Optimization of laser beams in FRAP experiments of microscopical objects

In fluorescence recovery after photobleaching (FRAP) experiments the sample is irradiated on a small spot, the diameter of which must be related to the sample size and the diffusion constant to be measured. This paper considers the conventional FRAP set-up where a laser beam is directed through a microscope vertical illuminator to the sample. The requirements of an intermediate optical system producing a Gaussian beam with a waist of given radius in the microscope object plane are considered, and the optical parameters determined.     

Mechanical device for morphological and orientation analysis of biological structures

A simple mechanical device for different kinds of morphological analysis is suggested. It consists of a binocular microscope, a luminiscent illuminator, a coordination table with a disc-chuck (0-360 degrees) and a nest for disposing the test systems and the light or electronmicroscopic negatives (6 x 9 and 9 x 12). The analysis is performed by means of visual scanning of the negatives, by counting the number of crossings of the structure with the test system knots and by registration of the measurements by a key recorder. For orientation analysis the microscope is supplied with a goniometer (0-360 degrees) and an oculometer with a cross of filaments.     

An Illuminator for Ultramicrotome Knife Orientation and Block Approach

The construction and operation of a simple, inexpensive illuminator that produces high quality illumination of the ultramicrotome knife edge and the edge to block face gap resembling dark field is described. Use of the illuminator greatly speeds knife adjustment and reduces the likelihood of specimen or knife edge damage. The illuminator uses a grain-of-wheat light bulb and an adjustable bulb holder fashioned from bent paper clips. The holder permits both lateral and axial adjustment of the bulb position, which is necessary to achieve satisfactory illumination with different specimens and knives. The illuminator, with slight modification, can be adapted for use on any ultramicrotome.     

Surface extensions of 3T3 cells towards distant infrared light sources

Using a specially designed phase-contrast light microscope with an infrared spot illuminator we found that approximately 25% of 3T3 cells were able to extend pseudopodia towards single microscopic infrared light sources nearby. If the cells were offered a pair of such light sources next to each other, 47% of the cells extended towards them. In the latter case 30% of the responding cells extended separate pseudopodia towards each individual light source of a pair. The strongest responses were observed if the infrared light sources emitted light of wavelengths in the range of 800-900 nm intermittently at rates of 30-60 pulses per min. The temperature increases of the irradiated spots can be shown to be negligible. The results suggest that the cells are able to sense specific infrared wavelengths and to determine the direction of individual sources.     

A microbial chip combined with scanning electrochemical microscopy.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was prepared by spotting a suspension of Escherichia coli on a polystyrene substrate by using a glass capillary pen. The respiration activity of the E. coli spot was imaged with SECM by mapping the oxygen concentration around the spot. The SECM images of the microbial chips clearly showed spots with lower reduction currents, indicating that E. coli in the spots uptake oxygen by respiration. The bactericidal effects of antibiotics (streptomycin and ampicillin) were measured using the E. coli-based microbial chip, and discussed in comparison with the minimum inhibitory concentration (MIC) determined by an agar plate dilution method.     

MEDLINE摘要本地下载与更新及癌基因表达数据的文本挖掘(英文)

近年来对生物医学文献的文本挖掘在功能基因组学研究中得到了广泛开展。为了更好的检索MEDLINE摘要,本文介绍利用Unix文本过滤命令实现了对摘要的自动下载和更新。同时,对癌基因表达数据,如癌的种类,癌基因表达情况,及与p53基因的关联等进行了初步的文本挖掘分析。     

A Use-Time Recorder for Application in Critical Microscopic Illumination

The recorder, controlled by a keyed lockable switch which activates a sealed lapsed-time meter and energizes a power outlet, is placed between the power mains electrical source and the illuminator, or other equipment, to be monitored. Electrical connection between the latter and the power outlet of the recorder is made with paired nonstandard locking connectors; one connector being attached to the power outlet and the other replacing the standard male convience plug on the power cord of the illuminator. The lockable switch maintains absolute control over use of the monitored equipment while the time meter provides an accurate record of use-time. Bypass of the locked recorder is prevented by the non-standard connectors. Total fabrication costs of the recorder are less than $25.00.     

An Evaluation of Exercise Pen Use by Circus Tigers (Pathera tigris tigris)

This study quantified the behavior of 11 tigers during periodic access to an exercise pen throughout the day and night. The study determined the amount of time spent in the pen and the percentage of time spent performing stereotypic pacing, normal locomotor behavior, and lying down while in the pen. Average access to the exercise pen was 10 hr 49 min overnight and 5 hr 30 min during the day. At night, the tigers spent 29.1% of their time in the exercise pen, during which they paced 19.6% and performed normal locomotor behavior for 23.1% of that time. By day, they spent 40.4% of their time in the exercise pen, during which they paced 10.0% and performed normal locomotor behavior 35.7% of that time. The tigers spent the rest of the time in the pen lying down. Overall, tigers will utilize an exercise pen, spending a greater percentage of time in the pen during the day than at night and also performing less stereotyped pacing than at night.     

A laser-light pulse counting method for automatic and sensitive counting of bacteria stained with acridine orange

A laser-light pulse counting method was developed and investigated for its ability automatically to count acridine orange-stained bacteria and microcolonies of bacteria on the surface of polycarbonate membrane filters. The system consisted of an Argon laser which delivered a 5 μm diameter spot of coherent 488 nm wavelength light which was raster scanned across a 5×2 mm area of the membrane. Fluorescence pulses of orange light ( ca 650 nm) were detected via a dichroic mirror and barrier filter with a photomultiplier tube. For microcolony preparations a good relationship between the number of light pulses and cell density down to below 10²/ml was observed, but at lower cell densities counting was not reliable, probably because of fluorescent debris. The method was also able to count single cells, but background auto-fluorescence and photo-bleaching produced a high and variable background signal in some samples.     

Differential cell analysis of cytocentrifuged bronchoalveolar fluid samples affected by the area counted

OBJECTIVE: To investigate variations in the differential cell counts between the quadrants of cytocentrifuged bronchoalveolar lavage (BAL) fluid preparations and to evaluate the diagnostic impact of these differences in interstitial lung diseases (ILD). STUDY DESIGN: BAL fluid samples obtained from 30 patients suspected of having ILD or pneumonia were cytocentrifuged and additionally stained with May-Grünwald-Giemsa stain. Two observers differentiated 200 cells in each quadrant as well as in a circular pattern around the center of the cytocentrifuge spot. RESULTS: Lymphocytes and alveolar macrophages were not randomly distributed on the cytocentrifuge spot. Ten samples of patients with histologically confirmed ILD were selected to test the diagnostic impact using a validated computer program. The predicted diagnosis did not correspond to the histologic diagnosis for one quadrant from 1 of these 10 samples (sarcoidosis instead of idiopathic pulmonary fibrosis), whereas the differential cell counts performed around the center of the cytocentrifuge spot provided the correct diagnosis in all cases. CONCLUSION: BAL fluid differential cell counts varied between the quadrants of the cytocentrifuge spot. The center of the cytocentrifuge spot appeared to be the most reliable area. Therefore, cell counting is recommended in a circular pattern around the center of the cytocentrifuge spot.     

Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons

Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.     

Optical readout of gold nanoparticle-based DNA microarrays without silver enhancement

We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on "Laser-Induced Scattering around a NanoAbsorber". It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.     

Handwriting: Hand–pen contact force synergies in circle drawing tasks

This study investigated synergistic actions of hand–pen contact forces during circle drawing tasks in three-dimensional (3D) space. Twenty-four right-handed participants drew thirty concentric circles in the counterclockwise (CCW) and clockwise (CW) directions. Three-dimensional forces acting on an instrumented pen as well as 3D linear and angular positions of the pen were recorded. These contact forces were then transformed into the 3D radial, tangential, and normal force components specific to circle drawing. Uncontrolled manifold (UCM) analysis was employed to calculate the magnitude of the hand–pen contact force synergy. Three hypotheses were tested. First, hand–pen contact force synergies during circle drawing are dependent on the angular position of the pen tip. Second, hand–pen contact force synergies are dependent on force components in circle drawing. Third, hand–pen contact force synergies are greater in CCW direction than CW direction. The results showed that the strength of the hand–pen contact force synergy increased during the initial phase of circle drawing and decreased during the final phase. The synergy strength was greater for the radial and tangential components as compared to the normal component. Also, the circle drawing in CW direction was associated with greater hand–pen contact force synergy than the CCW direction. The results of this study suggest that the central nervous system (CNS) prioritizes hand–pen contact force synergies for the force components (i.e., radial and tangential) that are critical for circle drawing. The CNS modulates hand–pen contact force synergies for preparation and conclusion of circle drawing, respectively.     

OBSERVATIONS ON NARCISSUS LEAF SCORCH IN SOUTH-WEST ENGLAND

Narcissus leaf scorch caused by Stagonospora Curtisii (Berk.) Sacc. is very common in south-west England, where it causes economic damage arising from flower spot and leaf decay. Information is given as to the varying susceptibility of many different varieties, a comparative measure of which is obtained by counting the primary infections.
No certain control measures can be recommended, but results of experiments on bulb disinfection and on spraying are given.     

Scent of a Ewe: transmission of a social cue by conspecifics affects sexual performance in male sheep.

Unlike males from other domestic species, domestic rams (Ovis aries) are not sexually stimulated, as determined by measuring sexual performance, following the opportunity to watch a copulating pair. Previously, we reported that aspects of ram sexual performance were improved when rams interacted with a male conspecific that had mated an estrous ewe. Whether the cues were gender-, estrous state-, or behavior-related was tested in this study. Sexually experienced rams were exposed to male pen mates that had interacted with an estrous ewe, a non-estrous ewe, an estrous ewe with a cloth perineal patch, or a ram, or that had been placed alone in a small pen. The rams were then tested for sexual performance. Rams performed more olfactory investigative behaviors toward pen mates that had interacted with a ewe, regardless of her estrous state, than toward a pen mate that had been exposed to another male. Rams exposed to pen mates that had interacted with a ewe also had shorter postejaculatory and interejaculation intervals and subsequently achieved more ejaculations in standardized sexual performance tests. Results from this experiment confirm that male-male interactions affect sexual performance in male sheep and that olfactory cues likely account for the transfer of information among individuals.     

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.     

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.     

The potential of a light spot,heat area,and novel object to attract laying hens and induce piling behaviour

Piling behaviour of laying hens often results in smothering or death due to suffocation. Mechanisms leading to piling are not yet understood though various potential factors have been suggested. In this experimental study, we predicted that the presence of a light spot, a novel object (metal foil), or a heat area within animal pens would increase animal numbers around the stimulus leading to piling behaviour. We presented the cues in a 4 × 2 Latin-square design in eight identical experimental pens including each 55 Lohmann Selected Leghorn hens. The cues were presented in two test areas per pen, at two bouts per day in the morning, consecutively for 5 days, over four periods (age: 20, 22, 24, 26 weeks). Each pen received a cue and control condition simultaneously (test areas without cue presentation) once. For a bout, each cue was presented for 35 min except for the light spot where the duration was 10 min. Birds’ responses to the cues during bout and non-bout times were video recorded and analysed for the first bout of each period. To assess the cues’ attractiveness, the number of hens during bout times was counted at predefined times within the test and control areas. To assess the cues’ effects on piling, we described piling behaviour (pile number, duration, animal numbers, trigger) in control and test areas during bout times. Furthermore, we described piling behaviour during bout times and non-bout times on the first day of the first period and fourth period. The best model explaining the number of hens included the interactions of treatment and bout time, and treatment and area. Over the bout’s time course, more hens were attracted to the light spot compared to the control condition, and more to test areas compared to control areas. In the novel object condition, more hens were drawn to the test areas compared to the control areas. Hens were not attracted to the heat area. Piling in bout times was observed twice when hens pecked at the novel object. During non-bout times, piling behaviour occurred frequently at midday and in the late morning compared to the afternoon, mostly in corners and mainly preceded by the mutual attraction of hens. Overall, hens were attracted to light spots and less so to the novel object though neither reliably induced piling behaviour. The occurrence of piling behaviour in non-bout times shows that more work is needed to understand mechanisms eliciting piling behaviour.     

A survey on management and housing of peri-parturient dairy cows and their calves

Housing and management around the time of calving impact dairy cow behaviour, health and welfare, but little is known about current practice. The aim was to provide an overview of current calving practice and the study describes the main calving housing and management based on replies to an online questionnaire by 42 dairy cattle experts in 28 countries, or regions, in Europe, Canada and USA. The survey suggests that in the majority of countries and regions included in this study, dairy cows typically calve in indoor calving facilities; either individual pens, group pens or a system where the cow is moved from a group pen into an adjacent individual pen before calving. Regarding individual calving pens, the survey suggests that in the majority of countries and regions included in this study, most pens have open sides and offer cows no opportunity to isolate, although research shows that a secluded corner of an individual pen creates a preferred calving site. Further, the survey suggests that when cows calve in individual calving pens or tie-stalls, they are often moved there with signs of imminent calving, although research shows that this practice increases the duration of calving and it is recommended to move cows before their expected calving time. Regarding group pens, none of the 42 respondents replied that group pens typically offer cows the opportunity to isolate at calving. Recent research suggests that when cows calve in a secluded area of a group calving pen, this reduces the risk of failure of passive transfer of immunity. Regarding calving facilities where group pens are combined with adjacent individual pens, this was reported to be the most typical in 10 of the 24 countries and regions with indoor calving sections covered by the survey. The same concerns regarding when the cow is moved from the group pen to an individual pen apply, as outlined above. Irrespective of pen type, the most frequently reported surface was deep bedded straw and the most frequent type of separation between pens was open sides. Cow-calf separation within 12 hours of birth, and thereafter individual housing of calves combined with milk feeding via a teat bucket or bar was indicated the most frequent management. The survey presents experts’ evaluations of current practice of housing and management of peri-parturient dairy cows and their calves, and suggests that there is a discrepancy between current calving management and housing and recommendations based on recent research.     

Review: Factors affecting fouling in conventional pens for slaughter pigs

This review assesses factors affecting fouling in conventional pens for slaughter pigs. Fouling of the pen happens when pigs change their excretory behaviour from occurring in the designated dunging area to the lying area. This can result in a lower hygiene, bad air quality, extra work for the farmer, disturbance of the pigs’ resting behaviour and an increase in agonistic interactions. A systematic search was conducted and results narrowed down to 21 articles. Four factors were found to affect fouling directly: insufficient space allowance, the flooring design of the pen, the thermal climate and pigs’ earlier experience. Further, these primary factors are affected by secondary factors such as the shape of the pen, the weight of the pigs and especially the heat balance of the pigs, which is affected by several tertiary factors including, for example, temperature, humidity and draught. Results indicate that the most important factor to control when trying to prevent fouling of a pen is the pen climate. An appropriate climate may be accomplished through floor cooling in the designated lying area, sprinklers above the designated dunging area and by ensuring a more optimal ambient temperature curve that also fits the weight of the pigs in different stages of the production. All in all, fouling of the pen in conventional slaughter pigs is a multifactorial problem, but it is important to focus on increasing the comfortability, and especially the climate, of the designated lying area.