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

Procedimento de imagem A configuração experimental é mostrado em detalhe na Figura 1. A LS fonte de luz serve tanto como iluminação para medições de absorção e como fonte de excitação para as medidas de fluorescência e é filtrada com uma estreita banda passante filtro de interferência de BPF. Um conjunto de fixos e variáveis ​​neutra ND filtros de densidade combinada com a presença de um obturador automático S permite controlar a quantidade de luz na amostra e para mantê-la baixa o suficiente para evitar qualquer fotobranqueamento. Iluminação uniforme da amostra é conseguido usando um expansor de feixe BE com um combinado de duas lentes do telescópio de Galileu e um difusor. O S amostra é imersa na solução de compensação e é mantida no lugar de um titular e rodado ao longo de seu eixo vertical por meio de um estágio de alta velocidade de rotação (Newport, PR50) com uma precisão absoluta de 0,05 graus. Três controladores manuais permitem distintas para o eixo vertical para amostra ser inclinado e ajustado em seu plano ortogonal. O sinal de transiluminação é detectada diretamente pela câmera CCD, o sinal de fluorescência é filtrado por um filtro de interferência de banda estreita passagem junto com um filtro longpass (Omega. Optical, Brattleboro, VT) e, em seguida, coletados com uma lente telecêntrica TL. Telecêntrica oferecem a vantagem de fornecer características únicas que as tornam ideais para tomografia projeção óptica. Na verdade, a presença de uma abertura parar localizado dentro do conjunto de lentes no ponto focal da lente assegura que os raios, que tornam a imagem do diafragma de abertura, vai viajar paralelo ao eixo óptico praticamente eliminando qualquer distorção de perspectiva e proporcionando a mesma ampliação para múltiplos planos dentro da profundidade telecêntrica. Preparação da Amostra O procedimento a seguir é seguido a fim de fixar o tecido / órgãos <ol><li> Amostras são fixadas em PFA para 8 horas a 4C. </li><li> Amostra é então lavadas em PBS por 15 minutos </li><li> A amostra é incorporado em um bloco de Agarose 0,8% </li><li> Agarose bloco é desidratado por uma série de 20% a 100% de soluções de etanol. Este procedimento série de desidratação de etanol ajuda a evitar qualquer retração assimétrica da amostra. </li><li> Finalmente a amostra é incubada 10 horas em etanol 100%, a fim de remover qualquer conteúdo de água da amostra. </li><li> Coloque a amostra em uma solução de 1:02 de álcool benzílico e benzoato de benzila, pelo tempo necessário para a amostra para limpar. Note que o tempo pode variar consideravelmente de amostra para amostra de algumas horas a vários dias. </li><li> Etanol em traços mínimos podem estar presentes no final do processo de compensação que deu origem ao sever artefatos nas reconstruções devido ao movimento térmico do álcool na solução de limpeza em si. , A fim de evitar esse problema, um ciclo de desmatamento no segundo recomendado. </li><li> Ponto 4 e 5 devem ser realizados no escuro para evitar o branqueamento de agentes de contraste fluorescente ou proteínas. </li></ol> Preparação da Amostra coração É importante remover qualquer conteúdo de sangue de qualquer tecido antes de ser trabalhada, a fim de evitar artefatos de alta absorção nas reconstruções. O seguinte procedimento pode ser seguido. <ol><li> Anestesiar o rato com uma injeção intraperitoneal (IP) de uma mistura de cetamina (90 mg / kg) e xilazina (10mg/kg). Nota: &quot;. Deste ponto em diante manter a amostra protegida da luz&quot; </li><li> Injetar 50 U de heparina (IP) e cinco minutos depois, realizar uma laparotomia longitudinal. </li><li> Abra a veia renal esquerda (LRV) e infundir 20ml de solução salina para a VCI. </li><li> O bloco de agarose deve ser desidratada, com quatro, incubações uma hora, em 20%. 40%, 60%, e etanol 80%, e, finalmente, as amostras são incubadas em etanol 100% por 10 horas (overnight). </li><li> Coração é então fixado após o procedimento geral para preparação de amostras Nota: &quot;. Deste ponto em diante manter a amostra protegida da luz&quot; </li></ol> Reconstruções absorção Reconstrução de absorção óptica na ausência de dispersão são, em geral, análogo ao X-CT e podem ser obtidas utilizando um algoritmo de retroprojeção filtrada comum Radon. As imagens são então levados a absorção por uma câmera CCD em transiluminação mais de 360 ​​projeções com um ângulo de um grau ao longo do eixo vertical. É conveniente para alinhar o eixo vertical do paralelo de amostra para a coluna de pixels do CCD. <ol><li> Coloque o bloco de agarose em uma câmara preenchida com solução de limpeza </li><li> Iluminam a amostra com um feixe colimado para ambos excitação e medições de transmissão </li><li> Rode a amostra mais de 360 ​​projeções com um ângulo de 1 grau. </li><li> Adquirir imagens em transiluminação em ambas as absorção (intrínseco) e comprimentos de onda de fluorescência. </li><li> Coloque um obturador para evitar a iluminação contínua e reduzir clareador. </li><li> Use afalgoritmo de retroprojeção iltered Radon tomograficamente para reconstruir a amostra </li></ol> Reconstruções de fluorescência O algoritmo usado para as reconstruções de absorção não é válido para a reconstrução da distribuição de fluorescência e, se não levados em conta, a contribuição absorção levará a artefatos grave. <ol><li> Proceder como indicado no &quot;Absoprtion Reconstruções&quot; seção. </li><li> Adquirir simultaneamente a absorção e medições de fluorescência </li><li> Combine os dados no campo normalizado imagens Nascido </li><li> Escrever as equações do modelo frente da propagação da luz nos comprimentos de onda intrínseca e de fluorescência. </li><li> Calcular matriz de pesos em uma malha discretizada para funções de Green do modelo frente </li><li> Inverter a matriz de pesos e multiplicá-lo pela detectado Normalized imagens campo Nascido para obter a reconstrução da distribuição de fluorescência no objeto </li></ol>

<ol>
	<li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R., Davidson, D. <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, 541-544 (2002).</li><li>Lee, K., Avondo, J., Morrison, H., Blot, L., Stark, M., Sharpe, J., Bangham, A., Coen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+plant+development+and+gene++in+three+dimensions+using+optical+projection+tomography.">Visualizing plant development and gene in three dimensions using optical projection tomography.</a> Plant Cell. 18, 2145-2156 (2006).</li><li>Oldham, M., Sakhalkar, H., Wang, Y. M., Guo, P. Y., Oliver, T., Bentley, R., Vujaskovic, Z., Dewhirst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+whole+rodent+organs+using+optical+computed+and+emission+tomography.">Three-dimensional imaging of whole rodent organs using optical computed and emission tomography.</a> J. Biomed. Opt. 12, 1-10 (2007).</li><li>Ntziachristos, V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+three-dimensional+fluorescence+reconstruction+of+diffuse+media+by+use+of+a+normalized+Born+approximation.">Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation.</a> Opt. Lett. 26, 893-895 (2001).</li></ol>

born normalization for fluorescence optical projection tomography for whole heart imaging

Tomografia projeção óptica é uma técnica de imagem tridimensional que foi recentemente introduzido como uma ferramenta de imagem principalmente em biologia do desenvolvimento e estudos de expressão gênica. A técnica torna amostra biológica opticamente transparentes pela primeira desidratando-los e depois colocar em uma mistura de álcool benzílico e benzoato de benzila na proporção de 2:1 (Babb ou Murray solução s aberto). A técnica torna amostras biológicas opticamente transparentes pela primeira desidratando-los em soluções de etanol graduada em seguida, colocando-os em uma mistura de álcool benzílico e benzoato de benzila na proporção de 2:1 (Babb ou solução Limpar Murray s) para limpar. Após o processo de compensação a contribuição de espalhamento na amostra pode ser muito reduzida e feitas quase insignificante enquanto a contribuição de absorção não pode ser eliminado completamente. Ao tentar reconstruir a distribuição de fluorescência da amostra sob investigação, esta contribuição afeta as reconstruções e leva, inevitavelmente, a artefatos de imagem e erros de quantificação .. Enquanto que a absorção pode ser reduzida ainda mais com uma permanência de semanas ou meses nos meios de compensação, isto levará a uma progressiva perda de fluorescência e de um tempo exageradamente longo amostra de processamento. Isto é verdade tanto para a reconstrução agentes de contraste exógeno (agentes de contraste molecular), bem como contraste endógeno (por exemplo, reconstruções de organismos geneticamente expressa proteínas fluorescentes).

Watch this Scientific Journal Video about Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging at JoVE.com

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Sugerimos uma abordagem Nascido normalizados para Tomografia projeção óptica (BnOPT), que representa as propriedades de absorção de amostras com imagens para obter resultados precisos e quantitativa de fluorescência reconstruções tomográficas. Usamos o algoritmo proposto para reconstruir a distribuição de sonda de fluorescência molecular dentro de órgãos de animais de pequeno porte.

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 ...

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JoVE Journal

Biology

10.3K visualizações.  Harvard Medical School. Sugerimos uma abordagem Nascido normalizados para Tomografia projeção óptica (BnOPT), que representa as propriedades de absorção de amostras com imagens para obter resultados precisos e quantitativa de fluorescência reconstruções tomográficas. Usamos o algoritmo proposto para reconstruir a distribuição de sonda de fluorescência molecular dentro de órgãos de animais de pequeno porte.

Vídeo: Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Cuidados e limpeza do microscópio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Biologia

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Componentes principais do microscópio de luz

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de fase e de interferência diferencial Contrast Microscopy (DIC)

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automático de análise de pulsação óptica de pequenos corações

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

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 ...

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 ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Longo prazo Cultura de Amostras de mama Humano do Câncer e sua análise por meio de tomografia óptica de Projeção

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imagens de fluorescência com um nanômetros Precisão (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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 ...

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.