Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

Mental chronometry using latency-resolved functional MRI

Vascular responses to neural activity are exploited as the basis of a number of brain imaging techniques. The vascular response is thought to be too slow to resolve the temporal sequence of events involved in cognitive tasks, and hence, imaging studies of mental chronometry have relied on techniques such as the evoked potential. Using rapid functional MRI (fMRI) of single trials of two simple behavioral tasks, we demonstrate that while the microvascular response to the onset of neural activity is delayed consistently by several seconds, the relative timing between the onset of the fMRI responses in different brain areas appears preserved. We examined a number of parameters that characterize the fMRI response and determined that its onset time is best defined by the inflection point from the resting baseline. We have found that fMRI onset latencies determined in this manner correlate well with independently measurable parameters of the tasks such as reaction time or stimulus presentation time and can be used to determine the origin of processing delays during cognitive or perceptual tasks with a temporal accuracy of tens of milliseconds and spatial resolution of millimeters.

Real-time visualization of intracellular hydrodynamics in single living cells

Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics.

Dynamic contrast-enhanced magnetic resonance imaging reveals stress-induced angiogenesis in MCF7 human breast tumors.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T2-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.

A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.

The Mice at play in the CALIFA survey. A case study of a gas-rich major merger between first passage and coalescence

We present optical integral field spectroscopy (IFS) observations of the Mice, a major merger between two massive (≳10^11 M_⊙) gas-rich spirals NGC 4676A and B, observed between first passage and final coalescence. The spectra provide stellar and gas kinematics, ionised gas properties, and stellar population diagnostics, over the full optical extent of both galaxies with ~1.6 kpc spatial resolution. The Mice galaxies provide a perfect case study that highlights the importance of IFS data for improving our understanding of local galaxies. The impact of first passage on the kinematics of the stars and gas has been significant, with strong bars most likely induced in both galaxies. The barred spiral NGC 4676B exhibits a strong twist in both its stellar and ionised gas disk. The edge-on disk galaxy NGC 4676A appears to be bulge free, with a strong bar causing its “boxy” light profile. On the other hand, the impact of the merger on the stellar populations has been minimal thus far. By combining the IFS data with archival multiwavelength observations we show that star formation induced by the recent close passage has not contributed significantly to the total star formation rate or stellar mass of the galaxies. Both galaxies show bicones of high ionisation gas extending along their minor axes. In NGC 4676A the high gas velocity dispersion and Seyfert-like line ratios at large scaleheight indicate a powerful outflow. Fast shocks (vs ~ 350 km s^-1) extend to ~6.6 kpc above the disk plane. The measured ram pressure (P/k = 4.8 × 10^6 K cm^-3) and mass outflow rate (~8−20 M_⊙ yr^-1) are similar to superwinds from local ultra-luminous infrared galaxies, although NGC 4676A only has a moderate infrared luminosity of 3 × 10^10 L_⊙. Energy beyond what is provided by the mechanical energy of the starburst appears to be required to drive the outflow. Finally, we compare the observations to mock kinematic and stellar population maps extracted from a hydrodynamical merger simulation. The models show little enhancement in star formation during and following first passage, in agreement with the observations. We highlight areas where IFS data could help further constrain the models.

Instabilidades MHD no Tokamak TCABR

Este trabalho descreve o estudo das instabilidades magneto-hidro-dinâmicas (MHD) comumente observadas nas descargas elétricas de plasma no tokamak TCABR, do Instituto de Física da USP. Dois diagnósticos principais foram empregados para observar essas instabilidades: um conjunto poloidal de 24 bobinas magnéticas (bobinas de Mirnov) colocadas próximas à borda do plasma e um medidor de emissões na faixa do Ultra Violeta e de raios X moles com 20 canais (sistema SXR), cujo circuito de condicionamento de sinais foi aprimorado como parte deste trabalho. Esses diagnósticos foram escolhidos porque fornecem informações complementares, uma vez que o sistema SXR observa a parte central da coluna de plasma, enquanto as bobinas de Mirnov detectam as instabilidades MHD na região mais externa da coluna. As informações coletadas por esses diagnósticos foram submetidas à análise espectral com resolução temporal e espacial, possibilitando determinar a evolução das características espectrais e espaciais das instabilidades MHD observadas. Essas análises revelaram que durante a etapa inicial da formação do plasma (quando a corrente de plasma ainda está aumentando) ilhas magnéticas com números de onda decrescente, identificadas como sendo modos kink de borda, são detectadas nas bobinas de Mirnov. Após a formação do plasma, quando os parâmetros de equilíbrio estão relativamente estáveis (platô), oscilações são detectadas tanto nas bobinas de Mirnov quanto no sistema de SXR, indicando a presença de instabilidades MHD em toda a coluna de plasma. Em geral as oscilações medidas nas bobinas de Mirnov tem baixa amplitude e correspondem a pequenas ilhas magnéticas que foram identificadas como sendo modos de ruptura (modos tearing). Por outro lado, as instabilidades na região central foram identificadas como dentes de serra, que correspondem a relaxações periódicas da região interna à superfície magnética com fator de segurança q=1 e que são acompanhadas de oscilações precursoras, cuja amplitude depende da fase do ciclo de relaxação. Devido à essa modulação de amplitude, aparecem picos de frequência satélite nos espectrogramas dos sinais do SXR. Além disso, devido ao fato dos ciclos de relaxação não serem sinusoidais, os harmônicos da frequência de relaxação também aparecem nesses espectrogramas. No entanto, em muitas descargas do TCABR, a intensidade das oscilações medidas nas bobinas de Mirnov aumentam significativamente durante o platô, com efeitos sobre a frequência de todas as instabilidades MHD, até mesmo sobre os dentes de serra localizados na região central da coluna. Em todos os casos, observou-se que durante o platô a frequência das ilhas magnéticas coincide com a frequência das oscilações precursoras do dente de serra, apesar de serem duas instabilidades distintas, localizadas em posições radiais muito diferentes. Essa coincidência de frequências possibilitou descrever a evolução em frequência de todas as oscilações detectadas em diversos diagnósticos com base em apenas duas frequências básicas: a dos ciclos de relaxação dente de serra e a das ilhas magnéticas.

Nesting In The Clouds: Evaluating And Predicting Sea Turtle Nesting Beach Parameters From Lidar Data

Humans' desire for knowledge regarding animal species and their interactions with the natural world have spurred centuries of studies. The relatively new development of remote sensing systems using satellite or aircraft-borne sensors has opened up a wide field of research, which unfortunately largely remains dependent on coarse-scale image spatial resolution, particularly for habitat modeling. For habitat-specialized species, such data may not be sufficient to successfully capture the nuances of their preferred areas. Of particular concern are those species for which topographic feature attributes are a main limiting factor for habitat use. Coarse spatial resolution data can smooth over details that may be essential for habitat characterization. Three studies focusing on sea turtle nesting beaches were completed to serve as an example of how topography can be a main deciding factor for certain species. Light Detection and Ranging (LiDAR) data were used to illustrate that fine spatial scale data can provide information not readily captured by either field work or coarser spatial scale sources. The variables extracted from the LiDAR data could successfully model nesting density for loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) sea turtle species using morphological beach characteristics, highlight beach changes over time and their correlations with nesting success, and provide comparisons for nesting density models across large geographic areas. Comparisons between the LiDAR dataset and other digital elevation models (DEMs) confirmed that fine spatial scale data sources provide more similar habitat information than those with coarser spatial scales. Although these studies focused solely on sea turtles, the underlying principles are applicable for many other wildlife species whose range and behavior may be influenced by topographic features.

Identificação de padrões de uso do solo urbano em São Paulo/SP utilizando parâmetros de variogramas.

As imagens de alta resolução espacial impulsionaram os estudos de Sensoriamento Remoto em ambientes urbanos, já que elas permitem uma melhor distinção dos elementos que compõem esse ambiente tão heterogêneo. Técnicas de Geoestatística são cada vez mais utilizadas em estudos de Sensoriamento Remoto e o variograma é uma importante ferramenta de análise geoestatística, pois permitem entender o comportamento espacial de uma variável regionalizada, neste caso, os níveis de cinza de uma imagem de satélite. O presente trabalho pretende avaliar a proposta metodológica que consiste em identificar padrões residenciais urbanos de três classes de uso e ocupação do solo por meio da análise dos valores apresentados pelos parâmetros, alcance, patamar e efeito pepita de um variograma. A hipótese é que os valores correspondentes a esses parâmetros representem o comportamento espectral padrão de cada classe e, portando, indicam que há um padrão na organização espacial de cada uma das classes. Para a presente pesquisa foram utilizadas imagem IKONOS 2002, e a classificação de uso e ocupação do solo da sub-bacia do córrego Bananal na bacia do Rio Cabuçu de Baixo em São Paulo SP. Amostras das imagens de cada classe foram extraídas e os valores de nível de cinza em cada pixel foram utilizados para calcular os variogramas. Após análise dos resultados obtidos, apenas o parâmetro alcance foi levado em consideração, pois é através desse parâmetro que se observa o grau de homogeneidade de cada amostra. Os valores de alcance obtidos nos cálculos dos variogramas identificaram com melhor precisão a classe Conjuntos Residenciais que é uma classe com padrões e características singulares, já a identificação das classes Ocupação Densa Regular e Ocupação Densa Irregular não obteve uma precisão boa, sendo que essas classes são similares em diversos aspectos.

Use of subpixel techniques in pocket cameras to measure vibrations and displacements

Presentación oral SPIE Photonics Europe, Brussels, 16-19 April 2012.

Measurement of wide frequency range structural microvibrations with a pocket digital camera and sub-pixel techniques

Analysis of vibrations and displacements is a hot topic in structural engineering. Although there is a wide variety of methods for vibration analysis, direct measurement of displacements in the mid and high frequency range is not well solved and accurate devices tend to be very expensive. Low-cost systems can be achieved by applying adequate image processing algorithms. In this paper, we propose the use of a commercial pocket digital camera, which is able to register more than 420 frames per second (fps) at low resolution, for accurate measuring of small vibrations and displacements. The method is based on tracking elliptical targets with sub-pixel accuracy. Our proposal is demonstrated at a 10 m distance with a spatial resolution of 0.15 mm. A practical application over a simple structure is given, and the main parameters of an attenuated movement of a steel column after an impulsive impact are determined with a spatial accuracy of 4 µm.

Repeatability, reproducibility, and accuracy of a novel pushbroom hyperspectral system

This study analyzes the repeatability, reproducibility and accuracy of a new hyperspectral system based on a pushbroom sensor as a means of measuring spectral features and color of materials and objects. The hyperspectral system consisted of a CCD camera, a spectrograph and an objective lens. An additional linear moving system allowed the mechanical scanning of the complete scene. A uniform overhead luminaire with daylight configuration was used to irradiate the scene using d:45 geometry. We followed the guidelines of the ASTM E2214-08 Standard Practice for Specifying and Verifying the Performance of Color-Measuring Instruments that define the standards and latest multidimensional procedures. The results obtained are analyzed in-depth and compared to those recently reported by other authors for spectrophotometers and multispectral systems. It can be concluded that hyperspectral systems are reliable and can be used in the industry to perform spectral and color readings with a high spatial resolution.

Análisis multitemporal del cambio de uso del suelo, en el Paisaje Terrestre Protegido Miraflor Moropotente Nicaragua, 1993-2011

El análisis multitemporal permite detectar cambios entre diferentes fechas de referencia, deduciendo la evolución del medio natural o las repercusiones de la acción humana sobre el medio. El propósito del estudio fue evaluar el cambio de uso del suelo en el Paisaje Terrestre Miraflor Moropotente en el período 1993-2011, a través de imágenes satelitales, a fin de determinar el estado de fragmentación del paisaje. Los cambios de usos de suelo fueron derivados de la clasificación de tres imágenes Landsat TM, con una resolución espacial de 30 metros tomadas en febrero de 1993, abril de 2000 y enero 2011. Se realizó una verificación en campo para la identificación de coberturas de suelo y la corroboración en las imágenes satelitales. La fragmentación se realizó con el cálculo de métricas e índices de fragmentación a nivel del paisaje. Los principales resultados muestran que los cambios de uso de suelo están determinados por la degradación antrópica, principalmente en la conversión de la vegetación nativa a espacios agrícolas y la expansión de la ganadería. El crecimiento demográfico y los monocultivos van ejerciendo presión sobre el bosque, transformando zonas de vocación forestal a cultivos agrícolas. Los cambios de cobertura han significado un paisaje fragmentado con diferentes grados de perturbación, que conllevan a una disminución de la superficie de hábitats naturales, reducción del tamaño de los fragmentos y aislamientos de los mismos.

Measurement of wide frequency range structural microvibrations with a pocket digital camera and sub-pixel techniques

Analysis of vibrations and displacements is a hot topic in structural engineering. Although there is a wide variety of methods for vibration analysis, direct measurement of displacements in the mid and high frequency range is not well solved and accurate devices tend to be very expensive. Low-cost systems can be achieved by applying adequate image processing algorithms. In this paper, we propose the use of a commercial pocket digital camera, which is able to register more than 420 frames per second (fps) at low resolution, for accurate measuring of small vibrations and displacements. The method is based on tracking elliptical targets with sub-pixel accuracy. Our proposal is demonstrated at a 10 m distance with a spatial resolution of 0.15 mm. A practical application over a simple structure is given, and the main parameters of an attenuated movement of a steel column after an impulsive impact are determined with a spatial accuracy of 4 µm.

Validación local de la temperatura superficial del mar del sensor MODIS en aguas someras del Mediterráneo occidental

La temperatura superficial del mar (SST) estimada a partir de los productos 11 μm diurnos y nocturnos y 4 μm nocturnos del sensor MODIS (Moderate Resolution Imaging Spectroradiometer) a bordo de la plataforma Aqua, han sido comparados con datos medidos in situ a tres profundidades diferentes (15, 50 y 100 cm) en una zona costera del Mediterráneo Occidental. Esta comparación ha permitido analizar la incertidumbre que existe en la estimación de este parámetro en aguas someras y próximas a la costa mediante imágenes de satélite de baja resolución espacial. Los resultados obtenidos demuestran que el producto diurno SST_11 μm, obtiene los estadísticos RMSE (error cuadrático medio) y r2 (coeficiente de correlación de Pearson) más ajustados con valores de 1°C y 0,96, respectivamente, para la profundidad 50 cm.