Damos graças especiais para Yves Couder para muitas discussões úteis. Agradecemos Atef Asnacios para a calibração das consolas e discussão. Agradecemos Lisa Willis, Elliot Meyerowitz e Oliver Hamant para a leitura crítica. Este trabalho foi financiado em parte pelo Human Frontier Science Program RGP0062/2005-C subvenção; Agence Nationale de la Recherche projeta&#39;&#39; Growpec,&#39;&#39; e&#39;&#39;&#39;&#39; Mechastem.

1. Prepare lâminas de vidro para montagem Amostra <ol><li> Prepare encaixar mídia agarose: 0,7% de agarose de baixa fusão em 10% manitol (em água). </li><li> Usando um instrumento de metal forte (por exemplo, ponta broca, cal), gravar uma área de 0,5 x 0,5 cm no centro de uma lâmina de vidro de microscopia. Ou em vez disso, colar um pequeno pedaço de lamela de vidro (cerca de 20 x 200 mm) para a lâmina de vidro com cola Araldite. NOTA: Este encrespa a superfície, a fim de facilitar a aderência da camada de agarose para assegurar que as varas de comunicação de agarose ou corrige para a corrediça. Este slide pode ser reutilizado. </li><li> Posicionar um gota de cerca de 20 ul de encaixar meios de agarose para a região gravada da placa de vidro. Isto dá uma película fina de agarose. </li><li> Guarde a lâmina de vidro em uma caixa úmido e esperar que os meios de comunicação de agarose solidificar. </li></ol> 2. Dissecando e montagem Amostras meristema <ol><li> Microdissect meristemas para imagi confocalng: remover os botões florais, um por um a partir do tronco, puxando-os para baixo com uma pinça ultrafinas. É importante remover todos os botões florais até os brotos que não apresentam sépalas (isto é, o primórdio P1 14). </li><li> Usando uma lâmina de barbear ou a ponta da pinça, cortar o meristema para longe da haste. O corte deve ser paralela à região da superfície do meristema, que deve ser medida com a AFM. O ápice obtida deve estar entre 300 mm e 600 mM alto para caber sob a ponta do AFM. </li><li> Empurre o ápice no filme de meios de agarose preparado no passo 1.1.4. Escolha uma posição sobre a lâmina de vidro / agarose e empurre tal que o meristema é tanto diretamente em contato com a lâmina de vidro e se projeta para fora da agarose. É crucial para posicionar o meristema no agarose, nos poucos segundos que se seguem o seu corte a partir da haste. NOTA: Este é, a fim de impedir que o meristema da secagem. Nesta fase, o meristema estará de pé em um crack porque a agarose fracturado mediante a aplicação de pressão. </li><li> Prepare vários meristemas desta forma para a imagiologia de lote, proporcionando que eles têm a mesma altura e são posicionados inferior a 500 mm a partir de um outro sobre a corrediça. Mantenha os meristemas preparados na caixa úmido enquanto dissecar mais amostras. NOTA: Limita a variação proveniente da calibração. Idealmente, 2 amostras de cada condição pode ser preparado e medido de forma aleatória. Até o momento não há nenhum efeito observado de imagem em lote na medição (stress induzido resposta). Isto pode ser devido à diluição de moléculas de sinalização de stress no agar e / ou a solução de montagem. Em alternativa, a resposta ao estresse é o mesmo, independentemente da quantidade de amostra preparada. </li><li> Antes de passar para o próximo passo, certifique-se que a fronteira entre o meristema e os botões florais é seco. Se não, retire o excesso de água com papel de filtro. Isto deve impedir que na fase seguinte, que o derretidoagarose inunda a amostra devido às forças de capilaridade. </li><li> Com uma pipeta de 20 mL, adicione o suficiente derretido montagem agarose para preencher o crack criou no passo 1.2.3. e cercar cada meristema. A agarose deve atingir o nível da cicatriz do botão de flor removido (primórdio). Para alcançar este objectivo, pode ser necessário adicionar agarose em cada extremidade da fenda. </li><li> Quando a agarose é adicionado, ele vai rapidamente inundar o crack; utilizando a pipeta, chupar parte da montagem de agarose fundida para garantir que o meristema é o ponto mais alto da corrediça. Isto corrige o meristema de modo que não pode mover-se durante o exame. </li></ol> 3. Montagem amostras de raízes ou hipocótilo <ol><li> Para raízes e hipocótilos, sem dissecção é necessário. No fina película de agarose na lâmina preparada, fazer um sulco ou diretamente acima de um etch no vidro ou ao longo de uma lamela de vidro. Posicione a raiz ou hipocótilo neste sulco garantindo que ele está em contato com ode vidro ao longo de todo o seu comprimento. </li><li> Antes de passar para o próximo passo, certifique-se de que a amostra é seca em sua superfície. Se não, retire o excesso de água com papel de filtro. </li><li> Adicionar derretido montagem agarose ao redor da amostra como no passo 1.2.6. </li></ol> 4. AFM Preparação e calibração de sensibilidade (para uma JPK NanoWizard AFM) <ol><li> Montar um cantilever na AFM. O balanço deve ser montado com penetrador em forma redonda. O raio vai determinar a, y, z x resolução e a profundidade de penetração no qual a medição exacta poderia ser arquivados. <ol><li> Para fazer medições meso-escala nanométrica na superfície da epiderme, use um penetrador rodada 50 raio nm; para fazer medições de mesoescala, use um raio mM penetrador rodada 0,5; para fazer medições em microescala (através 2-3 células), use um penetrador rodada 2,5 mM raio. </li></ol></li><li> Posicionar o laser na ponta do braço de suporte. Alinhar o laser para o meiodo captor através do espelho. </li><li> Coloque um copo limpo sob o AFM. NOTA: O seguinte é definido para transformar o sinal da posição do laser sobre o receptor de AFM em uma deformação do cantilever e, avaliando a constante do cantilever primavera, para traduzi-lo em uma força. </li><li> Aproximação com uma força-alvo &quot;set point&quot; de 1 V. </li><li> Selecione &quot;espectroscopia de força&quot;. Faça um recorte, definindo o máximo de &quot;set point em relação&quot; a 2 V, o &quot;estender velocidade&quot; a 40 mM / seg, eo &quot;comprimento z&quot; de 5 mm. </li><li> A partir do menu &quot;calibração manager&quot;, selecione a parte linear da curva para a frente para se ajustar à sensibilidade utilizando o botão &quot;select ajuste faixa&quot;. O &quot;ponto de ajuste relativa&quot; deve ser transformado de &quot;volt&quot; para &quot;meter&quot;. </li><li> A partir do menu &quot;Gerenciador de calibração&quot;, selecionar &quot;constante da mola&quot; para avaliar a elasticity do cantilever usando flutuações térmicas. Pressione o botão &quot;run&quot; para obter o enredo densidade espectral do cantilever. Encontre o pico de ressonância com a freqüência mais baixa. Coloque-o usando o botão &quot;selecionar ajuste faixa&quot;. NOTA 1: Para mais detalhes sobre o ajuste de flutuações térmicas, leia 15 Cook et al. NOTA 2: É preferível utilizar um método directo para avaliar a elasticidade do braço de suporte através de um braço de suporte de referência (ou material com rigidez conhecida). O usado aqui foi gentilmente doado por Atef Asnacios 16. </li><li> Adicione 200 ml de solução de manitol a 10% sob a ponta do cantilever. Realinhar o laser para o meio do captador usando o espelho. </li><li> Recalibrar a sensibilidade: a partir do menu &quot;calibração manager&quot;, clique no botão &quot;desconhecido&quot;; repita os passos 2.3 até 2.5. </li></ol> . 5 Aquisição de Dados: Módulo Cartografia aparente de Young <ol&gt; <li> Coloque as amostras montadas sob a AFM e adicionar uma gota de 500 mL de uma solução de manitol a 10% para a amostra. A solução de manitol plasmolyzes as células dentro de minutos. </li><li> Aproximação com uma força-alvo (&quot;set point&quot;), de 20 nN (&quot;set point&quot;, não deve ser superior a 3 V) </li><li> Recuo com um &quot;ponto de ajuste relativa&quot; de 200 nN, um &quot;estender velocidade&quot; de 40 mM / s, e &quot;comprimento z&quot; de 5 mm. </li><li> Ajustar o &quot;set point relativa&quot;, a fim de obter uma reentrância de 100 nm a 200 nm, para a montagem do cantilever ou 0,5 mm para o braço de suporte montado 1 mM / 5 um. </li><li> No modo de &quot;mapeamento de força&quot;, selecione uma região para analisar: por meristemas, 70 mM x 70 mM com uma resolução (&quot;pixels&quot;) de 64 x 64; para hipocótilos e das raízes, 100 um x 100 mm com uma resolução de 64 x 64. </li><li> Pressione Digitalizar para iniciar os experimentos. Salve a saída. </li&gt; </ol> . 6 Análise de Dados: Cálculos aparente módulo de Young <ol><li> Abra o arquivo de dados no software de análise de dados. </li><li> Selecione &quot;processamento em lote&quot; para aplicar a mesma análise para todas as curvas de um único mapa. </li><li> Seleccionar &quot;corrigir a altura para a flexão do braço de suporte&quot; de modo a extrair a profundidade de penetração. </li><li> Seleccionar &quot;módulo de função ajuste de Young&quot; que vai assumir a força é proporcional à área de reentrância. Defina a &quot;forma tip&quot; para parabólica para assumir que a forma é um recuo parabólica. </li><li> Indique o raio da ponta. </li><li> Escolha preferencialmente a curva &quot;retract&quot; para a análise, porque é menos sensível a topografia do que a curva &quot;estender&quot;. No entanto, se há aderência significativa da sonda durante a &quot;retração&quot;, use a curva &quot;estender&quot;, que é insensível a degola. </li><li> Conjuntoa relação de Poisson para 0,5. Imprensa em analisar e salvar a saída. </li></ol>

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+in+vitro+extensibility+of+growing+plant+cell+walls.">Measuring in vitro extensibility of growing plant cell walls.</a> Methods in molecular biology. 715, 291-303 (2011).</li><li>Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+plant+cell+wall+extension+(creep)+induced+by+acidic+pH+and+by+alpha-expansin.">Measuring plant cell wall extension (creep) induced by acidic pH and by alpha-expansin.</a> Journal of visualized experiments : JoVE. , 1263 (2009).</li><li>Durachko, D. a. n. i. e. l. M., C, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Plant+Cell+Wall+Extension+(Creep)+Induced+by+Acidic+pH+and+by+Alpha-Expansin.">Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin.</a> J. Vis. Exp.. , 25 (2009).</li><li>Suslov, D., Verbelen, J. P., Vissenberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onion+epidermis+as+a+new+model+to+study+the+control+of+growth+anisotropy+in+higher+plants.">Onion epidermis as a new model to study the control of growth anisotropy in higher plants.</a> Journal of experimental botany. 60, 4175-4187 (2009).</li><li>Parre, E., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+and+the+role+of+the+physical+properties+of+the+cell+wall+in+pollen+tube+growth+of+Solanum+chacoense.">Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense.</a> Planta. 220, 582-592 (2005).</li><li>Zerzour, R., Kroeger, J., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+growth+in+pollen+tubes+is+associated+with+spatially+confined+dynamic+changes+in+cell+mechanical+properties.">Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties.</a> Developmental biology. 334, 437-446 (2009).</li><li>Radotic, K., 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=Atomic+force+microscopy+stiffness+tomography+on+living+Arabidopsis+thaliana+cells+reveals+the+mechanical+properties+of+surface+and+deep+cell-wall+layers+during+growth.">Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.</a> Biophysical journal. 103, 386-394 (2012).</li><li>Milani, P., 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=In+vivo+analysis+of+local+wall+stiffness+at+the+shoot+apical+meristem+in+Arabidopsis+using+atomic+force+microscopy.">In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy.</a> The Plant journal : for cell and molecular biology. 67, 1116-1123 (2011).</li><li>Peaucelle, A., 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=Pectin-induced+changes+in+cell+wall+mechanics+underlie+organ+initiation+in+Arabidopsis.">Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis.</a> Current biology : CB. 21, 1720-1726 (2011).</li><li>Braybrook, S. A., Hofte, H., Peaucelle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mechanical+contributions+of+the+pectin+matrix:+Insights+for+cell+growth.">Probing the mechanical contributions of the pectin matrix: Insights for cell growth.</a> Plant signaling &amp; behavior. 7, 1037-1041 (2012).</li><li>Routier-Kierzkowska, A. L., 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=Cellular+force+microscopy+for+in+vivo+measurements+of+plant+tissue+mechanics.">Cellular force microscopy for in vivo measurements of plant tissue mechanics.</a> Plant physiology. 158, 1514-1522 (2012).</li><li>Agudelo, C. G., 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=TipChip:+a+modular,+MEMS-based+platform+for+experimentation+and+phenotyping+of+tip-growing+cells.">TipChip: a modular, MEMS-based platform for experimentation and phenotyping of tip-growing cells.</a> The Plant journal : for cell and molecular biology. 73, 1057-1068 (2013).</li><li>Miedes, E., 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=Xyloglucan+endotransglucosylase/hydrolase+(XTH)+overexpression+affects+growth+and+cell+wall+mechanics+in+etiolated+Arabidopsis+hypocotyls.">Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls.</a> Journal of experimental botany. 64, 2481-2497 (2013).</li><li>Byrne, M. E., 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=Asymmetric+leaves1+mediates+leaf+patterning+and+stem+cell+function+in+Arabidopsis.">Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis.</a> Nature. 408, 967-971 (2000).</li><li>Cook, S. M., 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=Practical+implementation+of+dynamic+methods+for+measuring+atomic+force+microscope+cantilever+spring+constants.">Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants.</a> Nanotechnology. 17, 20135-22145 (2006).</li><li>Desprat, N., Richert, A., Simeon, J., Asnacios, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creep+function+of+a+single+living+cell.">Creep function of a single living cell.</a> Biophysical journal. 88, 2224-2233 (2005).</li><li>Peaucelle, A., Braybrook, S., Hofte, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+mechanics+and+growth+control+in+plants:+the+role+of+pectins+revisited.">Cell wall mechanics and growth control in plants: the role of pectins revisited.</a> Frontiers in plant science. 3, 121 (2012).</li><li>Mittler, 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=ROS+signaling:+the+new+wave.">ROS signaling: the new wave.</a> Trends in plant science. 16, 300-309 (2011).</li><li>Braybrook, S. A., Peaucelle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechano-chemical+aspects+of+organ+formation+in+Arabidopsis+thaliana:+the+relationship+between+auxin+and+pectin.">Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin.</a> PLoS. 8, e57813 (2013).</li><li>Asnacios, A., Hamant, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanics+behind+cell+polarity.">The mechanics behind cell polarity.</a> Trends in cell biology. 22, 584-591 (2012).</li><li>Meister, A., 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=FluidFM:+combining+atomic+force+microscopy+and+nanofluidics+in+a+universal+liquid+delivery+system+for+single+cell+applications+and+beyond.">FluidFM: combining atomic force microscopy and nanofluidics in a universal liquid delivery system for single cell applications and beyond.</a> Nano letters. 9, 2501-2507 (2009).</li><li>Lintilhac, P. M., Wei, C., Tanguay, J. J., Outwater, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ball+tonometry:+a+rapid,+nondestructive+method+for+measuring+cell+turgor+pressure+in+thin-walled+plant+cells.">Ball tonometry: a rapid, nondestructive method for measuring cell turgor pressure in thin-walled plant cells.</a> Journal of plant growth regulation. 19, 90-97 (2000).</li><li>Kroeger, J. H., Zerzour, R., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+or+driving+force?+The+role+of+turgor+pressure+in+oscillatory+plant+cell+growth.">Regulator or driving force? The role of turgor pressure in oscillatory plant cell growth.</a> PloS one. 6, e18549 (2011).</li><li>Forouzesh, E., Goel, A., Mackenzie, S. A., Turner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+extraction+of+Arabidopsis+cell+turgor+pressure+using+nanoindentation+in+conjunction+with+finite+element+modeling.">In vivo extraction of Arabidopsis cell turgor pressure using nanoindentation in conjunction with finite element modeling.</a> The Plant journal : for cell and molecular biology. 73, 509-520 (2013).</li></ol>

Os autores não têm nada a revelar.

Nas plantas, a mudança de propriedades mecânicas desempenham um papel importante na orientação do crescimento e morfogénese. Até à data, tem havido um grande progresso em desvendar as redes genéticas e químicas que controlam o crescimento das plantas, mas nosso conhecimento de como essas redes contribuem e são afetadas por mudanças nas propriedades mecânicas é rudimentar. Este método deve permitir-nos para preencher esta lacuna, e por isso deve ser de grande interesse para os cientistas que estudam qualque...

Crescimento em plantas é conseguido pela expansão coordenada das paredes celulares rígidas que cercam cada uma e todas as células do organismo. Acumulando evidência indica que é através da modificação química da parede celular de plantas que controlar localmente essa expansão. A expansão é pensado para ser conduzido principalmente por pressão sobre as paredes das células, provocadas por uma elevada pressão de turgescência da célula; esta resposta de tensão para a pressão de turgescência é governada pelas propriedades mecânicas das paredes das células 1. Pouco se sabe sobre estas propriedades mecânicas e como eles mudam durante o desenvolvimento. Além disso, pouco se sabe sobre a forma como estas propriedades mecânicas são controlados e se opiniões contribuir para alterar a química da parede celular de um modo que aparentemente é coordenado através de um tecido. Se quisermos compreender a relação entre as alterações químicas e mecânicas na parede celular das plantas durante o desenvolvimento e, finalmente, como essas interações microscópicas governar uma plantaO crescimento macroscópico, um método que pode controlar as propriedades mecânicas das paredes das células no desenvolvimento de órgãos à escala celular ou de tecido é necessária. O método de microscopia de força atómica (AFM), descrito aqui, o qual é baseado em micrométricas ou nanométricas compressões de tecidos ou cavidades, foi desenvolvido para medir com precisão as características mecânicas de paredes celulares no desenvolvimento de órgãos simultaneamente com resoluções subcelulares e entre as regiões inteiras de tecido. Outros métodos têm uma resolução que é muito baixo ou muito alto: o extensômetro só é capaz de medir as propriedades mecânicas médias de um tecido inteiro na escala milimétrica 2-4, numa escala que é, por exemplo muito grande para medir os primeiros eventos organogênese; o microindenter pode fazer medições em resolução subcelular em escala nanométrica, mas é restrita a medição células isoladas e não grupos de células ou órgãos 5-7. Com a AFM, a exigirtecido d, celulares e subcelulares resoluções podem ser alcançados 8-10. Recentemente, vários protocolos têm sido desenvolvidos especificamente para medir mecânica do tecido de plantas que também poderiam ser utilizados 11, 12. Vamos apresentar aqui como avaliar a elasticidade do tecido através da medição do módulo aparente de Young 13. O módulo de elasticidade é utilizada para descrever a rigidez de um material. Durante a pequena deformação a força necessária para deformar um material é proporcional à área de reentrância. O módulo de Young é este coeficiente. No caso de um material homogéneo contínuo com o mesmo coeficiente será medido, independentemente do tipo de recuo (tamanho e forma) mas mudará com a velocidade da medição. No caso de a estrutura complexa do tecido de plantas, temos observado até agora que a força é proporcional à deformação, permitindo a determinação deum coeficiente de proporcionalidade que chamamos &quot;jovem módulo aparente&quot;. Em contraste a partir de meios contínuos nas plantas, este novo módulo aparente é sensível ao tamanho do recuo. Ele não corresponde ao novo módulos de uma parede celular puro. Ela descreve melhor a elasticidade do andaime da parede celular do tecido de.

<table border="0" cellpadding="0" cellspacing="0" width="850">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">NanoWizard</td>
			<td style="width:167px;">All the 3-generation are abele to do the work withe the same preferment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM stage</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">CellHesion</td>
			<td>Required for sample withe low topography (les then 11&#181;m between the lowest and the highest point in the aria of force scanning).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM optics</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">Top View Optics&#160;</td>
			<td>Very important in order to position the sample. Cold be replaces by long range a binocular or microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo Microscopes&#160;</td>
			<td style="width:348px;">Leica</td>
			<td style="width:119px;">M125</td>
			<td>Any type of stereo microscopes could do.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">150nm mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td style="width:119px;">R150-NCL-10</td>
			<td>To measure only the cell wall at the surface of the epidermis use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1&#181;m mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>SD-Sphere-NCH-S-10&#160;</td>
			<td>to measure the mechanics of the cell wall orthogonal to the surface of the epidermis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tipless cantiliver</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>TL-NCH-20&#160;</td>
			<td>to measure the local mechanics of the tissue (2-3 cell wide) use a 5&#181;m mounted cantilever. We attached a 5&#181;m borasilicate bead to a tipless cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5&#181;m silicon microspheres</td>
			<td style="width:348px;">Corpuscular</td>
			<td style="width:119px;">C-SIO-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aradilte&#160;</td>
			<td style="width:348px;">Bartik S.A. 77170 Coubet France</td>
			<td style="width:119px;"></td>
			<td>Aradilte for fixing the bead to the tip les cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;low melting Agarows</td>
			<td style="width:348px;">Fishersci Fair Lawn , new jersey 07410</td>
			<td style="width:119px;">BP160-100</td>
			<td>34-45 Gelation Temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">D-Mannitol</td>
			<td style="width:348px;">Sigma-Aldrich, 3050 Spruce Street, St Louis Mo 63103 USA)</td>
			<td style="width:119px;">M4125-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2&#160; Stainless Steel No. 5 Tweezers</td>
			<td style="width:348px;">Ideal-Tek 6828 Balerna Switzerland&#160;</td>
			<td align="right">951199</td>
			<td></td>
		</tr>
	</tbody>
</table>

afm-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions

Nós descrevemos um método recentemente desenvolvido para medir as propriedades mecânicas das superfícies dos tecidos vegetais através de microscopia de força atômica (AFM) micro / nano-recuos, para um JPK AFM. Especificamente, neste protocolo medimos o módulo de Young aparente das paredes celulares em resoluções subcelulares em todas as regiões de até 100 mm x 100 mm em meristemas florais, hipocótilos e raízes. Isto requer uma preparação cuidadosa da amostra, a correta seleção de micro-penetradores e profundidades recuo. Para ter em conta as propriedades da parede celular só, as medições são realizadas em soluções altamente concentradas de manitol de modo a plasmolyze as células e, assim, remover a contribuição da pressão de turgescência celular. Em contraste com outras técnicas existentes, por meio de diferentes penetradores e profundidades de entalhe, este método permite a medição simultânea multiscale, <em&gt; Ou seja em resoluções subcelulares e em centenas de células que compõem um tecido. Isso significa que agora é possível caracterizar espacialmente temporalmente as mudanças que ocorrem nas propriedades mecânicas da parede celular durante o desenvolvimento, permitindo que essas alterações sejam correlacionados com o crescimento e diferenciação. Isto representa um passo fundamental para compreender como as alterações celulares microscópicas coordenados trazer eventos morfogenéticos macroscópicos. No entanto, várias limitações permanecem: o método pode ser usado apenas em relativamente pequenas amostras (cerca de 100 um de diâmetro) e apenas em tecidos externos; o método é sensível à topografia do tecido; ele mede apenas certos aspectos das propriedades mecânicas complexas do tecido. A técnica está a ser desenvolvida rapidamente e é provável que a maioria destas limitações sejam ultrapassadas em breve.

Na Figura 1 apresenta-se típicas Jovens mapas de módulos de meristemas florais (Figuras 1a e 1b), jovens e velhos hipocotiledonar (Figuras 1C-F) e meristema da raiz (Figura 1G e 1H). Em todos os experimentos o penetrador é hemisférica, mas seu raio é diferente, de modo que diferentes resoluções espaciais podem ser alcançados Figuras 1C e 1D mostram resultados típicos para penetradores meso-esca...

Watch this Scientific Journal Video about AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions at JoVE.com

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapeamento baseado em AFM das propriedades elásticas de paredes celulares: em tecido, os celulares, e as Resoluções Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

afm-based-mapping-elastic-properties-cell-walls-at-tissue-cellular

Research

JoVE Journal

Biology

12.8K visualizações.  Université Paris Diderot. We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Vídeo: AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Ensaio Co Sedimentação; para a Análise da proteína de ligação a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Biologia

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Métodos única molécula para acompanhar as alterações nas propriedades elásticas bicamada

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte II: Os hidratos de carbono

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Um canal de gravação de patch-clamp anexado-Cell

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tensão e cálcio Dual Channel Mapping Optical do Cultivadas HL-1 Atrial miócitos Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissecção da Auditivo Bulla em ratos pós-natais: Isolamento dos ossos do ouvido médio e da análise histológica

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Imagem celular ao vivo super-resolução de estruturas subcelulares

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

Quantificação de Densidades Celulares e Propriedades Antigênicas usando Levitação Magnética

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...