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>

<ol>
	<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+Microscopy.">ImageJ for Microscopy.</a> BioTechniques. 43 (S1), 25-30 (2007).</li><li>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Th&#233;venaz, P., Ruttimann, U. 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. Exp. (36), (2010).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <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+extrusion+from+the+epidermis+of+developing+zebrafish.">Live imaging of cell extrusion from the epidermis of developing zebrafish.</a> Journal of Visualized Experiments: JoVE. (52), (2011).</li><li>Benard, E. L., vander Sar, A. M., Ellett, F., Lieschke, G. J., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments: JoVE. (61), (2012).</li><li>Nguyen-Chi, 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=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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

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><tbody><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Low gelling temperature agarose</td><td>Sigma-Aldrich</td><td>A9414-25G</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Electron Microscopy Sciences</td><td>0208-4-PO</td><td></td></tr><tr><td>Disposable 3 ml graduated</td><td>Samco</td><td>212</td><td></td></tr><tr><td>Polyethylene transfer pipette</td><td></td><td></td><td></td></tr><tr><td>9cm bacterial grade Petri dishes</td><td>Greiner Bio One</td><td>632180</td><td></td></tr><tr><td>Fluorinated ethylene propylene (FEP) tubing</td><td>Bola</td><td>S1815-04</td><td></td></tr><tr><td>Zeiss LSM-710 Confocal microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>W Plan-Apochromat 20x/1.0 DIC Objective</td><td>Zeiss</td><td>421452-9600-000</td><td></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

Universidad de Cartagena UDEC

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

Picometer-Precision Atomic Position Tracking through Electron Microscopy

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns with sub-angstrom resolution. With this significant progress, advanced image quantification and analysis are still at the early stages. In this work, we present the complete pathway for the metrology of atomic resolution scanning transmission electron microscopy (STEM) images. This includes (1) tips for acquiring high-quality STEM images; (2) denoising and drift-correction for enhancing measurement accuracy; (3) obtaining initial atomic positions; (4) indexing the atoms based on unit cell vectors; (5) quantifying the atom column positions with either 2D-Gaussian single peak fitting or (6) multi-peak fitting routines for slightly overlapping atomic columns; (7) quantification of lattice distortion/strain within the crystal structures or at the defects/interfaces where the lattice periodicity is disrupted; and (8) some common methods to visualize and present the analysis.
Furthermore, a simple self-developed free MATLAB app (EASY-STEM) with a graphical user interface (GUI) will be presented. The GUI can assist in the analysis of STEM images without the need for writing dedicated analysis code or software. The advanced data analysis methods presented here can be applied for the local quantification of defect relaxations, local structural distortions, local phase transformations, and non-centrosymmetry in a wide range of materials.

This work presents a workflow for atomic position tracking in atomic resolution transmission electron microscopy imaging. This workflow is performed using an open-source Matlab app (EASY-STEM).

Picometer-Precision Rastreamento de posição atômica através de microscopia eletrônica

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns ...

Química

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

Aplicações de imobilização de tecidos drosophila com coágulos fibrinas para imagem ao vivo

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

Biologia do Desenvolvimento

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Abordagens de visualização e análise tridimensional para estudar alongamento axial de vertebrados e segmentação

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imagem altamente multiplexada e de super resolução de células T usando madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Imunologia e Infecção

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Controle da adesão celular usando técnicas de padronização de hidrogel para aplicações em microscopia de força de tração

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Bioengenharia

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

As amostras de teste para otimizar STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Biologia

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Simultânea Multicolor Imagem de Estruturas Biológicas com fluorescência Fotoativação Localização Microscopia

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Imagem 4-Dimensional da Morfogense da Copa Óptica de Zebrafish

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Neurociência

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Tridimensional Super Resolution Microscopia de filamentos de actina F por Interferometric fotoativado Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to chromatic aberration and the imperfect alignment of cameras, images acquired in each channel may be shifted, and magnified, as well as rotated relative to each other in any of the three dimensions. With the classical calibration method, chromatic shifts are measured by multicolor beads attached to the surface of a coverslip, and a number of software are available to measure the chromatic shifts from such calibration samples. However, chromatic aberration can vary with depth, change with observation conditions and be induced by the biological sample itself, thus hindering determination of the true amount of chromatic shift in the sample of interest and across the volume. Correcting chromatic shifts at higher accuracy is particularly relevant for super-resolution microscopy where only slight chromatic shifts may affect quantitative analyses and alter the interpretation of multicolor images. We have developed an open-source software Chromagnon and accompanying methods to measure and correct 3D chromatic shifts in biological samples. Here we provide a detailed application protocol that includes special requirements for sample preparation, data acquisition, and software processing to measure chromatic shifts in biological samples of interest.

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.