C. Vinegoni reconhece o apoio do National Institutes of Health (NIH) conceder 1-RO1-EB006432.

Os estoques de Drosophila melanogaster utilizadas neste experimento são as seguintes: <ul style="list-style-type:none;"><li> elav-Gal4 (FBti0002575) </li><li> ap-Gal4md544 (FBal0051787) </li><li> UAS-srcEGFP (FBti0013990) </li><li> w1118 (FBal0018186) </li></ul> Para todos os experimentos, o sistema UAS-Gal4 foi usada para superexpressão GFP no tecido de interesse (ou seja, as glândulas salivares ou os discos de asa). Mais especificamente, as moscas fêmeas transgênicas com o Gal4 apropriadas foram cruzados com companheiros de UAS-EGFP transgênicos. As cruzes foram criados a 25 ° C em uma incubadora umidificada. Quando as pupas começar a preencher os frascos (cerca de 5-6 dias após o início da cruz), eles foram selecionados para GFP fluorescência em fase de pré-pupa branco (0-1 hr após a formação pupário) e cobradas por tomografia. <ol><li> Configurar cruza apropriado e traseiro a 25 ° C </li><li> 5-6 dias após o início da cruz, selecione progênie fluorescentes para imagens. Use um pincel molhado para remoção suave das prepupas em branco dos lados do frasco. </li><li> Coloque prepupas em uma gota de água ou PBS colocado numa placa de Petri para remover partículas de alimentos e a cola que cobre o caso pupal e faz o espécime autofluorescent. Limpe suavemente com um pincel. Se envelhecimento da pupa é necessário, local escolhido prepupas branco numa placa de Petri com papel toalha úmido (câmara úmida) ou para os lados de uma célula limpa com comida voar. Isso irá garantir que não há umidade suficiente, para permitir o desenvolvimento adequado do animal. </li></ol> Drosophila Montagem <ol><li> Cole a parte inferior do caso pupal em um tubo capilar de vidro (800 mícrons de diâmetro), tais que o eixo da mosca anterior / posterior está orientado paralelo ao tubo. </li><li> Fixar o tubo capilar verticalmente em um estágio de rotação. </li><li> Ajustar a inclinação do eixo de rotação do palco para que o eixo de rotação a ser paralela às colunas de pixel da imagem CCD. </li></ol> Modelo de determinação Encaminhar <ol><li> Corrigir uma pupa em um capilar de vidro e montar como descrito anteriormente </li><li> Encha um tubo capilar de sílica (100 microns, OD) com um corante fluorescente (Cy5.5) </li><li> Insira o tubo capilar no interior da pupa em 45 graus com relação ao eixo anterior / posterior </li><li> Iluminar o pupa com um feixe de laser de excitação com uma lente de abertura numérica baixo. Coletar imagens de fluorescência transiluminação mais de 360 ​​graus de rotação da pupa ao longo de seu eixo vertical. </li><li> Ajustar os dados experimentais com um modelo apropriado para a frente </li></ol> Glândulas Salivares imagem <ol><li> Montar uma pupa expressando GFP nas glândulas salivares como descrito anteriormente </li><li> Iluminar com um feixe de excitação e recolher em transiluminação do sinal de fluorescência da GFP mais de 360 ​​graus de rotação da pupa ao longo de seu eixo vertical. </li><li> Reconstruir os dados adquiridos utilizando o modelo para a frente </li></ol> Time Lapse-Imaging <ol><li> Montar uma GFP prepupa branco expressando nos discos asa imaginal </li><li> Manter o ambiente úmido e de imagem em temperatura ambiente para evitar a desidratação da pupa. </li><li> Coloque um obturador para evitar a iluminação contínua </li><li> Iluminar com um feixe de excitação e recolher em transiluminação do sinal de fluorescência da GFP mais de 360 ​​graus de rotação da pupa ao longo de seu eixo vertical. </li><li> Reconstruir os dados adquiridos utilizando o modelo para a frente. </li></ol> Histologia <ol><li> Dissecar CNS glândulas salivares / discos de olho em 1x PBS. </li><li> Fix tecidos em formol 4%, 1x PEM (Pipes 100mM, EGTA 1 mM, 1mM MgCl 2) por 20min em temperatura ambiente. </li><li> Remover fixação e montagem em Vectashield com DAPI (Vector) </li><li> Imagem com um microscópio fluorescente. </li></ol> Time-Lapse Histologia <ol><li> Coletar prepupas branco e estágio-los em uma câmara úmida. </li><li> Coletar pupas em diferentes estágios de desenvolvimento </li><li> Prick caso pupal duas vezes (0 hrs após a formação pupário) com uma agulha fina (pino inseto 000, FST) antes da fixação. </li><li> Fix pupas em formol 8%, 1x PEM por 6-8 horas em temperatura ambiente com agitação suave. </li><li> Após a remoção de fixação e lavagem com PBS 1x, corte a pupa fixa com uma lâmina afiada em duas partes perpendicular ao eixo A / P; </li><li> Submergir as duas peças em Vectashield com DAPI (Vector) e montagem em lâminas de lamela (Lab-Tek) para geração de imagens. </li><li> Aquisição de imagens com um microscópio de fluorescência confocal. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="figura 1" border="0" /> Figura 1. Por favor, <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">clique aqui</a> para ver alÄrger versão da figura 1.

<ol>
	<li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light:+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light: the evolution of whole-body photonic imaging.</a> Nat. Biotechnol. 23, 313-320 (2005).</li><li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat. Methods. 4, 331-336 (2007).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Nat. Methods. 5, 45-47 (2008).</li></ol>

The authors have nothing to disclose.

In vivo reconstruções do caso pupal e as glândulas salivares expressando GFP-de um D. melanogaster prepupa (Escala de bar, a 500 microns) com a histologia correspondente (azul, coloração DAPI; verde, GFP fluorescência) é mostrado na Figura 1 (a). Time-lapse série de imagem de D. discos melanogaster asa imaginal é mostrado na Fig.1 (b). As imagens são adquiridas a partir de um único espécime vivo, em quatro momentos diferentes (0,3.0, 3.5 e 6,5 horas). Na primeira projeção uma col...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Visualizando a formação de órgãos de desenvolvimento, bem como progession e tratamento da doença, muitas vezes depende fortemente da capacidade de interrogar opticamente alterações moleculares e funcionais em organismos vivos intactos. A maioria dos métodos existentes de imagens ópticas são inadequados para imagens em dimensões que se situam entre os limites de penetração de microscopia óptica moderna (0,5-1mm) e os limites de difusão-imposta de macroscopia óptica (&gt; 1cm) [1]. Assim, muitos organismos modelo importante, insetos por exemplo, embriões de animais ou nas extremidades de pequenos animais, permanecem inacessíveis para a in-vivo de imagens ópticas. Embora haja um interesse crescente para o desenvolvimento de métodos de resolução de nanômetros de imagem óptica, não tem havido muitos esforços bem sucedidos na melhoria da profundidade de penetração de imagem. A capacidade de executar in-vivo de imagens de microscopia para além dos limites é, de fato encontrou-se com as dificuldades associadas com o espalhamento de fótons presente nos tecidos. Esforços recentes para embriões imagem inteira, por exemplo, [2,3] necessitam de tratamento químico especial da amostra, para limpá-las de dispersão, um procedimento que os torna adequados apenas para pós-mortem de imagem. Estes métodos entanto evidência a necessidade de amostras maiores do que as imagens normalmente permitido por microscopia de dois fótons ou confocal, especialmente em biologia do desenvolvimento e na descoberta de medicamentos. Nós desenvolvemos uma nova técnica de imageamento óptico chamado Mesoscópica Fluorescência Tomografia [4], que apropriado para não-invasivos de imagem in-vivo em dimensões de 1mm-5mm. O método de resolução de trocas de profundidade de penetração, mas oferece desempenho sem precedentes tomográfica de imagem e que foi desenvolvido para aumentar o tempo como uma nova dimensão em observações biologia do desenvolvimento (e possivelmente outras áreas da pesquisa biológica) por transmitir a capacidade de imagem da evolução da fluorescência tagged respostas ao longo do tempo. Como tal, pode acelerar os estudos de dependências morfológicas ou funcionais em mutações genéticas ou estímulos externos, e podem importante, capturar a imagem completa do desenvolvimento ou a função do tecido, permitindo a visualização de lapso de tempo longitudinal do organismo, mesmo em desenvolvimento. A técnica utiliza um microscópio de laboratório modificados e multi-projecção de iluminação para coletar dados em 360 graus de projeções. Aplica-se a simplificação de Fermi Fokker-Plank solução da equação de transporte de fótons, combinados com princípios geométricos óptica, a fim de construir um esquema de inversão realista adequados para a faixa de mesoscópica. Isso permite que os in-vivo visualização de todo o corpo de não-transparente estruturas tridimensionais em amostras de até vários milímetros de tamanho. Nós demonstramos o desempenho in vivo da técnica de imagem por estruturas tridimensionais de desenvolver tecidos Drosophila in-vivo e seguindo a morfogênese das asas na opaco Drosophila pupas em tempo real ao longo de seis horas consecutivas.

Watch this Scientific Journal Video about Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila at JoVE.com

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscópica fluorescência tomografia opera além dos limites de penetração de tecido de corte de microscopia de fluorescência. A técnica é baseada na projeção multi-iluminação e uma descrição do transporte de fótons. Demonstramos in-vivo visualização de corpo inteiro em 3D da morfogênese da GFP-expressando asa em discos imaginais<em&gt; Drosophila melanogaster.</em

Mesoscópica de fluorescência para Tomografia In-vivo Imaging de desenvolvimento Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

mesoscopic-fluorescence-tomography-for-vivo-imaging-developing

Research

JoVE Journal

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K visualizações.  Massachusetts General Hospital. Mesoscópica fluorescência tomografia opera além dos limites de penetração de tecido de corte de microscopia de fluorescência. A técnica é baseada na projeção multi-iluminação e uma descrição do transporte de fótons. Demonstramos in-vivo visualização de corpo inteiro em 3D da morfogênese da GFP-expressando asa em discos imaginais Drosophila melanogaster.

Vídeo: Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Eletroporação única célula in vivo dentro do cérebro intacto desenvolvimento

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Biologia

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Normalização nascido para Tomografia Projeção de fluorescência óptica para imagens de todo o coração

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparação de Desenvolvimento e Adultos Drosophila Brains e retina de imagens ao vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo Centrifugação de Embriões Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análise rápida e Exploração de microscopia de fluorescência Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Síntese de nanopartículas de núcleo dopado com óxido de ferro Ga 68para tomografia por emissão de pósitrons Dual/imagem latente de ressonância magnética de (T1)

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.