply that deformation, ischemia, and reperfusion all contribute to the damage process during prolonged loading, although their importance varies with time. The critical deformation threshold and period of ischemia that cause muscle damage will certainly vary between individuals. These variations are related to intrinsic factors, such as pathological state, which partly explain the individual susceptibility to the development of DTI and highlight the need for regular assessments of individual subjects.

Ischemia-reperfusion injury in rat skeletal muscle assessed with T2-weighted and dynamic contrast-enhanced MRI.

Pressure ulcers are localized areas of soft tissue breakdown due to mechanical loading. Susceptible individuals are subjected to pressure relief strategies to prevent long loading periods. Therefore, ischemia-reperfusion injury may play an important role in the etiology of pressure ulcers. To investigate the inter-relation between postischemic perfusion and changes in skeletal muscle integrity, the hindlimbs of Brown Norway rats were subjected to 4-h ischemia followed by 2-h reperfusion. Dynamic contrast-enhanced MRI was used to examine perfusion, and changes in skeletal muscle integrity were monitored with T2-weighted MRI. The dynamic contrast-enhanced MRI data showed a heterogeneous postischemic profile in the hindlimb, consisting of areas with increased contrast enhancement (14-76% of the hindlimb) and regions with no-reflow (5-77%). For T2, a gradual increase in the complete leg was observed during the 4-h ischemic period (from 34 to 41 msec). During the reperfusion phase, a heterogeneous distribution of T2 was observed. Areas with increased contrast enhancement were associated with a decrease in T2 (to 38 msec) toward preischemic levels, whereas no-reflow areas exhibited a further increase in T2 (to 42 msec). These results show that reperfusion after prolonged ischemia may not be complete, thereby continuing the ischemic condition and aggravating tissue damage.

Noise effects and filtering in controlled light exposure microscopy.

Phototoxicity and photobleaching are major limitations of fluorescence live-cell microscopy. A straightforward way to limit phototoxicity and photobleaching is reduction of the excitation light dose, but this causes loss of image quality. In confocal fluorescence microscopy, the field of view is illuminated uniformly whereas in controlled light exposure microscopy, illumination is controlled per pixel on the basis of two illumination strategies. The controlled light exposure microscopy foreground strategy discriminates between bright and weak foreground. Bright foreground pixels are illuminated with a reduced light dose resulting in limited excitation of fluorophores and consequently limited phototoxicity and photobleaching. The controlled light exposure microscopy background strategy discriminates between foreground and background. Pixels that are judged to be background are also illuminated with a reduced light dose. The latter illumination strategy may introduce artefacts due to the stochastic character of photon flow. These artefacts are visible as erratic 'darker pixels' in the foreground with a lower pixel value than the neighbouring pixels. This paper describes a special adaptive image processing filter that detects and corrects most of the 'darker pixels'. It opens the possibility to use controlled light exposure microscopy even in high noise (low signal to noise ratio) imaging to further reduce phototoxicity and photobleaching.

Four-dimensional telomere analysis in recordings of living human cells acquired with controlled light exposure microscopy.

Telomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their three-dimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non-random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere-binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.

Evaluation of quality of life in people with aphasia using a Dutch version of the SAQOL-39.

PURPOSE: To examine the quality of life (QoL) of people with aphasia and to study the influence of variables such as age, time post onset and (degree of) social support on the QoL of aphasic persons. METHOD: We compared the scores of an aphasic population (N = 43) with those of a healthy control group (N = 43) and of a group of patients with brain lesions without neurogenic communication disorders (N = 43) on a Dutch version of the Stroke and Aphasia Quality of Life-scale (SAQoL-39) and on a social support questionnaire. In half of the aphasic group, the SAQoL-39 was re-administered 8 months after the first testing. RESULTS: People with aphasia obtained significantly lower scores for QoL measures compared with both other groups. Especially, communicative and psychosocial factors seem to influence these results. Older people with aphasia scored lower than younger persons and women tend to evaluate their QoL somewhat more negatively than men. Persons who had aphasia for more than 6 months tended to have higher QoL-scores compared with those who had become aphasic more recently. After 8 months, the retested group scored significantly higher on communication and on psychosocial functioning than on first testing. CONCLUSIONS: Gathering information on QoL after suffering from stroke and from aphasia can lead to a better understanding of the problems involved. The clinical use of instruments such as the SAQoL-39 can probably contribute to a more patient oriented rehabilitation, whereby the focus not only lie in improving linguistic skills but also on reducing the impairments and the handicaps that accompany aphasia and thus on increasing QoL.

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle.

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics.

Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy.

Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.

Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. METHODS: A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. RESULTS: In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. CONCLUSIONS: Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.

Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.

Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (CLEM), a simple imaging approach that reduces photobleaching and phototoxicity two- to tenfold, depending on the fluorophore distribution in the object. By spatially controlling the light-exposure time, CLEM reduces the excitation-light dose without compromising image quality. We show that CLEM reduces photobleaching sevenfold in tobacco plant cells expressing microtubule-associated GFP-MAP4 and reduces production of reactive oxygen species eightfold and prolongs cell survival sixfold in HeLa cells expressing chromatin-associated H2B-GFP. In addition, CLEM increases the dynamic range of the fluorescence intensity at least twofold.

SISL (ScreeningsInstrument Schisis Leuven): assessment of cleft palate speech, resonance and myofunction.

This paper presents an assessment protocol for the evaluation and description of speech, resonance and myofunctional characteristics commonly associated with cleft palate and/or velopharyngeal dysfunction. The protocol is partly based on the GOS.SP.ASS'98 and adapted to Flemish. It focuses on the relevant aspects of cleft type speech necessary to facilitate assessment, adequate diagnosis and management planning in a multi-disciplinary setting of cleft team care.

A potential link between oral status and hearing impairment: preliminary observations.

Some previous studies suggest an association between tooth loss and hearing loss. The aim of this study is to assess the relation between oral status and hearing acuity. Forty-eight patients (mean age: 64.7 years) were allocated to four groups: one was wearing complete dentures in both jaws, another had shortened dental arches, a third had full dental arches in both jaws and the last lacked any occlusal stops (i.e. no occlusal vertical dimension, because of the absence of teeth or occlusal pairs). Audiological testing was performed in a noise-free chamber. Air and bone conduction were checked at different frequencies and the air-bone gap was determined. After correction for age and gender, a difference in air and bone conduction because of the oral status was found for low and for high frequencies while no significant differences were (P < 0.05) found for the air-bone gap. The number of teeth, number of occluding tooth pairs and presence or lack of occlusal vertical dimension, was significantly related to the gradient of hearing loss (P < 0.05). The discrepancy in hearing loss between complete denture wearers and patients without any occlusal vertical dimension, strengthens the hypothesis that it is the lack of the latter that is associated with hearing loss. At what level hearing loss occurs, needs further investigation.

Four-dimensional imaging of chromatin dynamics during the assembly of the interphase nucleus.

Large-scale chromatin organization is likely to play an important role in epigenetic control of gene expression. This implies that after mitosis the correct chromatin organization must be re-established in the nuclei of the two daughter cells. Here we analyze the dynamic behavior of chromatin during the transition from late anaphase to G1 in dividing HeLa cells, which express green fluorescent protein-tagged histone H2B. Time-lapse confocal microscopy was used to image the movement and the decondensation of chromatin as cell division progresses. Typically, time series of over 100 three-dimensional images (4D images) were collected, spanning a time period of up to three hours. Special care was taken to avoid photodamage, since cell cycle progression is exquisitely sensitive to photochemical damage. Quantitative analysis of the 4D images revealed that during the anaphase to G1 transition the movement of chromatin domains relative to other chromatin is remarkably limited. Chromatin dynamics can best be described as a radial expansion of the cluster of chromosomes that is present in late anaphase. We find that decondensation occurs in two phases. First a rapid decondensation by about a factor of two, followed by a slower phase in which part of the chromatin does not decondense any further, whereas the remaining chromatin decondenses further about two fold.

The influence of oral implant-supported prostheses on articulation and myofunction.

The aim of the present research was to assess articulation and myofunction in patients wearing fixed or removable prostheses supported by oral implants. 164 edentulous patients with implant supported prostheses were divided in four subgroups, dependent on their dental status, and compared to control groups of forty five subjects having a natural dentition. More than fifteen articulatory and myofunctional parameters were evaluated. The results showed that subjects with prostheses on implants tend to have more articulation problems than controls. Especially patients with a complete fixed prosthesis on implants in the upper jaw seemed to experience problems pronouncing /s/ and /z/. There also seemed to be influences of age and hearing factors.