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.

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.

Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells.

Cyclins are cell cycle regulators whose proteins oscillate dramatically during the cell cycle. Cyclin steady-state mRNA levels also fluctuate, and there are indications that both their rate of transcription and mRNA stability are under cell cycle control. Here, we demonstrate the transcriptional regulation of higher eukaryote cyclins throughout the whole cell cycle with a high temporal resolution. The promoters of two Arabidopsis cyclins, cyc3aAt and cyc1At, mediated transcriptional oscillation of the beta-glucuronidase (gus) reporter gene in stably transformed tobacco BY-2 cell lines. The rate of transcription driven by the cyc3aAt promoter was very low during G1, slowly increased during the S phase, peaked at the G2 phase and G2-to-M transition, and was down-regulated before early metaphase. In contrast, the rate of the cyc1At-related transcription increased upon exit of the S phase, peaked at the G2-to-M transition and during mitosis, and decreased upon exit from the M phase. This study indicates that transcription mechanisms that seem to be conserved among species play a significant role in regulating the mRNA abundance of the plant cyclins. Furthermore, the transcription patterns of cyc3aAt and cyc1At were coherent with their slightly higher sequence similarity to the A and B groups of animal cyclins, respectively, suggesting that they may fulfill comparable roles during the cell cycle.

Desenvolvimento e caracterização do filtro óptico de interferência variável para detectores de alta resolução espectral e biossensores.

O presente trabalho está fundamentado no desenvolvimento de uma metodologia e/ou uma tecnologia de obtenção e caracterização de filtros ópticos de interferência de banda passante variável [C.M. da Silva, 2010] e de banda de corte variáveis, constituídos por refletores dielétricos multicamadas de filmes finos intercalados por cavidades de Fabry-Perot não planares com espessuras linearmente variáveis, que apresentam a propriedade do deslocamento linear da transmitância máxima espectral em função da posição, isto é, um Filtro de Interferência Variável (FIV). Este método apresenta novas e abrangentes possibilidades de confecção de filtros ópticos de interferência variável: lineares ou em outras formas desejadas, de comprimento de onda de corte variável (passa baixa ou alta) e filtros de densidade neutra variável, através da deposição de metais, além de aplicações em uma promissora e nova área de pesquisa na deposição de filmes finos não uniformes. A etapa inicial deste desenvolvimento foi o estudo da teoria dos filtros ópticos dielétricos de interferência para projetar e construir um filtro óptico banda passante convencional de um comprimento de onda central com camadas homogêneas. A etapa seguinte, com base na teoria óptica dos filmes finos já estabelecida, foi desenvolver a extensão destes conhecimentos para determinar que a variação da espessura em um perfil inclinado e linear da cavidade entre os refletores de Bragg é o principal parâmetro para produzir o deslocamento espacial da transmitância espectral, possibilitando o uso de técnicas especiais para se obter uma variação em faixas de bandas de grande amplitude, em um único filtro. Um trabalho de modelagem analítica e análise de tolerância de espessuras dos filmes depositados foram necessários para a seleção da estratégia do \"mascaramento\" seletivo do material evaporado formado na câmara e-Beam (elétron-Beam) com o objetivo da obtenção do filtro espectral linear variável de características desejadas. Para tanto, de acordo com os requisitos de projeto, foram necessárias adaptações em uma evaporadora por e-Beam para receber um obliterador mecânico especialmente projetado para compatibilizar os parâmetros das técnicas convencionais de deposição com o objetivo de se obter um perfil inclinado, perfil este previsto em processos de simulação para ajustar e calibrar a geometria do obliterador e se obter um filme depositado na espessura, conformação e disposição pretendidos. Ao final destas etapas de modelagem analítica, simulação e refinamento recorrente, foram determinados os parâmetros de projeto para obtenção de um determinado FIV (Filtro de Interferência Variável) especificado. Baseadas nos FIVs muitas aplicações são emergentes: dispositivos multi, hiper e ultra espectral para sensoriamento remoto e análise ambiental, sistemas Lab-on-Chip, biossensores, detectores chip-sized, espectrofotometria de fluorescência on-chip, detectores de deslocamento de comprimento de onda, sistemas de interrogação, sistemas de imageamento espectral, microespectrofotômetros e etc. No escopo deste trabalho se pretende abranger um estudo de uma referência básica do emprego do (FIV) filtro de interferência variável como detector de varredura de comprimento de ondas em sensores biológicos e químicos compatível com pós processamento CMOS. Um sistema básico que é constituído por um FIV montado sobre uma matriz de sensores ópticos conectada a um módulo eletrônico dedicado a medir a intensidade da radiação incidente e as bandas de absorção das moléculas presentes em uma câmara de detecção de um sistema próprio de canais de microfluidos, configurando-se em um sistema de aquisição e armazenamento de dados (DAS), é proposto para demonstrar as possibilidades do FIV e para servir de base para estudos exploratórios das suas diversas potencialidades que, entre tantas, algumas são mencionadas ao longo deste trabalho. O protótipo obtido é capaz de analisar fluidos químicos ou biológicos e pode ser confrontado com os resultados obtidos por equipamentos homologados de uso corrente.

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.

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.

Remote Sensing of Vegetation Change Across a Latitudinal Gradient in the Canadian Arctic

Global air surface temperatures and precipitation have increased over the last several decades resulting in a trend of greening across the Circumpolar Arctic. The spatial variability of warming and the inherent effects on plant communities has not proven to be uniform or homogeneous on global or local scales. We can apply remote sensing vegetation indices such as the Normalized Difference Vegetation Index (NDVI) to map and monitor vegetation change (e.g., phenology, greening, percent cover, and biomass) over time. It is important to document how Arctic vegetation is changing, as it will have large implications related to global carbon and surface energy budgets. The research reported here examined vegetation greening across different spatial and temporal scales at two disparate Arctic sites: Apex River Watershed (ARW), Baffin Island, and Cape Bounty Arctic Watershed Observatory (CBAWO), Melville Island, NU. To characterize the vegetation in the ARW, high spatial resolution WorldView-2 data were processed to create a supervised land-cover classification and model percent vegetation cover (PVC) (a similar process had been completed in a previous study for the CBAWO). Meanwhile, NDVI data spanning the past 30 years were derived from intermediate resolution Landsat data at the two Arctic sites. The land-cover classifications at both sites were used to examine the Landsat NDVI time series by vegetation class. Climate variables (i.e., temperature, precipitation and growing season length (GSL) were examined to explore the potential relationships of NDVI to climate warming. PVC was successfully modeled using high resolution data in the ARW. PVC and plant communities appear to reside along a moisture and altitudinal gradient. The NDVI time series demonstrated an overall significant increase in greening at the CBAWO (High Arctic site), specifically in the dry and mesic vegetation type. However, similar overall greening was not observed for the ARW (Low Arctic site). The overall increase in NDVI at the CBAWO was attributed to a significant increase in July temperatures, precipitation and GSL.

Detection of low energy antimatter with emulsions

Emulsion detectors feature a very high position resolution and consequently represent an ideal device when particle detection is required at the micrometric scale. This is the case of quantum interferometry studies with antimatter, where micrometric fringes have to be measured. In this framework, we designed and realized a new emulsion based detector characterized by a gel enriched in terms of silver bromide crystal contents poured on a glass plate. We tested the sensitivity of such a detector to low energy positrons in the range 10–20 keV . The obtained results prove that nuclear emulsions are highly efficient at detecting positrons at these energies. This achievement paves the way to perform matter-wave interferometry with positrons using this technology.

Sea-surface temperature reconstruction for sediment core GeoB1023-5

Sediment core GeoB 1023-5 from the eastern South Atlantic was investigated at high temporal resolution for variations of sea-surface temperature (SST) during the past 22 kyr, using the alkenone (UK'37) method. SSTs increased by 3.5°C from about 18°C during the Last Ice Age (21±2 cal kyr BP) to about 21.5°C at 14.5 cal kyr BP. This warming trend associated with the deglaciation phase was followed by a cooling event with lowest SSTs near 20°C, persisting for about 1000 years between 13 and 12 cal kyr BP. The SSTs then continued to increase to about 22.5°C at the Holocene climatic optimum at 7 cal kyr BP, and decreased again during the Late Holocene to a core-top value of 19.8°C that is comparable to modern annual mean SST values. When compared with alkenone SST records from the eastern North Atlantic, our SST record indicates continuous warming throughout the deglaciation phase in the Benguela Current, while its northern counterpart, the Canary Current, experienced prominent cooling during 'Heinrich Event 1' (H1). On the other hand, for the time period corresponding to the 'Younger Dryas' (YD) cooling event, the Benguela SST record exhibits a cold-temperature interval that corresponds to that observed in the eastern North Atlantic SST records. This observation suggests that interhemispheric climate response in Atlantic eastern boundary current systems was different with respect to the two abrupt climate events associated with Termination I. For the H1, the eastern South Atlantic SST record strongly supports the hypothesis that an 'anti-phase' thermal behavior in South Atlantic surface waters was forced by the slowdown of the North Atlantic Deep Water formation during cold spells in the North Atlantic. In contrast, the abrupt cooling in the eastern South Atlantic coincident with the YD period was probably induced by more vigorous global atmospheric circulation, enhancing the upwelling intensity in both eastern boundary current systems. This atmospheric control may have overridden any effect caused by changes in thermohaline circulation on the South Atlantic SSTs during the YD, which leads to the assumption that the thermohaline circulation was already much closer to its interglacial mode during the YD than during the H1.