Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy.

The authors have developed a live-cell multimodality microscope combining epifluorescence with digital holographic microscopy; it has been implemented with a decoupling procedure allowing to separately measure from the quantitative phase important cell parameters including absolute volume, shape and integral intracellular refractive index. In combination with the numerous different specific fluorescent cellular probes, this multimodality microscopy can address important issues in cell biology. This is demonstrated by the study of intracellular calcium homeostasis associated with the change in cell volume, which play a critical role in the excitotoxicity-induced neuronal death.

Axial sub-nanometer accuracy in digital holographic microscopy

We present state-of-the-art dual-wavelength digital holographic microscopy (DHM) measurement on a calibrated 8.9 nm high chromium thin step sample and demonstrate sub-nanometer axial accuracy. By using a modified DHM reference calibrated hologram (RCH) reconstruction method, a temporal averaging procedure and a specific dual-wavelength DHM arrangement, it is shown that specimen topography can be measured with an accuracy, defined as the axial standard deviation, reduced to at least 0.9 nm. Indeed for the first time to the best of our knowledge, it is reported that averaging each of the two wavefronts recorded with real-time dual-wavelength DHM can provide up to 30% spatial noise reduction for the given configuration. Moreover, the presented experimental configuration achieves a temporal stability below 0.8 nm, thus paving the way to Angström range for dual-wavelength DHM.

Reproducibility of species lists, visual cover estimates and frequency methods for recording high mountain vegetation

Question: When multiple observers record the same spatial units of alpine vegetation, how much variation is there in the records and what are the consequences of this variation for monitoring schemes to detect change? Location: One test summit in Switzerland (Alps) and one test summit in Scotland (Cairngorm Mountains). Method: Eight observers used the GLORIA protocols for species composition and visual cover estimates in percent on large summit sections (>100 m2) and species composition and frequency in nested quadrats (1 m2). Results: The multiple records from the same spatial unit for species composition and species cover showed considerable variation in the two countries. Estimates of pseudoturnover of composition and coefficients of variation of cover estimates for vascular plant species in 1m x 1m quadrats showed less variation than in previously published reports whereas our results in larger sections were broadly in line with previous reports. In Scotland, estimates for bryophytes and lichens were more variable than for vascular plants. Conclusions: Statistical power calculations indicated that, unless large numbers of plots were used, changes in cover or frequency were only likely to be detected for abundant species (exceeding 10% cover) or if relative changes were large (50% or more). Lower variation could be reached with the point methods and with larger numbers of small plots. However, as summits often strongly differ from each other, supplementary summits cannot be considered as a way of increasing statistical power without introducing a supplementary component of variance into the analysis and hence the power calculations.

Label-free cytotoxicity screening assay by digital holographic microscopy.

Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

Holographic collisions in confining theories

We study the gravitational dual of a high-energy collision in a confining gauge theory. We consider a linearized approach in which two point particles traveling in an AdS-soliton background suddenly collide to form an object at rest (presumably a black hole for large enough center-of-mass energies). The resulting radiation exhibits the features expected in a theory with a mass gap: late-time power law tails of the form t −3/2, the failure of Huygens" principle and distortion of the wave pattern as it propagates. The energy spectrum is exponentially suppressed for frequencies smaller than the gauge theory mass gap. Consequently, we observe no memory effect in the gravitational waveforms. At larger frequencies the spectrum has an upward-stairway structure, which corresponds to the excitation of the tower of massive states in the confining gauge theory. We discuss the importance of phenomenological cutoffs to regularize the divergent spectrum, and the aspects of the full non-linear collision that are expected to be captured by our approach.

Positional stability of holographic optical traps

The potential of digital holography for complex manipulation of micron-sized particles with optical tweezers has been clearly demonstrated. By contrast, its use in quantitative experiments has been rather limited, partly due to fluctuations introduced by the spatial light modulator (SLM) that displays the kinoforms. This is an important issue when high temporal or spatial stability is a concern. We have investigated the performance of both an analog-addressed and a digitally-addressed SLM, measuring the phase fluctuations of the modulated beam and evaluating the resulting positional stability of a holographic trap. We show that, despite imparting a more unstable modulation to the wavefront, our digitally-addressed SLM generates optical traps in the sample plane stable enough for most applications. We further show that traps produced by the analog-addressed SLM exhibit a superior pointing stability, better than 1 nm, which is comparable to that of non-holographic tweezers. These results suggest a means to implement precision force measurement experiments with holographic optical tweezers (HOTs).

Holographic optical tweezers combined with back-focal-plane displacement detection

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.

Automated segmentation of multiple red blood cells with digital holographic microscopy.

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

Effect of selective recording on estimates of heritability

En este trabajo se analiza el efecto de la selección de datos sobre las estimaciones de heredabilidad. Se estimó el valor de heredabilidad del tamaño de camada en una población porcina en la que los datos correspondientes a las cerdas más viejas eran una muestra seleccionada. Las estimaciones se obtuvieron usando distintos conjuntos de datos derivados de toda la información disponible. Esos conjunto de datos se compararon evaluando su capacidad predictiva. Se vio que las estimaciones de heredabilidad obtenidas utilizando todos los datos disponibles correspondían a valores infraestimados. También se simuló un carácter materno y se generó un conjunto de datos seleccionados eliminando aquellos correspondientes a las hembras sin padres conocidos. Distintos modelos, habitualmente empleados cuando no existe selección de registros, se consideraron para estimar el valor de heredabilidad. Los resultados mostraron que ninguno de esos modelos ofrecía estimaciones insesgadas. Sólo los modelos que tenían en cuenta el efecto de la selección sobre la media residual y la media y varianza genéticas ofrecían estimaciones poco sesgadas. Sin embargo, para poder aplicarlos se debe conocer la selección realizada. El problema de la selección de datos es difícil de abordar cuando se desconoce cual es el proceso de selección que se ha realizado en una población.

Phenomenological models of holographic superconductors and hall currents

We study general models of holographic superconductivity parametrized by four arbitrary functions of a neutral scalar field of the bulk theory. The models can accommodate several features of real superconductors, like arbitrary critical temperatures and critical exponents in a certain range, and perhaps impurities or boundary or thickness effects. We find analytical expressions for the critical exponents of the general model and show that they satisfy the Rushbrooke identity. An important subclass of models exhibit second order phase transitions. A study of the specific heat shows that general models can also describe holographic superconductors undergoing first, second and third (or higher) order phase transitions. We discuss how small deformations of the HHH model can lead to the appearance of resonance peaks in the conductivity, which increase in number and become narrower as the temperature is gradually decreased, without the need for tuning mass of the scalar to be close to the Breitenlohner-Freedman bound. Finally, we investigate the inclusion of a generalized ¿theta term¿ producing Hall effect without magnetic field.

Holographic isotropization linearized

The holographic isotropization of a highly anisotropic, homogeneous, strongly coupled, non-Abelian plasma was simplified in ref. [1] by linearizing Einstein"s equations around the final, equilibrium state. This approximation reproduces the expectation value of the boundary stress tensor with a 20% accuracy. Here we elaborate on these results and extend them to observables that are directly sensitive to the bulk interior, focusing for simplicity on the entropy production on the event horizon. We also consider next-to-leading-order corrections and show that the leading terms alone provide a better description of the isotropization process for the states that are furthest from equilibrium.