Gostaríamos de agradecer a Gaby Martins e os organizadores do workshop EMBO2010 3D Desenvolvimento de imagem onde este trabalho começou e todos os contribuintes para os projetos de Fiji e ImageJ.

1. 4D Time-lapse experimentos com imagens As definições utilizadas para aquisição de imagem será diferente, dependendo do equipamento utilizado. A capacidade de microscopia confocal para a secção opticamente uma amostra depende de uma série de factores: o comprimento de onda de excitação, o tamanho do furo de pino, abertura numérica da objectiva, o índice de refracção da amostra e do meio no qual a amostra é incorporado. O tamanho do orifício confocal seleccionado irá determinar a espessura da secção óptica recolhido. Um orifício menor produzirá uma secção óptica mais fina aumentando a resolução do eixo z, mas reduzindo a quantidade de luz captada. Um orifício maior irá aumentar a espessura da secção óptica, reduzindo resolução eixo-z, mas aumentando a quantidade de luz captada. Outros fatores a serem considerados durante a coleta de dados 4D antes de correção incluem: <ol><li> Otimizar velocidade de digitalização de remover, ou limite, deriva durante a captura de um único ponto de tempo. Portanto, o tempo necessário para a recolha de imagens deve ser uma pequena fração do intervalo entre os pontos temporais. </li><li> Perfeitamente estruturas repetitivas, ou grelhas, não são adequados como estruturas de registo, uma vez que não é possível determinar a correcção necessária. Contas aleatoriamente distribuídos ou estruturas irregulares permitirá registro inequívoco. </li><li> Se o arrastamento da esperada, aumentar a área de leitura e os limites superiores e inferiores de focagem, a fim de garantir que a área de interesse permanece no interior da pilha de imagens. </li><li> Além de garantir que haja resolução espacial adequada para resolver as estruturas de interesse, definir a taxa de amostragem para fornecer resolução temporal para os eventos dinâmicos em estudo. Nota: O tempo entre a imagem pontos temporais devem, portanto, ser pelo menos metade do-intervalo de tempo entre eventos repetidos regulares e diminuir para eventos irregulares. </li></ol> 2. Abrindo o confocal Dataset O pacote de código aberto Fiji é uma distribuição do programa ImageJ, que contém os plug-ins pré-instalados para realizar inúmeros processos em dados coletados a partir de experimentos de microscopia. O software fornece fácil atualização arquitetura plug-in e inclui uma cópia do correto 3D deriva plug-in usado para esse protocolo. O software suporta a importação de uma vasta gama de formatos de imagem de microscopia de propriedade através da utilização de Bio-Formatos de importação plug-in do Aberto da Microscopia Ambiente. <ol><li> Instale o programa Fiji (http://fiji.sc/Downloads). </li><li> Carregue o conjunto de dados adquiridos usando o plugin do Importador LOCI Bio-Formatos. </li><li> Se o conjunto de dados é maior do que a memória alocada para o programa, selecione a opção &quot;Use pilha virtual&quot; dentro da seção de gerenciamento de memória. </li></ol> 3. Correção de Deriva de um objeto 3D em Pós-Processamento Durante o curso de um lapso de tempo prolongadoexperimentar uma amostra pode mover-se, mesmo quando incorporado. Para corrigir qualquer movimento e para permitir que os eventos de migração fotografada a ser analisada, de pós-processamento de imagens pode ser executada. Todo o processamento pós imagem deve ser claramente descrito na metodologia de qualquer análise derivado deste trabalho. <ol><li> Uma vez que o conjunto de dados foi carregado, execute o plug-in correto deriva 3D. </li><li> Se houver vários canais de imagem, selecione o canal a ser usado para registrar as imagens. Este deve, idealmente, representam uma estrutura estática dentro da amostra, em vez de quaisquer elementos migratórias ou móveis. No entanto, se isso não for possível o canal com o mínimo de movimento deve ser escolhido. </li><li> Se a memória RAM disponível no sistema utilizado para esta análise é inferior ao dobro do tamanho do conjunto de dados original, seleccionar a opção de pilha virtual utilização. Isto irá armazenar o hyperstack registrada como uma seqüência de imagens, em vez de salvar o arquivo para a memória RAM. <ol><li> Selecione uma pasta para o plug-in de saída individual corrigido imaarquivos Ges. Um arquivo de imagem separado será criado para cada canal em cada posição z. </li></ol></li><li> O plug-in, em seguida, conduzir uma análise de correlação de fase de pares entre cada ponto de tempo para determinar a necessária correcção que é então aplicada ao conjunto de dados. </li></ol>

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	<li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 42-42 (2004).</li><li>Collins, T. 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E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE transactions on image processing : a publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Kuglin, C. D., Hines, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+correlation+image+alignment+method.">The phase correlation image alignment method.</a> Proceedings of the IEEE, International Conference on Cybernetics and Society. , 163-165 (1975).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+motility+and+actin+cytoskeleton+of+individual+neurons+and+neural+crest+cells+in+zebrafish+embryos.">Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.</a> J VIs. 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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=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. 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Os autores declaram que não têm interesses financeiros concorrentes.

A nossa capacidade de usar o software de pós-processamento para corrigir desvio da amostra de conjuntos de dados obtidos a partir de experimentos de microscopia de lapso de tempo prolongados é limitado por uma série de fatores. A capacidade para discernir deriva contra movimento migratório de uma amostra é dependente dos marcadores celulares utilizados. Marcadores celulares que são ou amplamente expressas dentro de uma amostra ou não estão envolvidos em eventos migratórios durante a aquisição de imagem propor...

Imagiologia confocal é amplamente usado em biologia celular e de desenvolvimento para seguir os movimentos da célula e alterações na morfologia. Capturar uma série de secções ópticas em diferentes planos focais permite a geração de um modelo tridimensional (3D) de uma amostra, que pode ser estendido em quatro dimensões (4D), criando uma série de lapso de tempo de conjuntos de dados 3D. A geração de conjuntos de dados 4D permite a medição detalhada de movimentos e comportamentos celulares. Em experimentos de lapso de tempo de longo prazo, é comum observar o movimento da amostra. Isto pode ser causado por pequenas imprecisões no controlo da fase de hardware e de posições focais. Enquanto em outros casos, a deriva é um resultado de movimentos induzidos pelo crescimento da amostra ou flexibilidade dentro da mídia de montagem da amostra. Existem métodos para compensar ou limitar esses movimentos, incluindo melhorias nos sistemas com foco de hardware e aumento da rigidez do meio de montagem. No entanto, estas abordagens não pode ser aplicado em muitos casos devido à imagiologia set-se obrigados a fornecer as condições adequadas para a manutenção de amostras e crescimento. Soluções de software de código aberto que existem para a correção de movimento em 2D ao longo do tempo, através do uso das StackReg e TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/) 5 plugins no ImageJ ou Fiji, mas estes não podem ser aplicada a conjuntos de dados 4D. Para corrigir o desvio da amostra, temos desenvolvido um plug-in (Correto deriva 3D) para utilizar a plataforma de processamento de imagens de código aberto, Fiji 1. Nosso plug-in é capaz de realizar o registro de correlação de fase para corrigir movimento que ocorre como resultado da deriva amostra em experimentos de time-lapse tridimensionais. Correlação de fase 6 é um método computacional para determinar a tradução eficiente entre as imagens. O plug-in descrito aqui utiliza a biblioteca fase de correlação desenvolvido por Preibisch et al. 7. Em experiências de multi-canal, o plug-in utiliza um canal para determinaçãone a correção requerida. Esta correcção é aplicada então a quaisquer canais adicionais resultantes, o conjunto de dados de registo do 4D. No sistema modelo de peixe-zebra é possível realizar imagiologia de lapso de tempo ao longo de um período de várias horas ou mesmo vários dias 8. Um método comum para a montagem do peixe-zebra é para incorporar o embrião vivo anestesiados em baixo agarose ponto de fusão (0,8-1,5%), restringindo seu movimento 9-11. Enquanto o movimento é restringido o crescimento da amostra continua a ocorrer, o que resulta em que as células dentro do campo de vista da posição de deslocamento. A fim de seguir o movimento das células dentro do embrião, é necessário em primeiro lugar para corrigir o movimento de toda a amostra. Este protocolo foi desenvolvido com espécimes de peixe-zebra, e tem sido utilizado para o desenvolvimento de imagem somito 12, mas pode ser aplicada a qualquer conjunto de dados confocal 4D.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

A geração de quatro dimensões (4D) de conjuntos de dados confocal; consistindo de sequências de imagens em 3D ao longo do tempo; proporciona um excelente método para capturar comportamentos celulares envolvidos em processos de desenvolvimento. A capacidade de controlar e seguir os movimentos de células é limitado pelos movimentos das amostras que ocorrem devido à deriva da amostra ou, em alguns casos, o crescimento durante a aquisição de imagem. Células de rastreamento em conjuntos de dados afetados pelo desvio e / ou crescimento irá incorporar esses movimentos em qualquer análise da posição da célula. Isto pode resultar no movimento aparente de estruturas estáticas dentro da amostra. Portanto antes de rastreamento de celular, qualquer amostra de desvio deve ser corrigido. Usando o open source Fiji distribuição 1 de ImageJ 2,3 e as ferramentas loci incorporados 4, desenvolvemos o correto 3D deriva plug-in para remover movimento amostra errônea em conjuntos de dados confocal. Este protocolo compensa efetivamente para a tradução da amostra ou alterações no po focalção, utilizando correlação de fase para registrar cada ponto de tempo de um de quatro conjuntos de dados dimensionais confocal, mantendo a capacidade de visualizar e medir movimentos celulares sobre as experiências de lapso de tempo prolongados.

No peixe-zebra em desenvolvimento, as células musculares rápidas fundir em fibras multinucleadas das 19 horas após a fertilização (20 - palco somite) 13. A fim de visualizar o movimento de núcleos e de fusão das células do músculo que efectuados 4D confocal imaging com lapso de tempo utilizando uma estirpe transgénica que expressa a proteína fluorescente verde (GFP) sob o controlo do promotor de actina-α esquelético etiquetar todas músculo As células 14 e injectado ARN que codifica p...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Microscopia de lapso de tempo permite a visualização de processos de desenvolvimento. Crescimento ou deriva de amostras durante a aquisição de imagem reduz a capacidade de acompanhar e medir movimentos celulares durante o desenvolvimento de forma precisa. Descreve-se o uso de open source software de processamento de imagem para corrigir desvio tridimensional da amostra ao longo do tempo.

Correção Deriva de exemplo a seguir 4D confocal tempo-lapso

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K visualizações.  Monash University.  Microscopia de lapso de tempo permite a visualização de processos de desenvolvimento. Crescimento ou deriva de amostras durante a aquisição de imagem reduz a capacidade de acompanhar e medir movimentos celulares durante o desenvolvimento de forma precisa. Descreve-se o uso de open source software de processamento de imagem para corrigir desvio tridimensional da amostra ao longo do tempo. 

Vídeo: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imagem bioluminescência para avaliação das respostas imune após o implante do tecido do coração Engineered (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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 ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse imagens de fluorescência de crescimento de raiz Arabidopsis com Manipulação acelerada do meio root usando o RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Amostra Estratégias Preparação para Mass Spectrometry imagem da cultura celular 3D Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Um chip microfluídico para ICPMS Amostra Introdução

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisão celular dos mamíferos em matrizes 3D através de microscopia de reflexão Confocal quantitativa

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelos avançados de fígado 3D para testes de genotoxicidade in vitro após exposição nanomaterial de longo prazo

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

Medição contínua do ruído biológico em Escherichia Coli Usando microscopia de lapso de tempo

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Espólio-Temporal Em Vivo Imagem de Sistemas de Entrega de Drogas Oculares usando Microendoscopia a laser confocal de fibra óptica

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Uma câmara flexível para imagens de células vivas com lapso de tempo com microscopia de espalhamento Raman estimulada

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...