On-line three-parameter data uptake, analysis, and display device for flow cytometry and other applications

Multiparameter flow cytometric measurements are of growing interest in the study of complex features of biological cells. With state of the art instrumentation, three-parameter (3-P) data handling is relatively complicated and time consuming and the display methods are not satisfactory. As an alternative, an interactive 3-P analyzing module, Cytomic 123 is described, which displays 3-P fields during and immediately after data uptake in the form of a cubic array of 32,768 channels. The fields can be randomly rotated by hardware and software. The event frequencies in the field are primarily visualized by brightness modulation of the display dots. Additionally, the display of the field may be confined to user selected ranges of event frequencies, which may also be superposed to mixed frequency displays. A set of preprogrammed functions is available for the following tasks: (a) uptake of 3-P histograms combined with on-line control of the transducer pulses, (b) automatic uptake of a series of 2-P time correlated histograms in the cube, (c) generation and numerical evaluation of sections and projections of cube histograms, (d) interactive generation and evaluation of spatial subfields for integration, or as sorting matrix by successive erosion of section planes, or reprojection of projection windows, and (e) isometric display of sections and projections and exchange of data sets with other Cytomic modules or other data systems, especially the Cytomic 12 module, whose 2-P capabilities can be used. The module is built with low cost Z80 microprocessor eurocards. A standard oscilloscope serves as a display unit.     

Strain and strain rate parametric imaging. A new method for post processing to 3-/4-dimensional images from three standard apical planes. Preliminary data on feasibility, artefact and regional dyssynergy visualisation

Background

We describe a method for 3-/4D reconstruction of tissue Doppler data from three standard apical planes, post processing to derived data of strain rate/strain and parametric colour imaging of the data. The data can be displayed as M-mode arrays from all six walls, Bull's eye projection and a 3D surface figure that can be scrolled and rotated. Numerical data and waveforms can be re-extracted.

Methods

Feasibility was tested by Strain Rate Imaging in 6 normal subjects and 6 patients with acute myocardial infarction. Reverberation artefacts and dyssynergy was identified by colour images. End systolic strain, peak systolic and mid systolic strain rate were measured.

Results

Infarcts were visualised in all patients by colour imaging of mid systolic strain rate, end systolic strain and post systolic shortening by strain rate. Reverberation artefacts were visible in 3 of 6 normals, and 2 of 6 patients, and were identified both on bull's eye and M-mode display, but influenced quantitative measurement. Peak systolic strain rate was in controls minimum -1.11, maximum -0.89 and in patients minimum -1.66, maximum 0.02 (p = 0.04). Mid systolic strain rate and end systolic strain did not separate the groups significantly.

Conclusion

3-/4D reconstruction and colour display is feasible, allowing quick visual identification of infarcts and artefacts, as well as extension of area of post systolic shortening. Strain rate is better suited to colour parametric display than strain.     

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections—achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.     

Scintigraphy,Ultrasound and CT Scanning of the Liver ,

Isotope examination of the liver depends on the functional activity of the liver phagocytes, while ultrasound and CT scanning display the anatomical structure. Cold areas on an isotope scan may be due to impaired function or space-occupying lesions. The method is nonspecific and does not differentiate between cysts, abscesses and metastases. Both ultrasound and CT scanning can differentiate space-occupying lesions with a high degree of accuracy so that both techniques can be used to improve the accuracy and specificity of the radioisotope examination. CT scanning of the liver is limited by relatively slow data acquisition and the small differences in X-ray absorption within soft tissues unless contrast agents are used. In comparison, ultrasonic data are rapidly collected and displayed and liver consistency is imaged without contrast media or ionizing radiation. Diffuse abnormalities of the liver, such as cirrhosis, cannot be detected by CT scanning but are apparent on ultrasound examination. In addition, equipment purchase and maintenance costs for ultrasound are a fraction of those for CT scanning. Experience to date at Yale indicates that ultrasound and CT scanning are complementary and supplementary to isotope examination of the liver but that ultrasound in most patients produces better resolution and enhanced tissue differentiation at considerably less cost.     

Display behaviour and dewlap colour as predictors of contest success in brown anoles

Many animals display visual signals in male contests for access to females and territories. These visual signals can be multimodal and stimulate different aspects of a signal receiver's visual system. Over two summers, we tested whether aspects of behaviour and dewlap colour might function as signals that predict contest success when males compete for access to either mates or territories in male brown anole lizards. We found that behaviour (PC1, a correlated composite of head‐bob, push‐up, and dewlap extension frequency) and an aspect of dewlap colour (PC3, the relative amounts of ultraviolet, yellow, orange, and red of the dewlap margin) were retained in the minimum adequate model predicting contest success across years and social contexts. Winners showed significant differences in behaviour (winners displayed more) and dewlap margin PC3 (winners had lower PC3 scores) compared to contest losers. These findings suggest that display behaviour and dewlap colour might serve as signals indicating a male's ability to win contests for access to females and territories. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 646–655.     

Open, reconfigurable cytometric acquisition system: ORCAS.

A digital signal processing (DSP)-based digital data acquisition system has been developed to support novel flow cytometry efforts. The system flexibility includes how it detects, captures, and processes event data. Custom data capture boards utilizing analog to digital converters (ADCs) and field programmable gate arrays (FPGA) detect events and capture correlated event data. A commercial DSP board processes the captured data and sends the results over the IEEE 1394 bus to the host computer that provides a user interface for acquisition, display, analysis, and storage. The system collects list mode data, correlated pulse shapes, or streaming data from a variety of detector types using Linux, Mac OS X, and Windows host computers. It extracts pulse features not found on commercial systems with excellent sensitivity and linearity over a wide dynamic range. List mode data are saved in FCS 3.0 formatted files while streaming or correlated waveform data are saved in custom format files for postprocessing. Open, reconfigurable cytometric acquisition system is compact, scaleable, flexible, and modular. Programmable feature extraction algorithms have exciting possibilities for both new and existing applications. The recent availability of a commercial data capture board will enable general availability of similar systems.     

Using digital photography to study animal coloration

In understanding how visual signals function, quantifying the components of those patterns is vital. With the ever-increasing power and availability of digital photography, many studies are utilizing this technique to study the content of animal colour signals. Digital photography has many advantages over other techniques, such as spectrometry, for measuring chromatic information, particularly in terms of the speed of data acquisition and its relatively cheap cost. Not only do digital photographs provide a method of quantifying the chromatic and achromatic content of spatially complex markings, but also they can be incorporated into powerful models of animal vision. Unfortunately, many studies utilizing digital photography appear to be unaware of several crucial issues involved in the acquisition of images, notably the nonlinearity of many cameras' responses to light intensity, and biases in a camera's processing of the images towards particular wavebands. In the present study, we set out step-by-step guidelines for the use of digital photography to obtain accurate data, either independent of any particular visual system (such as reflection values), or for particular models of nonhuman visual processing (such as that of a passerine bird). These guidelines include how to: (1) linearize the camera's response to changes in light intensity; (2) equalize the different colour channels to obtain reflectance information; and (3) produce a mapping from camera colour space to that of another colour space (such as photon catches for the cone types of a specific animal species).  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 211–237.     

Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows

The purpose of this study was to compare embryo quality of lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue beef cows (BB) and to identify factors that are associated with embryo quality in LHFC and NLHFH. After superovulation and embryo recovery at Day 7, embryos (n=727 from 47 LHFC, 27 NLHFH and 50 BB) were scored morphologically for quality, colour and developmental stage. Blood samples and data concerning parity, age, milk production and management were collected. Data were compared univariably between the three groups. A multivariable regression model was built with quality and colour of the LHFC and NLHFH embryos as dependent variables. Only 13.1% of LHFC embryos were categorized as excellent compared to 62.5% and 55.0% of the embryos in NLHFH and BB, respectively. Almost none of the NLHFH or BB embryos displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC embryos. Only 4% of all LHFC embryos reached blastocyst stage compared to 23.2% and 17.3% in NLHFH and BB. Based on the multivariable regression analysis, "physiological status" (lactating or not) together with the serum total protein concentration of LHFC and NLHFH, was significantly associated with embryo quality and colour. Thus, LHFC display an inferior embryo quality compared to NLHFH and BB. Producing milk or not seems to be significantly associated with embryo quality. Therefore, reduced embryo quality on Day 7 following AI, could be an important factor in the subfertility problem in modern high-yielding dairy cows.     

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.     

Eight-parameter PC-AT based flow cytometric data system

An 8-parameter flow cytometric data system is described using an IBM-AT compatible personal computer (PC) and a commercial analog to digital conversion (ADC) board. A dedicated pulse processing interface adapts the flow cytometric pulses to the ADC board and controls the number of parameters to be taken up and the trigger conditions. The trigger thresholds are automatically held at a level immediately above the noise level. For the timing of kinetic measurements a linear voltage ramp of adjustable rise time is available. A built-in precision voltage source can be used for an overall calibration. The data system is operated by software written in assembly language. Data may be collected and processed in 1-8-parameter listmode or 1-3-parameter histogram mode. Functions are available for graphical color displays, numerical integration, multiparameter gating, and printing.     

Effect of sex and bright coloration on survival and predator‐induced wing damage in an aposematic lantern fly with startle display

1. Aposematic coloration in prey promotes its survival by conspicuously advertising unpalatability to predators. Although classical examples of aposematic signals involve constant presentation of a signal at a distance, some animals suddenly display warning colours only when they are attacked. 2. Characteristics of body parts suddenly displayed, such as conspicuous coloration or eyespot pattern, may increase the survival of the prey by startling the predator, and/or by signalling unpalatability to the predators at the moment of attack. 3. The adaptive value of such colour patterns suddenly displayed by unpalatable prey has not been studied. We experimentally blackened the red patch in the conspicuous red–white–black hindwing pattern displayed by an unpalatable insect Lycorma delicatula White (Hemiptera: Fulgoridae) in response to predator's attack. 4. There was no evidence that the presence of the red patch increased prey survival over several weeks. We hypothesise that predators generalised from the red–white–black patches on the hindwings of unpalatable L. delicatula to any similar wing display as a signal of unpalatability. Because a higher proportion of males than females stay put at their resting sites, displaying their wings in response to repeated attacks by predators, wing damage was more frequent in males than in females. 5. To our knowledge, this is the first experimental test of an adaptive role of aposematic signals presented by unpalatable prey during sudden displays triggered by direct predatory attack.     

Wintering Snowy Owls Bubo scandiacus integrate plumage colour,behaviour and their environment to maximize efficacy of visual displays

For a comprehensive understanding of the evolution of animal signals, it is necessary to understand how the performance of visual displays is maximized to get the most possible attention from receivers. We assessed whether the white plumage of Snowy Owls Bubo scandiacus functioned as a social signal and, if so, how coloration and behavioural adaptations enhance signal efficacy. Signalling theory predicts that: (1) the colour properties of plumage should vary across the body, with the brightest parts being those involved in visual display performance; (2) specific displays calling attention to or enhancing detection or conspicuousness to conspecifics should be evident; and (3) location of the signallers should be such that signal efficacy is optimized. All three predictions were supported. The brightest areas of the plumage (particularly the face, throat and upper breast) were always unspotted, and white is particularly effective in open habitats characteristic of this species. The birds displayed a specific posture and orientated toward the sun preferentially on sunny days, and Owls with the whitest (least spotted) plumage displayed more and signalled more frequently from perches on the ground, where albedo from the snow may enhance the visual display. Snowy Owls integrate coloration, behaviour and environment through habitat selection to maximize the efficacy of their visual displays.     

Decoupling vigour and quality in the autumn colours game: Weak individuals can signal, cheating can pay

According to the coevolution theory, autumn colours are a warning signal to insects, signalling the level of chemical defences or availability of nutrients. Because in the original model tree vigour and defences were positively correlated, it is not clear whether signalling would still be stable when they are decoupled, and the fact that weak trees often display bright autumn colours is usually presented as evidence against the coevolution theory. I show that in a theoretical model of insect-tree coevolution, signalling is still stable when vigour and defences are decoupled. Weak trees can signal. Moreover, partial cheating is possible. The different equilibria depend on the importance of vigour and defences against insect attack, of vigour in the production of the signal, and of pleiotropic effects between colour and defences. These results provide precise predictions that can be used for planning future empirical test.     

EpiCollect: Linking Smartphones to Web Applications for Epidemiology,Ecology and Community Data Collection

Background

Epidemiologists and ecologists often collect data in the field and, on returning to their laboratory, enter their data into a database for further analysis. The recent introduction of mobile phones that utilise the open source Android operating system, and which include (among other features) both GPS and Google Maps, provide new opportunities for developing mobile phone applications, which in conjunction with web applications, allow two-way communication between field workers and their project databases.

Methodology

Here we describe a generic framework, consisting of mobile phone software, EpiCollect, and a web application located within www.spatialepidemiology.net. Data collected by multiple field workers can be submitted by phone, together with GPS data, to a common web database and can be displayed and analysed, along with previously collected data, using Google Maps (or Google Earth). Similarly, data from the web database can be requested and displayed on the mobile phone, again using Google Maps. Data filtering options allow the display of data submitted by the individual field workers or, for example, those data within certain values of a measured variable or a time period.

Conclusions

Data collection frameworks utilising mobile phones with data submission to and from central databases are widely applicable and can give a field worker similar display and analysis tools on their mobile phone that they would have if viewing the data in their laboratory via the web. We demonstrate their utility for epidemiological data collection and display, and briefly discuss their application in ecological and community data collection. Furthermore, such frameworks offer great potential for recruiting ‘citizen scientists’ to contribute data easily to central databases through their mobile phone.     

Genetics of polymorphism in the land snail,Cepaea nemoralis

The results of 40 single-pair matings of the landsnailCepaea nemoralis, mostly concerning the rarer banding patterns are presented. A total of 3678 F₁ individuals were obtained, 2315 of which were scored when adult, the remainder as juveniles.The presence or absence of band 3 in banded snails does not seem to be genetically determined. It is shown that several banding patterns (e.g. 12300, 02300, 00340, 003300, 00305) should be classified as 00300. The presence or absence of extra (satellite-) bands seems to be multifactorially controlled. It is suggested that pattern 10345 is dominant over 12345 and that it is determined either by an allele of the threebanded locus, recessive to pattern-allele 00345, or by a different locus but then epistatically suppressed by the gene for 00345. Pattern 02345 in some cases is genetically 00345 and in other cases 12345. Fusion of bands 4 and 5 is multifactorially controlled, and is perhaps influenced by, or linked to, the loci for colour and three-bandedness. Fusion of bands 1 and 2 seems to be also multifactorially controlled. This fusion is strongly correlated with fusion (45), and is independent of colour. Pale is recessive to dark body colour. The latter occurs in several shades which are multifactorially controlled. Reddish body colour is recessive to yellowish, and the gene concerned is linked with that for shell colour. Variation in the expression of dominance was found at the loci for 00300, 00345, 10345, reddish body colour and pale body colour.     

A generalised mimicry system involving angiosperm flower colour, pollen and bumblebees’ innate colour preferences

Flower colour is a major advertisement signal of zoophilous plants for pollinators. Bees, the main pollinators, exhibit innate colour preferences, which have often been attributed to only one single floral colour, though most flowers display a pattern of two or several colours. The existing studies of floral colour patterns are mostly qualitative studies. Using a model of bee colour vision we quantitatively investigate two questions: whether or not component colours of floral colour patterns may mimic pollen signals, and whether or not bumblebees exhibit innate preferences for distinct parameters of naturally existing floral colour patterns. We analysed the spectral reflectances of 162 plant species with multicoloured flowers and inflorescences, distiniguishing between inner and outer colours of floral colour patterns irrespective of the particular structures so coloured.We found that:– The inner colour of radially symmetrical flowers and inflorescences and of zygomorphic flowers appears less diverse to bees than the peripheral colour.– The inner colour of most radial flowers and inflorescences as well as the inner colour of a large number of non-related zygomorphic flowers appears to bees to be very similar to that of pollen.– Bumblebees (Bombus terrestris) exhibit innate preferences for two-coloured over single-coloured dummy flowers in a spontaneous choice test.– Bumblebees exhibit innate preferences for dummy flowers with a large over those with a small centre area.– Bumblebees exhibit innate preferences for dummy flowers with a centre colour similar to that of pollen over those with another centre colour.Our findings support the hypotheses that the inner component of floral colour patterns could be interpreted as a generalised and little recognised form of mimicry of the colour of visually displayed pollen, that bumblebees exhibit innate preferences regarding colour and size parameters of floral colour patterns, and that these correspond to visually displayed pollen. These findings together suggest a prominent role of floral colour patterns in advertisement to and guidance of naive flower visitors.     

Quantification of Clustering in Joint Interspike Interval Scattergrams of Spike Trains

Joint interval scattergrams are usually employed in determining serial correlations between events of spike trains. However, any inherent structures in such scattergrams that are often seen in experimental records are not quantifiable by serial correlation coefficients. Here, we develop a method to quantify clustered structures in any two-dimensional scattergram of pairs of interspike intervals. The method gives a cluster coefficient as well as clustering density function that could be used to quantify clustering in scattergrams obtained from first- or higher-order interval return maps of single spike trains, or interspike interval pairs drawn from simultaneously recorded spike trains. The method is illustrated using numerical spike trains as well as in vitro pairwise recordings of rat striatal tonically active neurons.     

Chameleons communicate with complex colour changes during contests: different body regions convey different information

Many animals display static coloration (e.g. of feathers or fur) that can serve as a reliable sexual or social signal, but the communication function of rapidly changing colours (as in chameleons and cephalopods) is poorly understood. We used recently developed photographic and mathematical modelling tools to examine how rapid colour changes of veiled chameleons Chamaeleo calyptratus predict aggressive behaviour during male–male competitions. Males that achieved brighter stripe coloration were more likely to approach their opponent, and those that attained brighter head coloration were more likely to win fights; speed of head colour change was also an important predictor of contest outcome. This correlative study represents the first quantification of rapid colour change using organism-specific visual models and provides evidence that the rate of colour change, in addition to maximum display coloration, can be an important component of communication. Interestingly, the body and head locations of the relevant colour signals map onto the behavioural displays given during specific contest stages, with lateral displays from a distance followed by directed, head-on approaches prior to combat, suggesting that different colour change signals may evolve to communicate different information (motivation and fighting ability, respectively).     

Eddy-correlation system for long-term monitoring of fluxes of heat, water vapour and CO2

An eddy-correlation system is presented that was designed with special focus on long-term measurements of turbulent fluxes in the atmospheric boundary layer. It consists of a SOLENT sonic anemometer, a fast temperature sensor, and a LI-COR LI 6262 closed-path infrared gas analyser. The use of a fast temperature sensor turned out to be necessary because of errors in the sound virtual temperature measured by the sonic anemometer at high wind speeds. The components are combined with special attention paid to protection against lightning and other environmental stresses. The data acquisition program SOLCOM runs on standalone systems or in a network environment and performs ‘quasi on-line’ data processing, on-line graphical display of single data and fluxes, and on-line correction of the raw data. Raw data can be stored continuously on DAT tapes. All data handling can be done by remote access, thus only a minimum amount of m situ maintenance is required. Power spectra of vertical and longitudinal wind speed, air temperature, air humidity and carbon dioxide concentration showed to follow the -2/3 law quite well. There was some noise in the high frequency range of the carbon dioxide spectrum. However, the corresponding cross spectra with the vertical wind component showed less deviation from a straight line in the high frequency range. The sum of convective heat fluxes and soil heat flux showed good agreement with the measured net radiation for several months and it was concluded that the system described here constitute a good platform for long-term flux measurements over forest.