This work was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.

Atenção: O funcionamento de um scanner de tomografia de raios-X requer tanto a formação de radiação em geral, e treinamento de segurança de radiação específica do instrumento. Todos os procedimentos correspondentes relevantes para o laboratório do experimentador deve ser seguido. Imagem 1. Raiz Nota: Esta etapa descreve a imagem de um espécime grama mantidos no seu solo original em um vaso de plástico tubular (um tubo de plástico com um diâmetro de 40 mm, uma altura de 210 mm e espessura de parede de cerca de 2 mm). <ol><li> Coloque plantas envasadas no manipulador de amostra do instrumento a uma distância desejada para ampliação alvo. Para obter uma planta de um suporte de um diâmetro de 2 polegadas, a amostra a distância fonte deve ser de cerca de 3 polegadas (7 cm). </li><li> Ajuste as configurações de digitalização de raios-X para atingir a cor ideal (nível de cinza) o contraste da imagem detector in. Nota: Estas definições estão disponíveis no software de controle de instrumento utilizado. <ol><li> configurações de energia de raios-X Set; 85 kV e 190 uA foram utilizados neste exameple. </li><li> Definir tempo de exposição. Aqui, utilizou-se um tempo de exposição relativamente longo de 1 seg para uma melhor relação sinal-ruído. </li><li> Defina o número de projeções e quadros por projeção; 4 quadros por projecção para um total de 3.142 projeções é sugerido para boas estatísticas de dados. </li><li> Executar uma correção de sombreamento usando as condições de medição indicados acima, selecionando a guia &quot;correção Shading&quot;, e clicar em &quot;Criar&quot;. Nota: A correcção de sombreado compensa a variação na resposta dos pixels do dispositivo de imagem quando iluminado com um conjunto de fluxo constante de raios-X. O processo leva imagens em branco (com a amostra removida do caminho do feixe) com o feixe de raios-X é ligado e com o feixe de desligado. Esta correção é aplicada a todas as imagens recolhidas. </li><li> Selecione a opção &quot;Minimizar artefactos anel&quot; (também chamado &quot;modo de vaivém&quot;); a amostra será rodado em passos angulares, enquanto as imagens de projeção são adquiridos. Isto leva a DATa aquisição a uma taxa mais lenta, mas ajuda a eliminar artefactos anel. </li><li> Inicie a digitalização, clicando no botão &quot;Acquire&quot; sob a guia Acquisition (com as configurações descritas acima, coleção de imagens levará cerca de 4 horas). </li></ol></li></ol> Reconstrução 2. Dados Nota: Esta seção descreve a reconstrução de dados de volume 3D a partir de imagens RAW (radiografias da tomografia computadorizada). <ol><li> Carregar os dados em bruto para o programa. </li><li> Compare primeira e última imagem (que deve ser quase idêntico ao da última imagem é tomada depois de uma rotação da amostra 360 o) para garantir que o espécime não se mexeu ou as configurações de digitalização não se alterou durante a aquisição de dados. </li><li> Calcular o centro de rotação (COR), selecionando a guia &quot;centro de rotação&quot;, e clicar em &quot;Iniciar&quot;; opções de uso &quot;Automatic&quot; COR encontrar com &quot;alta qualidade&quot; precisão, e &quot;Dual&quot; (superior e inferior) slice seleição para o cálculo COR. </li><li> Selecione o volume da amostra a ser reconstruída: selecione a guia &quot;Volume&quot;, e editar as janelas de seleção de volume usando as miniaturas. </li><li> Execute reconstrução para criar o arquivo de volume contendo dados 3D, clicando em &quot;Iniciar&quot;. </li></ol> 3. Data Processing / Segmentação Nota: Esta seção descreve as medidas a serem tomadas para preparar os dados reconstruídos para processamento adicional na RooTrak programa para rastrear raízes como eles se ramificam através do solo, e isolar as raízes de qualquer material circundante para produzir uma pilha de imagens binárias de apenas o própria raiz. <ol><li> Processamento de dados de volume em ImageJ para preparar um RooTrak pilha de processável: <ol><li> Carregar o arquivo de volume em ImageJ. </li><li> Otimizar o contraste de imagem entre raiz e solo, ajustando as configurações de brilho e contraste (Clique na imagem / Ajustar / Brilho / Contraste). Quando a região de interesse dentro da imagem é visível e Claramente distinguíveis, as configurações são consideradas otimizado. </li><li> Salvar como uma pilha de imagens em JPEG, BMP ou PNG. </li></ol></li><li> Processamento na RooTrak para o segmento de raiz: <ol><li> Carregar a pilha de em RooTrak (vá em &quot;Ferramentas&quot;, e pressione &quot;Tracker&quot;). </li><li> Definir pontos de semente dentro de root: clique vários pontos dentro de cada uma das secções de raiz pertinentes visíveis na vista superior fatia dos dados de volume. </li><li> Defina os parâmetros Tracker &quot;Suavidade&quot; e &quot;similaridade&quot; para 0,3 e 0,8, respectivamente. </li><li> Executar a função de rastreamento. Esta seguirá a raiz da fatia de imagem superior todo o caminho até a fatia inferior. </li><li> Depois de ver os dados de volume, selecionar o número de fatias de acordo com o volume de dados utilizável; Neste caso, o acompanhamento foi parado em fatias 200, equivalente a uma profundidade de 6,2 mm, em que as fronteiras de raiz adoeceu-definido (a imagem da raiz rastreados começaram a misturar-se que do solo). Note opilha de imagens produzidas serão salvos automaticamente para onde quer que o diretório de saída foi criado. </li></ol></li></ol> 4. Volume e Análise de Superfície Nota: Esta etapa descreve a geração de malha isosurface da pilha imagem criada por RooTrak. <ol><li> Converter pilha de imagens de RooTrak em um formato de imagem binária em ImageJ. Selecione &quot;Processo&quot;, então &quot;Binário&quot;, depois &quot;Make Binary&quot;. </li><li> Use o plugin ImageJ open-source, BoneJ, para criar a malha triangular; em ImageJ selecione &quot;Plugins&quot;, então &quot;BoneJ&quot;, então &quot;isosuperfície&quot;. </li><li> Defina &quot;Resampling&quot; e &quot;Threshold&quot; a 6 e 120, respectivamente (as configurações padrão). Marque a opção &quot;Mostrar superfície&quot;, e pressione o botão &quot;OK&quot;. </li><li> Sobre o &quot;visualizador 3D&quot;, clique na guia Arquivo, em seguida, em &quot;superfícies de Exportação&quot;, em seguida, salvar como &quot;STL (binary)&quot;. </li><li> Abrir imeshJ, selecione o arquivo STL e digite o tamanho voxel em microns. Clique em &quot;CÁrea de Superfície alculate &quot;para adquirir área de superfície total de raízes amostra em mm 2. Da mesma forma, clique em&quot; Calcular Volume &quot;para obter volume total raiz amostra em mm3. </li></ol>

<ol>
	<li>McKenzie, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rhizosphere:+An+Ecological+Perspective.">The Rhizosphere: An Ecological Perspective.</a> Eur. J. Soil Sci. 59 (2), 416-417 (2008).</li><li>Farrar, J., Hawes, M., Jones, D., Lindow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+roots+control+the+flux+of+carbon+to+the+rhizosphere.">How roots control the flux of carbon to the rhizosphere.</a> Ecology. 84 (4), 827-837 (2003).</li><li>Gregory, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roots+rhizosphere+and+soil:+the+route+to+a+better+understanding+of+soil+science?.">Roots rhizosphere and soil: the route to a better understanding of soil science?.</a> Eur. J. Soil Sci. 57 (1), 2-12 (2006).</li><li>Philippot, L., Raaijmakers, J. M., Lemanceau, P., van der Putten, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+back+to+the+roots:+the+microbial+ecology+of+the+rhizosphere.">Going back to the roots: the microbial ecology of the rhizosphere.</a> Nat. Rev. Microbiol. 11 (11), 789-799 (2013).</li><li>Gregory, P. J., Hutchison, D. J., Read, D. B., Jenneson, P. M., Gilboy, W. B., Morton, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+roots+with+high+resolution+X-ray+micro-tomography.">Non-invasive imaging of roots with high resolution X-ray micro-tomography.</a> Plant and Soil. 255 (1), 351-359 (2003).</li><li>Mairhofer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RooTrak:+Automated+Recovery+of+Three-Dimensional+Plant+Root+Architecture+in+Soil+from+X-Ray+Microcomputed+Tomography+Images+Using+Visual+Tracking.">RooTrak: Automated Recovery of Three-Dimensional Plant Root Architecture in Soil from X-Ray Microcomputed Tomography Images Using Visual Tracking.</a> Plant Physiol. 158 (2), 561-569 (2012).</li><li>Anderson, S. H., Hopmans, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soil-Water-Root Processes: Advances in Tomography and Imaging. , (2013).</li><li>Mairhofer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovering+complete+plant+root+system+architectures+from+soil+via+X-ray+mu-Computed+Tomography.">Recovering complete plant root system architectures from soil via X-ray mu-Computed Tomography.</a> Plant Methods. 9, 8 (2013).</li><li>Lorensen, W. E., Cline, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marching+cubes:+a+high+resolution+3D+surface+construction+algorithm.">Marching cubes: a high resolution 3D surface construction algorithm.</a> Comput. Graph. 21 (4), 163-169 (1987).</li><li>Lobet, G., Draye, X., Perilleux, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+online+database+for+plant+image+analysis+software+tools.">An online database for plant image analysis software tools.</a> Plant Methods. 9 (38), (2013).</li><li>Schmidt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iRoCS+Toolbox+-+3D+analysis+of+the+plant+root+apical+meristem+at+cellular+resolution.">The iRoCS Toolbox - 3D analysis of the plant root apical meristem at cellular resolution.</a> Plant J. 77 (5), 806-814 (2014).</li><li>Galkovskyi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GiA+Roots:+software+for+the+high+throughput+analysis+of+plant+root+system+architecture.">GiA Roots: software for the high throughput analysis of plant root system architecture.</a> BMC Plant Biol. 12, 116 (2012).</li><li>Clark, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+Root+Phenotyping+with+a+Novel+Imaging+and+Software+Platform.">3-Dimensional Root Phenotyping with a Novel Imaging and Software Platform.</a> Plant Physiol. 156, 455-465 (2011).</li><li>. RootSnap! Available from: <a target="_blank" href="https://www.cid-inc.com">https://www.cid-inc.com</a> (2013)</li><li>Arsenault, J. L., Pouleur, S., Messier, C., Guay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WinRHIZO&#8482;+a+root-measuring+system+with+a+unique+overlap+correction+method.">WinRHIZO&#8482; a root-measuring system with a unique overlap correction method.</a> HortSci. 30, 906-906 (1995).</li></ol>

We have nothing to disclose.

Uma combinação de tomografia computadorizada por raios X e vários programas de código aberto provou ser uma combinação poderosa de forma não invasiva amostras de raízes de plantas imagem, dados de raiz segmento, e extrair informação quantitativa (área de superfície e volume) a partir dos dados 3D. A nossa capacidade para visualizar e medir características está sempre limitada pela resolução da digitalização, bem como por limitações do software RooTrak. No entanto, resolução de digitalização foi s...

Roots, como parte da rizosfera 2-5, representam uma parte &quot;invisível&quot; da biologia da planta, desde o solo torna difícil para as raízes de imagem não-invasiva 6, 7. No entanto, estudar o crescimento das raízes e interação dentro do ambiente do solo é fundamental para a compreensão o crescimento das raízes / planta e ciclagem de nutrientes, o que por sua vez afetam arborização, segurança alimentar e climática. Raios-X a tomografia computadorizada (XCT) provou ser uma ferramenta valiosa para a imagiologia não invasivo de amostras de raízes de plantas em seus ambientes locais 8. A fim de medir o desenvolvimento de raiz e alterações dimensionais sob diferentes condições, e ser capaz de comparar dados de diferentes conjuntos de dados / espécimes, é preciso extrair informação quantitativa a partir dos dados de tomografia. A segmentação dos dados de raiz a partir do que o solo circundante, isto é, o isolamento da imagem de raiz de todo o resto em torno dele (incluindo, por exemplo, uma planta vizinha) é um passo crítico antes Accuanálise de tamanho de taxa pode ser feito. No entanto, uma abordagem limite simples é muitas vezes inviável para os dados de raiz. Os desafios associados com as raízes das plantas de imagem no solo incluem variações nas propriedades de atenuação de raios-X do material de raiz, ea sobreposição de valores de atenuação entre raízes e do solo causada pela água e matéria orgânica. Estas questões foram soberbamente abordada recentemente pelo Mairhofer et al. em seu visual ferramenta de acompanhamento RooTrak 7, 9. O próximo passo após uma segmentação de sucesso é a determinação precisa do volume de raiz e área de superfície. O volume pode ser estimado pela contagem do número de voxeis e multiplicando por tamanho em cubos dos voxels como mostrado anteriormente 7. Para uma determinação mais precisa da área de superfície da raiz e do volume, a iso-superficie do sistema radicular segmentado pode ser representado por uma malha de triângulos, usando um algoritmo conhecido como marcha cubos 10. O open-source ImageJ 11 pode ser empregado para aproximar thvolume de raiz e com base no algoritmo Marching Cubes. Para o melhor de nosso conhecimento, apenas um número limitado de software de código aberto dedicado a calcular os dados de volume / superfície à base de tomografia para espécimes de raiz no intervalo centímetro e acima está disponível 12. Um software de fonte aberta que olhou para 13 centra-se no crescimento radicular e destina-se a características celulares que permitem a análise do volume quantitativo em resolução de uma única célula. Alguns softwares de código aberto dedicado a sistemas de raiz toda a 14 é excelente para sistemas radiculares tubular de pequeno diâmetro com base na aproximação que a sua forma é realmente tubular. No entanto, alguns trabalhos com imagens em 2D e são incapazes de lidar com 3D pilhas 14. Além disso, a forma aproximação tubular pode não ser válida quando os sistemas radiculares com superfícies ásperas e formas não uniformes, tais como os de árvores, são estudadas. Outra abordagem 15 usa duas dimensões (2D) sequências de imagens de rotação inovadora contornando the precisa de um scanner CT caro. Ele mede, registros e exibe raiz comprimentos sistema. O software testámos daqueles só está disponível comercialmente 16-18; um não parece ser capaz de lidar com imagem 3D pilhas 16, o segundo é uma ferramenta de área foliar e medição do comprimento radicular 17, enquanto o terceiro é baseado na análise de cor 18. Com base nesse levantamento, sugerimos que uma opção sem custo que se aproxima da superfície eo volume da raiz, independentemente da sua forma a partir de dados de tomografia 3D é desejável. Com base na RooTrak livremente disponível e ImageJ, temos desenvolvido um programa, chamado imeshJ (ver arquivo de código Suplementar), que processa uma malha isosurface (arquivo de estereolitografia superfície) gerado a partir de dados de raiz segmentados, e calcula a área de volume e superfície da raiz, fazendo cálculos geométricos simples sobre os dados de malha de índice triângulo. Aqui nós relatamos um método que combina o uso de imagens XCT,reconstrução dos dados e visualização (software CT Pro 3D e VG Studio), a segmentação da raiz da amostra do solo nos dados 3D (ImageJ software open-source e RooTrak), e extração das informações de superfície e volume a partir de uma malha triangular (ImageJ eo imeshJ código de computador).

<table>
	<tbody>
		<tr>
			<td>X-Tek/Metris XTH 320/225 kV&#160;</td>
			<td>Nikon Metrology</td>
			<td>n/a</td>
			<td>X-ray tomography scanner</td>
		</tr>
		<tr>
			<td>Inspect X</td>
			<td>Nikon Metrology</td>
			<td>n/a</td>
			<td>Instrument control software</td>
		</tr>
		<tr>
			<td>CT Pro 3D</td>
			<td>Nikon Metrology</td>
			<td>n/a</td>
			<td>Reconstruction software, version XT 2.2</td>
		</tr>
		<tr>
			<td>VG Studio MAX</td>
			<td>Visual Graphics GmbH</td>
			<td>n/a</td>
			<td>Visualization software for 3D volumes, version 2.1.5</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Open-source</td>
			<td>n/a</td>
			<td>Image processing and analysis software, version 1.6</td>
		</tr>
		<tr>
			<td>RooTrak</td>
			<td>Open-source</td>
			<td>n/a</td>
			<td>Root segmentation software, version 0.3.1-b1 beta</td>
		</tr>
		<tr>
			<td>imeshJ</td>
			<td>EMSL</td>
			<td>n/a</td>
			<td>MATLAB script developed by the authors</td>
		</tr>
		<tr>
			<td>Prairie dropseed grass sample</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Sample obtained from ground in residential area</td>
		</tr>
	</tbody>
</table>

extracting metrics for three-dimensional root systems: volume and surface analysis from in-soil x-ray computed tomography data

As raízes das plantas desempenham um papel fundamental nas interações planta-solo-micróbio que ocorrem na rizosfera, bem como processos com implicações importantes para as alterações climáticas e manejo da cultura. informações de tamanho quantitativo em raízes em seu ambiente nativo é inestimável para estudar o crescimento das raízes e processos ambientais que envolvem plantas. De raios-X de tomografia computadorizada (XCT) foi demonstrado ser uma ferramenta eficaz para a verificação da raiz in situ e análise. Nosso objetivo foi desenvolver uma ferramenta sem custos e eficiente que se aproxima da superfície eo volume da raiz, independentemente da sua forma a partir de dados tridimensional tomografia (3D). A estrutura de raiz de um dropseed Prairie (heterolepis Sporobolus) espécime foi fotografada usando XCT. A raiz foi reconstruído, e a estrutura de raiz primária foi extraído a partir dos dados usando uma combinação de software licenciado e de código aberto. Uma malha poligonal isosurface foi então criado para facilitar a análise. Nós desenvolvemos tele autônomo aplicativo imeshJ, gerada em MATLAB 1, para calcular o volume de raiz e área de superfície da malha. As saídas de imeshJ são a área da superfície (em mm 2) e o volume (em mm 3). O processo, que utiliza uma combinação única de ferramentas de imagem para análise quantitativa da raiz, está descrito. Uma combinação de XCT e software de código aberto provou ser uma combinação poderosa de forma não invasiva amostras de raízes de plantas imagem, dados de raiz segmento, e extrair informação quantitativa a partir dos dados 3D. Esta metodologia de processamento de dados 3D devem ser aplicáveis ​​a outros sistemas de material / amostra onde há conectividade entre os componentes de atenuação de raios-X semelhantes e dificuldades surgem com a segmentação.

A amostra que consiste em duas hastes de relva nativa Prairie dropseed (Sporobolus heterolepis) e o solo original em torno dele foi feita a partir de uma área residencial e colocado num pequeno suporte em forma de tubo visto na Figura 1. O tamanho reconstruída do voxel de dados foi de aproximadamente 31 um x 31 um x 31 um. O arquivo de volume reconstruído foi usada para criar uma pilha de imagens a partir de uma orientação selecionada (vista superior) utili...

Watch this Scientific Journal Video about Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data at JoVE.com

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Extraindo Métricas para sistemas radiculares tridimensionais: Análise de volume e de superfície de In-solo X-ray tomografia computadorizada de dados

Uma metodologia para a obtenção de informação sobre a estrutura de raiz visual e quantitativa a partir de dados de raios-X de tomografia computadorizada adquiridos em solo é apresentada.

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

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8.5K visualizações.  Environmental Molecular Sciences Laboratory.  Uma metodologia para a obtenção de informação sobre a estrutura de raiz visual e quantitativa a partir de dados de raios-X de tomografia computadorizada adquiridos em solo é apresentada. 

Vídeo: Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

Ambiente

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Um Procedimento volume pequeno para Concentração Viral da Água

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Uso de Chironomidae (Diptera) Pupal Exuviae como um Bioavaliação Protocolo Rápido para massas de água de superfície flutuante

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Monólito escavação para Coupled Hidrológicos, geoquímicos e microbiológicos Investigações

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Modificação e Aplicação de uma Folha Blower-vac para Campo Amostragem de Artrópodes

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocolo para Microplásticos Amostragem na superfície do mar e análise de amostras

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Vegetated Sistemas de Tratamento para Remoção de Contaminantes Associados à Toxicidade de Águas Superfícies na Agricultura e Escorrências Urbanas

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

Dispersão de nanomateriais em meios aquosos: para otimização de protocolo

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

Distribuição de estresse durante a compressão fria de rochas e minerais agregados usando difração de raio x do Synchrotron-baseado

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Isolamento e análise de comunidades microbianas na rizosfera, solo e raízes em experiências de grama perene

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...