Esta pesquisa foi financiada pelo Fundo de Ciência Austríaco (FWF): P23681-B16.

1. Preparação de materiais de plantas <ol><li> Utilize qualquer folhas ou plantas inteiras de material sujeito planta (Hosta spp. Ou Phaseolus vulgaris). </li></ol> 2. Preparação de soluções aquosas contendo Ice nucleação ativos (INA) Bactérias <ol><li> Culturas da bactéria INA, Pseudomonas syringae (Estirpe Cit-7) em placas de Petri a 25 ° C em agar Pseudomonas F preparada com 10 g / L de glicerol a 100% por direcção do fabricante. </li><li> Após culturas cresceram suficientemente, lugar a 4 ° C até ser necessário, mas manter a 4 ° C durante dois dias antes de garantir um elevado nível de actividade de nucleação de gelo. </li><li> Raspe bactérias de uma única placa a partir da superfície do agar com um plástico, descartáveis ​​ou reutilizáveis ​​espátula de metal no momento da utilização e em lugar de 10-15 ml de água desionizada numa cuvete descartável de 25 ml. A concentração deve estar no intervalo de 1 x 07-1 outubro x 10 9 · </sup&gt; Ml -1. A solução aparece nublado. Não há necessidade de se confirmar a concentração, utilizando um hemocitómetro ou um espectrofotómetro, tal como concentração precisa ser apenas aproximados. </li><li> Vortex a cuvete para um mínimo de 10 segundos para distribuir as bactérias. Nota: A concentração específica da mistura INA resultante não é importante e o protocolo descrito irá fornecer mais do que um nível adequado de actividade de nucleação de gelo. Esta mistura de bactérias INA e água será usada mais tarde nos experimentos de nucleação. </li></ol> 3. configuração de uma experiência de congelação <ol><li> Coloque a câmara de infravermelhos de alta resolução (SC-660) no interior da caixa de protecção de acrílico de modo a que os projectos de lente através de uma abertura na parte da frente da caixa e os fios de ligação da câmara a uma saída do dispositivo portátil ou gravação através da abertura traseira da caixa . Fixe a tampa da caixa e coloque a caixa de dentro da câmara ambiental ou freezer em um local que vai tudoow o material sujeito planta a ser visto. <ol><li> Fornecer um fundo escuro em torno do material vegetal, forrando as paredes da câmara com papel de construção preto para impedir a interferência de energia infravermelha refletida. </li><li> Montar a câmara com iluminação LED para minimizar o aquecimento da fonte de luz quando gravar imagens em comprimentos de onda visíveis é necessária. Apenas um mínimo de iluminação, tal como um armário de luz operado por bateria ou outro dispositivo de LED pequeno, é necessário para as plantas a ser visível pela câmara. <ol><li> Uma vez que as imagens visíveis do material sujeito planta são tomadas, desligue a iluminação LED. Distribua todas as conexões externas com fio (conexão firewire de computador, cabo de alimentação, etc.) para a câmera através de uma porta ou outra abertura na câmara. </li></ol></li><li> Preencha qualquer espaço extra na porta ou abertura com material isolante de espuma para evitar ou reduzir os gradientes de temperatura dentro da câmara. Ajustar a temperatura inicial da câmara a 1 ° C. </li></ol></li><li> Alinhe plantas ou partes de plantas para que o material vegetal é no campo de visão da câmera-e o material vegetal é visível na tela de visualização remota ou dentro do software escolhido. </li><li> Permitir que as plantas atinjam a 1 ° C durante 30 min a 1 hora, dependendo do tamanho do material de planta, antes de iniciar uma experiência de congelação controlada. Isto garante que a temperatura da planta não vai ficar para trás da temperatura do ar por diversos graus uma vez que o experimento de congelação é iniciada. A equilibração é conseguida quando a temperatura do material vegetal é a menos de 0,5 ° C de temperatura do ar. <ol><li> Coloque uma camada de isolamento de isopor no topo do solo de vasos de plantas se as plantas em vaso são utilizados. Uma vez que as plantas têm equilibrada, iniciar o arrefecimento da câmara. Nota: A camada de isolamento sobre a superfície do solo do vaso reduz a quantidade de perda de calor continuou a partir do vaso ao ar que envolve a planta, e impede que as raízes de freezing, dado que isto não ocorrem tipicamente durante um evento de geada na natureza devido ao enorme reservatório de calor residual presente no solo. </li></ol></li><li> Defina os parâmetros da câmera desejados (paleta de cor, faixa de temperatura, áreas específicas de interesse, etc.), como discutido na 3.4.1-3.4.4. <ol><li> Selecione a paleta de arco-íris para exibir as variações de temperatura enquanto visualiza a imagem ao vivo. </li><li> Definir a amplitude de temperatura de 5 ° C por ajustamento da barra de temperatura localizado logo abaixo da imagem no software. </li><li> Escolha a escala linear (algoritmo), para converter os dados de infravermelhos para a imagem de cor falsa, tal como definido pela paleta seleccionados (arco-íris) e definir o intervalo de temperatura de 5 ° C e para controlar automaticamente com base na imagem. Alternativamente, ajustar o intervalo definido manualmente durante a realização do experimento. <ol><li> Utilizar a temperatura de um ponto específico ou uma temperatura média dentro da área definida de interesse fornecida pelo softwsão. Recuperar os dados de temperatura de todos os pixels da sequência de vídeo gravado ou da informação embutido no arquivo de imagem. Figura 3 mostra uma tela típica de dentro de software ResearchIR. </li></ol></li><li> Posicionar um cursor num local dentro do tecido da planta que representa um ponto específico de interesse. Definir a área de interesse como pontos (1 -3 pixels de tamanho), caixas, linhas, círculos ou elipses. Várias combinações de pontos ou formas pode ser localizada sobre a imagem. </li></ol></li><li> Gravação de uma seqüência de vídeo <ol><li> Ajuste a câmera para gravar a 60 Hz e para a gravação de ser parado manualmente. </li><li> Indicar o local no computador ou disco rígido externo onde o ficheiro de vídeo gravado será colocado. </li><li> Iniciar a gravação. Nota: A gravação de um disco rígido externo é altamente recomendável uma vez que grandes arquivos de vídeo serão gerados. Arquivos de vídeo gravados podem ser mais tarde editada para conter somente a parte que contém o neinformações neces-. Isto irá reduzir significativamente o tamanho do arquivo. </li><li> Diminuir a temperatura da câmara de incrementos de 0,5 -1,0 ° C. Espere até que a temperatura da planta equilibra com a temperatura do ar e, em seguida, baixar a temperatura novamente por 0,5-1,0 ° C. Dependendo da massa de tecido da planta a ser observada e a sua morfologia, de equilíbrio pode levar de 10 a 15 min. Assim, dando uma velocidade de arrefecimento de cerca de 4 ° C / h. </li><li> Continuar dessa maneira até que a planta congela e observações estão concluídas. Terminar a gravação quando o processo de congelação foi concluída. Nota: O tecido da planta se ter equilibrado com a temperatura do ar quando o material de planta e de fundo são da mesma cor, uma vez que estão à mesma temperatura. Uma vez que a temperatura de fundo e a temperatura do tecido da planta são os mesmos, pode ser difícil de visualizar o material vegetal até a temperatura diminuir de novo e não há diferencial de temperatura entre o tecido da planta e umatemperatura IR. </li></ol></li></ol>

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A água tem a capacidade de supercool a temperaturas bem abaixo de 0 ° C e a temperatura a que a água irá congelar podem ser bastante variável. 36 A temperatura limite para a super-ref rigeração de água pura é de cerca de -40 ° C e é definido como o ponto de nucleação homogénea. Quando a água congela a temperaturas mais altas do que -40 ° C que é provocada pela presença de heterogénea nucleadores que permitem pequenas embriões de gelo para formar o qual, em seguida, servir como um catalisad...

Congelamento temperaturas que ocorrem quando as plantas estão em crescimento activo pode ser letal, particularmente se a planta tem tolerância ao congelamento pouca ou nenhuma. Tais eventos geada muitas vezes têm efeitos devastadores sobre a produção agrícola e também podem desempenhar um papel importante na formação da estrutura da comunidade em populações naturais de plantas, especialmente em alpino, ecossistemas sub-árcticas e árcticas 1-6. Os episódios de geadas da Primavera graves tiveram grandes impactos sobre a produção de frutos nos EUA e América do Sul nos últimos anos 7-9 e foram exacerbadas pelo início precoce de tempo quente seguido por mais típicas baixas temperaturas médias. O clima quente no início induz botões para se abrirem, ativando o crescimento de novos brotos, folhas e flores todos os que têm muito pouca ou nenhuma tolerância geada 1,3,10-12. Tais padrões climáticos erráticos foram relatados para ser um reflexo direto das mudanças climáticas em curso e espera-se que seja um padrão de tempo comum para as foresfuturo eeable 13. Os esforços para proporcionar técnicas de gestão econômica, eficazes e respeitadores do ambiente ou agrotóxicos que podem fornecer tolerância a geadas aumento tiveram sucesso limitado por uma série de razões, mas isso pode ser atribuído em parte à natureza complexa do congelamento tolerância e mecanismos de prevenção de congelamento nas plantas. 14 Os mecanismos adaptativos associados com a sobrevivência geada em plantas têm sido tradicionalmente divididos em duas categorias, a tolerância ao congelamento e evitar o congelamento. A categoria anterior está associada com mecanismos bioquímicos regulados por um conjunto específico de genes que permitem que as plantas para tolerar as tensões associadas com a presença e o efeito desidratante de gelo nos seus tecidos. Enquanto a última categoria é normalmente, mas não exclusivamente, associado a aspectos estruturais de uma planta que determinam se, quando e onde as formas de gelo em uma planta 14. Apesar da prevalência de evitar congelamento como um anúnciomecanismo aptive, pouca pesquisa tem sido dedicada nos últimos tempos para a compreensão dos mecanismos subjacentes e regulação de evitar congelamento. O leitor é remetido para uma revisão recente 15 para maiores detalhes sobre este assunto. Enquanto a formação de gelo a baixas temperaturas pode parecer um processo simples, muitos factores que contribuem para a determinação da temperatura à qual o gelo nucleia em tecidos de plantas e como se propaga dentro da planta. Parâmetros como a presença de extrínseco e intrínseco nucleadores de gelo, contra eventos nucleação homogênea heterogêneos, térmico-histerese (anticongelante) proteínas, a presença de açúcares específicos e outras osmolytes, e uma série de aspectos estruturais da planta pode desempenhar um importante papel no processo de congelação em plantas. Colectivamente, estes parâmetros influenciam a temperatura à qual uma planta congela, onde o gelo é iniciada e como ele cresce. Eles podem também afectar a morfologia dos cristais de gelo resultantes.Vários métodos têm sido utilizados para estudar o processo de congelação em plantas sob condições de laboratório, incluindo espectroscopia de ressonância magnética nuclear (RMN) 16, ressonância magnética (MRI) 17, crio-microscopia 18-19, e microscopia eletrônica de varredura de baixa temperatura (LTSEM ). 20 Congelamento de plantas inteiras em ambientes de laboratório e campo, no entanto, tem sido principalmente monitorizadas com termopares. A utilização de termopares para estudar congelamento baseia-se na libertação de calor (entalpia de fusão), quando a água passa por uma transição de fase do estado líquido para um sólido. A congelação é então gravada como um evento exotérmico. 21-23 Mesmo que os termopares são o método típico de escolha no estudo de congelação em plantas, a sua utilização tem muitas limitações que limitam a quantidade de informação obtida durante a ocorrência da congelação. Por exemplo, com termopares é difícil quase impossível de determinar onde o gelo é iniciado em plantas, como se propaga,se propaga a uma taxa ainda, e se alguns tecidos permanecem livres de gelo. Avanços em termografia infravermelha de alta resolução (Hrit) 24-27, no entanto, têm aumentado significativamente a capacidade de obter informações sobre o processo de congelação em plantas inteiras, especialmente quando usado em um modo diferencial de imagem. 28-33 No presente relatório, descrevem a utilização desta tecnologia para o estudo de vários aspectos do processo de congelação e vários parâmetros que afectam onde e gelo e em que a temperatura é iniciada em plantas. Um protocolo irá ser apresentada que vai demonstrar a capacidade da bactéria-nucleação de gelo-activo (INA), Pseudomonas syringae (Cit-7), para actuar como um nucleador extrínseca iniciar a congelação em uma planta herbácea a, uma temperatura abaixo de zero elevado. De alta resolução Infrared Camera O protocolo e exemplos documentados neste relatório utilizar uma alta resolução infravermelhoradiómetro de vídeo. O radiómetro (Figura 1) fornece uma combinação de imagens de espectro visível e infravermelho e dados de temperatura. A resposta espectral da câmera é na faixa de 7,5 a 13,5 mm e fornece 640 x 480 pixels de resolução. Espectro visível imagens geradas pelo construído em câmara pode ser fundido com IR-imagens em tempo real, o que facilita a interpretação de imagens complexas, térmicas. A gama de lentes para a câmera pode ser usada para fazer close-up e observações microscópicas. A câmera pode ser usada em modo stand-alone, ou interface e controlada com um laptop usando software proprietária. O software pode ser utilizado para se obter uma variedade de dados térmicos incorporados nos vídeos gravados. É importante notar que uma grande variedade de radiómetros infravermelhos estão disponíveis comercialmente. Portanto, é essencial que o pesquisador discutir a sua aplicação pretendida com um engenheiro de produto experiente e que o investigador testar a capacidade de qualquer específic radiômetro para fornecer as informações necessárias. O radiômetro de imagem usados ​​no protocolo descrito é colocado em uma caixa de acrílico (Figura 2) isoladas com isopor i n fim de dissuadir a exposição a condensação durante os protocolos de aquecimento e esfriamento. Essa proteção não é necessária para todas as câmeras ou aplicações.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Infrared Camera</td>
			<td>FLIR</td>
			<td>SC-660</td>
			<td>Many models available depending on application</td>
		</tr>
		<tr>
			<td>Infrared Analytical Software</td>
			<td>FLIR</td>
			<td>ResearchIR 4.10.2.5</td>
			<td align="right">$3,500</td>
		</tr>
		<tr>
			<td rowspan="2">Pseudomonas syringae (strain Cit-7)</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"><a href="mailto:icelab@berkeley.edu">Kindly provided by Dr. Steven Lindow, University of California&#160; Berkeley icelab@berkeley.edu</a></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Pseudomonas Agar F</td>
			<td>Fisher Scientific</td>
			<td>DF0448-17-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of high-resolution infrared thermography (hrit) for the study of ice nucleation and ice propagation in plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Ice-atividade de nucleação do gelo + bactéria, Pseudomonas syringae (estirpe Cit-7) Uma gota de 10 ul de água e 10 ml de água contendo P. syringae (Cit-7) foram colocados sobre a superfície abaxial de uma folha Hosta (Hosta spp.) (Figura 4). Tal como ilustrado, a gota de água contendo as bactérias INA congelou primeiro e era responsável por induzir a folha para congelar enquanto a gota de água sobre a superfície da folha permaneceu desco...

Watch this Scientific Journal Video about The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants at JoVE.com

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

O uso de alta resolução Termografia Infravermelha (Hrit) para o Estudo da Ice Ice nucleação e propagação de plantas

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

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Environment

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

9.5K visualizações.  Agricultural Research Service (USDA-ARS), Kearneysville, WV. Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Vídeo: The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expressão da proteína recombinante a partir da celulase Cel5A<em&gt; Trichoderma reesei</em&gt; Em Plantas de tabaco

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Ambiente

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Aperfeiçoamento da taxa de crescimento inicial das plantas agrícolas usando campos magnéticos estáticos

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

O uso de uma bateria de métodos químicos e ecotoxicológicos para a avaliação da eficácia dos Processos de Tratamento de Águas Residuais remover estrogénica Potência

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Técnicas empíricas, metagenômicas e computacionais iluminam os mecanismos pelos quais os fungicidas comprometem a saúde das abelhas

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Automatizado, de alta resolução Sistema de Coleta Móvel para a análise de azoto isotópica de NO<sub&gt; x</sub

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

A calibração e a utilização de sensores de capacitância de monitorar o conteúdo de água do caule em árvores

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

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) ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Protocolo para a produção de vídeo infravermelho tridimensional de congelamento em plantas

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Acompanhamento a longo prazo de vídeo de animais aquáticos, Cohoused: um estudo de caso da atividade locomotora diária do lagostim (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.