Tim Daley e Andrew Smith forneceu muitas discussões computacionais úteis durante os primeiros dias; Tim Daley escreveu o rotate_translate_cylindrical programa necessário para o protocolo 5. recursos computacionais foram fornecidos pelo High Performance Cluster Computing na Universidade do Sul da Califórnia. Este trabalho foi financiado pelo NIH conceder # GM098536 (MDD).

Todos os procedimentos e pessoal foram aprovados pela universidade do instituto do sul da Califórnia para Animal Care e do Comitê Use (IACUC), protocolo nº 11394. 1. Baculum Dissecção e Preparação <ol><li> Euthanize um rato macho sexualmente maduros através de dióxido de carbono sobre-exposição, de acordo com protocolos estabelecidos pelo Comité pertinente Institucional animal Cuidado e Uso (IACUC). </li><li> Coloque o animal em decúbito dorsal, e prolongar o pénis através da aplicação de pressão com os polegares lateralmente à abertura do prepúcio. </li><li> Uma vez que o pénis é prolongada, alargar o tecido através do prepúcio, tanto quanto possível. </li><li> Com uma tesoura, corte o proximal do corpo do pénis à glande do pénis onde o baculum reside. </li><li> Transfira os pénis dissecados para um tubo de 1,7 ml e adicionar 200 mL de água da torneira. Certifique-se de que o pênis está totalmente submersa no líquido. </li><li> Incubar o tecido em água a ~ 50 ° C durante 3-5 dias. </li><li&gt; Após incubação adequada, remova o tecido circundante do báculo, usando uma pinça sob um microscópio de dissecação. Gentilmente esguichar 70% de etanol para empurrar restante tecido e limpar o osso. </li><li> Colocar o báculo dissecados para um novo tubo de microcentrifugação com a tampa aberta. Deixe tampão aberto O / N a secar óssea. </li></ol> Digitalização 2. MicroCT <ol><li> Pressione um suporte cilíndrico digitalização microCT em um tijolo de espuma de florista para criar um cilindro de espuma de florista. </li><li> Extraia o cilindro de espuma de florista e cortar fatias ~ 2-5 cm de espessura. </li><li> Empurrar bacula seca na espuma florista, em torno da periferia de uma fatia individual a minimizar a interferência durante a verificação. A orientação precisa dos ossos deve ser comentou permitindo a identificação adequada das amostras individuais no Protocolo 4. </li><li> Delicadamente, coloque a fatia com os ossos incorporados no suporte para microCT. </li><li> Adquirir scans microCT. No caso de bacula rato 14 </sup&gt;, Foi utilizado um scanner uCT50 (Scanco Medical AG, Bruttisellen, Suíça) na USC Molecular Imaging Center sob as seguintes configurações: 90 kVp, 155 mA, 0,5 filtro mm Al, 750 projecções por 180 (cobertura de 360), tempo de exposição de 500 ms e tamanho voxel de 15,5 mm. </li></ol> 3. Processamento MicroCT: Convertendo um Stack .dcm para um arquivo .xyz Individual NOTA: Cada varredura microCT produz uma pilha de .dcm, ou &quot;DICOM&quot;, arquivos que representam fatias de imagem obtidos através do objeto. Toda a geometria computacional jusante requer arquivos .xyz planas, que é simplesmente um arquivo de texto que contém quatro colunas - X, Y e Z coordenadas de cada pixel, e a intensidade do pixel, que vão desde -5000 (preto) para 5000 (branco). Um limiar do pixel acima de 3.000 geralmente funciona bem como um limiar para a definição de ossos. <ol><li> Instalar Python (www.python.org) e os módulos Python COMANDOS, DICOM, Pylab, SYS e Numpy. </li><li> Open &quot;01_process_dicom.py &quot;{} Figshare com qualquer editor de texto. Na seção Variáveis, caminho de mudança, limites de pixels, e nomes de diretórios, conforme necessário. </li><li> Execute &quot;01_process_dicom.py python&quot;. Progress será impresso para a tela. Dentro de cada diretório nomeado na etapa 3.2, um novo arquivo é chamado; por exemplo, directory_name.PT3000.xyz, onde PT3000 indica o limiar do pixel indicado no passo 3.2. </li></ol> 4. Processamento MicroCT: Segmentando-out individual Specimen .xyz Arquivos <ol><li> Instale R (https://www.r-project.org/) com o RGL biblioteca. </li><li> Abra o arquivo &#39;02_segment_dicoms.r&#39; {} Figshare com qualquer editor de texto. Na seção Variáveis, mudar o nome do caminho para apontar para o arquivo .xyz criado no Protocolo n.º 3 acima. </li><li> De dentro R, execute a &quot;fonte ( &#39;02_segment_dicoms.r&#39;)&quot; comando (sem as aspas). </li><li> Após a imagem tridimensional do .xyz criado no Protocolo n.º 3 aparecer, digite o number de espécimes no arquivo geral .xyz. Em seguida, rotular e selecionar os pontos de cada amostra, utilizando as funções de rolagem e zoom. NOTA: No fundo, arquivos .xyz separadas será feita para cada espécime. Estes aparecem em um diretório chamado, por exemplo, XYZ_FILES_PT3000, onde PT3000 indica o limite de pixel usado. </li></ol> 5. &quot;Alinhar&quot; Specimen .xyz arquivos para Coordenadas Comum. <ol><li> Abra o script &quot;03_transform.py&quot; Python {Figshare}, que exige a mattdean_modules.py adicional módulo {Figshare}, bem como duas aplicações autónomas: &quot;rotate_translate_cylindrical&quot; (https://github.com/timydaley/dean_cylindrical_tranform) e &quot;qconvex&quot; (www.qhull.org/html/qconvex.htm) que são usados ​​por este script. </li><li> Na seção Variáveis, identificar os nomes caminho completo para mattdean_modules.py, rotate_path e qconvex_dir. Além disso, identificar o caminho completo para o diretório que contém o .xy indivíduoarquivos z criou na etapa 4. </li><li> 03_transform.py executado, o que cria um novo arquivo por espécime com o sufixo .TRANSFORMED.xyz. </li></ol> 6. &quot;fatiamento&quot; Specimen Alinhados .xyz Arquivos identificar Semi-marcos. <ol><li> Abrir e executar o script Python &quot;04_identify_landmarks.py&quot; {} Figshare. Na seção Variáveis, identificar nomes de caminho completo para o diretório que contém os arquivos .TRANSFORMED.xyz. Este script identifica 802 semi pontos de referência que pode ser usado para quantificar o tamanho e forma da estrutura. </li></ol>

<ol>
	<li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrics+morphometrics.">Geometrics morphometrics.</a> Annu. Rev. Anthropol. 36, 261-281 (2007).</li><li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. 6, (2005).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2012).</li><li>Bookstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Morphometric tools for landmark data: geometry and biology. , (1991).</li><li>Rohlf, F. J., Marcus, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Revolution+in+Morphometrics.">A Revolution in Morphometrics.</a> Trends. Ecol. Evol. 8 (4), 129-132 (1993).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D., Fink, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2004).</li><li>Rohlf, F. J., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensions+of+the+Procrustes+method+for+the+optimal+superimposition+of+landmarks.">Extensions of the Procrustes method for the optimal superimposition of landmarks.</a> Syst. Zool. 39 (1), 40-59 (1990).</li><li>Gunz, P., Mitteroecker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semilandmarks:+a+method+for+quantifying+curves+and+surfaces.">Semilandmarks: a method for quantifying curves and surfaces.</a> Hystrix. 24 (1), 103-109 (2013).</li><li>Gunz, P., Ramsier, M., Kuhrig, M., Hublin, J. J., Spoor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+bony+labyrinth+reconsidered,+introducing+a+comprehensive+geometric+morphometric+approach.">The mammalian bony labyrinth reconsidered, introducing a comprehensive geometric morphometric approach.</a> J. Anat. 220 (6), 529-543 (2012).</li><li>Mitteroecker, P., Gunz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+geometric+morphometrics.">Advances in geometric morphometrics.</a> Evol. Biol. 36 (2), 235-247 (2009).</li><li>Bookstein, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landmark+methods+for+forms+without+landmarks:+morphometrics+of+group+differences+in+outline+shape.">Landmark methods for forms without landmarks: morphometrics of group differences in outline shape.</a> Med. Im. Anal. 1 (3), 225-243 (1997).</li><li>Gunz, P., Mitteroecker, P., Bookstein, F., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. , 73-98 (2005).</li><li>Oxnard, C., O'Higgins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+Clearly+Needs+Morphometrics.+Does+Morphometrics+Need+Biology?.">Biology Clearly Needs Morphometrics. Does Morphometrics Need Biology?.</a> Biological Theory. 4 (1), 84-97 (2009).</li><li>Schultz, N. 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=The+genetic+basis+of+baculum+size+and+shape+variation+in+mice.">The genetic basis of baculum size and shape variation in mice.</a> G3. 6 (5), 1141-1151 (2016).</li><li>Schultz, N. G., Lough-Stevens, M., Abreu, E., Orr, T. J., Dean, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculum+was+gained+and+lost+multiple+times+during+mammalian+evolution.">The baculum was gained and lost multiple times during mammalian evolution.</a> Integr Comp Biol. 56 (4), 644-656 (2016).</li><li>Dines, J. 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=Sexual+selection+targets+cetacean+pelvic+bones.">Sexual selection targets cetacean pelvic bones.</a> Evolution. 68 (11), 3296-3306 (2014).</li><li>Adams, D. C., Ot&#225;rola-Castillo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=geomorph:+an+R+package+for+the+collection+and+analysis+of+geometric+morphometric+shape+data.">geomorph: an R package for the collection and analysis of geometric morphometric shape data.</a> Methods Ecol. Evol. 4 (4), 393-399 (2013).</li></ol>

The authors declare that they have no competing financial interests.

As etapas críticas do protocolo acima são: 1) dissecar o bacula, 2) coleta as imagens microCT, 3) converter a saída microCT para um arquivo plano de coordenadas XYZ, 4) segmentar a nuvem de pontos de cada espécime, 5) transformar cada amostra para um padronizado sistema de coordenadas, e 6) que define semi-marcos. Estes passos são facilmente modificado para acomodar diferentes objectos. Estes métodos podem provavelmente ser aplicado a qualquer objecto que, pelo menos, não é, essencia...

O campo de morfometria inclui uma diversidade de métodos para quantificar o tamanho e a forma da forma biológica, uma etapa fundamental na investigação científica 1, 2, 3, 4, 5, 6. Tradicionalmente, a análise estatística de tamanho e forma começa pela identificação de pontos de referência em uma estrutura biológica, e depois medir distâncias lineares, ângulos e proporções, que podem ser analisadas num quadro multivariada. -Marco com base geométrica Morfometria é uma abordagem que mantém a posição espacial de pontos, preservando informações geométricas de coleta de dados por meio de análise e visualização 5. Análise Procrustes Generalizada (GPA) pode ser aplicada para remover a variação na localização, dimensão e rotação dos pontos de referência para produzir um alinhamento entre amostras que a Minimizes suas diferenças ao quadrado - o que resta é a forma dissimilaridade 7. Um conceito importante de qualquer análise morfométrica é homologia, ou a ideia de que se pode identificar com segurança marcos representando características biologicamente significativas e discretas que correspondem entre amostras ou estruturas. Por exemplo, crânios humanos têm processos homólogos, forame, suturas, e dutos que podem permitir análises morfométricas. Infelizmente, a identificação de pontos de referência correspondente é difícil em muitas estruturas biológicas, especialmente aqueles com superfícies lisas ou curvas 8, 9, 10. Nós abordar este problema a seguir utilizando geometria computacional. O fluxo geral é para gerar uma análise tridimensional do objecto que pode ser representada como uma nuvem de pontos, e em seguida rodar e que transformar nuvem de pontos de modo a que todos specimens são orientados em um sistema de coordenadas comum. Então nós definimos matematicamente semi-marcos de regiões específicas do objeto. Discretas semi pontos de referência colocadas em tais regiões são biologicamente arbitrário 11. Realização GPA e as análises estatísticas subsequentes podem produzir artefatos indesejáveis 8, 12, porque os marcos colocados arbitrariamente pode não ser biologicamente homóloga. Portanto, permitimos que estas meias-marcos para matematicamente &quot;slides&quot;. Este procedimento minimiza a diferença de potencial entre as estruturas. Como argumentei o algoritmo de correr aqui utilizada é apropriada para quantificar regiões anatômicas semelhantes faltam facilmente identificados marcos 3, 6, 8, 10, 11, 12 correspondente. Esses métodos têm o seu limitations 13, mas deve ser adaptável para objectos de tamanho e forma diferentes. Aqui, nós ilustrar como este método foi aplicado em um estudo recente da baculum rato 14, um osso no pênis que tem sido ganhou e perdeu várias vezes independentes durante a evolução dos mamíferos 15. Discute-se a dissecção e preparação de um osso específico, o báculo (Protocolo 1), a geração de imagens microCT (protocolo 2), ea conversão destas imagens para um formato que permite que toda a geometria computacional jusante (Protocolos 3 e 4). Após estas etapas, cada amostra é representado por ~ 100K xyz coordenadas. Em seguida, a pé através de uma série de transformações que efetivamente alinhar todas as amostras em uma orientação comum (Protocolo 5), em seguida, definir semi-marcos de espécimes alinhados (Protocolo 6). Protocolos 1-4 deve ser semelhante, independentemente do objeto analisado. O protocolo nº 5 e Protocolo 6 são specamente projetado para um báculo, mas é nossa esperança que, detalhando estes passos, os investigadores podem imaginar modificações que seriam relevantes para o seu objeto de interesse. Por exemplo, foram aplicadas modificações destes métodos para estudar ossos pélvicos baleia e costelas 16.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting scissors</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">470106-338</td>
			<td style="width:167px;">Most sizes should work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting Forceps, Fine Tip, Curved</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">82027-406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">1.7 mL microcentrifuge tube</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">87003-294</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Absolute Ethanol</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:79px;">CAS 64-17-5</td>
			<td>To be diluted to 70% for dissections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Floral Foam</td>
			<td style="width:287px;">Wholesale Floral</td>
			<td style="width:79px;">6002-48-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">uCT50 scanner&#160;</td>
			<td colspan="2">Scanco Medical AG, Bruttisellen, Switzerland</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection, microct scanning and morphometric analyses of the baculum

morfometria modernos fornece métodos poderosos para quantificar o tamanho e forma variação. Um requisito básico é uma lista de coordenadas que definem marcos; no entanto, tais coordenadas devem representar estruturas homólogas em toda espécimes. Enquanto muitos objetos biológicos consistem de marcos facilmente identificadas para satisfazer a hipótese de homologia, muitos não têm tais estruturas. Uma possível solução é matematicamente lugar semi-marcos em um objeto que representam a mesma região morfológica através de espécimes. Aqui, nós ilustrar um gasoduto recentemente desenvolvido para definir matematicamente semi-marcos do rato baculum (osso do pênis). Os nossos métodos deve ser aplicável a uma vasta gama de objectos.

As coordenadas xyz das meias-marcos produzidos no Protocolo 6 pode ser diretamente importado para qualquer morfometria geométrica análise baseada no marco 17. O oleoduto computacional supra foi aplicado para estudar bacula rato 14, bem como pélvica baleia e as costelas 16. Mais detalhes sobre a definição computacional de semi-marcos são apresentados aqui, em uma tentativa de ajudar os pesquisadores a visualizar...

Watch this Scientific Journal Video about Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum at JoVE.com

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Muitas estruturas biológicas falta marcos facilmente definíveis, o que torna difícil a aplicação de métodos morfométricos modernos. Aqui, ilustramos métodos para estudar o báculo rato (um osso do pénis), incluindo dissecção e digitalização microCT, seguido por métodos computacionais para definir semi-chave que são utilizadas para quantificar a forma e tamanho variação.

Dissecação, digitalização MicroCT e morfométricas Análises do Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

dissection-microct-scanning-and-morphometric-analyses-of-the-baculum

Research

JoVE Journal

Bioengineering

7.5K visualizações.  University of Southern California (USC).  Muitas estruturas biológicas falta marcos facilmente definíveis, o que torna difícil a aplicação de métodos morfométricos modernos. Aqui, ilustramos métodos para estudar o báculo rato (um osso do pénis), incluindo dissecção e digitalização microCT, seguido por métodos computacionais para definir semi-chave que são utilizadas para quantificar a forma e tamanho variação. 

Vídeo: Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Preparação de amostras, Imaging, and Analysis Protocolos para Faca de ponta microscopia de varredura

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Bioengenharia

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Vídeo taxa de microscopia de varredura confocal e Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Avaliação de Biomateriais para ampliação vesical utilizando análises cistométricos Em modelos de roedores Vários

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Preparação de Ganglia para Análises Eletrofisiológicas e Moleculares de Neurônios Únicos

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Grande área varredura sonda Nanolitografia facilitada pelo alinhamento automatizado e sua aplicação para fabricação de substrato para estudos de cultura celular

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Digitalização de slides automatizado e segmentação em tecidos fluorescente-rotulado, usando um sistema de análise de Widefield alto teor

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodal volumétrico Retinal Imaging pelo oblíquo digitalização Laser Oftalmoscopia (oSLO) e a tomografia de coerência óptica (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

Morfométricos protocolo para a avaliação objetiva do comportamento de blastocisto durante a vitrificação e aquecimento passos

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Avaliação de comportamentos fotossintéticos por medições simultâneas de reflectância foliar e análises de fluorescência de clorofila

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Micromatrizes de biopartículas para análise quimiotática e molecular de swarming de nêutrons humanos in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...