C. Vinegoni reconoce el apoyo de los Institutos Nacionales de Salud (NIH) de subvención 1-SR1-EB006432.

Procedimiento de imágenes El montaje experimental se muestra en detalle en la figura 1. A LS fuente de luz sirve como luz para las mediciones de absorción y como fuente de excitación para medidas de fluorescencia y se filtra con una estrecha banda de interferencia de pase BPF filtro. Un conjunto de fijos y variables de densidad neutra ND filtros combinados con la presencia de un obturador automático S permiten controlar la cantidad de luz en la muestra y que lo mantenga lo suficientemente bajo como para evitar cualquier photobleaching. Iluminación uniforme de la muestra se logra mediante el uso de un expansor de haz SER con una combinación de dos lentes del telescopio de Galileo y un difusor. El S muestra se sumerge en la solución de compensación y se mantiene en su lugar en un titular y girar sobre su eje vertical por medio de una etapa de alta velocidad de rotación (Newport, PR50) con una precisión absoluta de 0,05 grados. Tres controladores manuales distintos para permitir que el eje vertical de la muestra que se inclina y se ajusta en su plano ortogonal. La señal de la transiluminación es detectado directamente por la cámara CCD, la señal de fluorescencia se filtra a través de un filtro de interferencias de banda estrecha de paso, junto con un filtro de paso largo (Omega. óptico, de Brattleboro, VT) y luego recogió con una lente telecéntrico TL. Objetivos telecéntricos ofrecen la ventaja de proporcionar características únicas que los hacen ideales para tomografía por proyección óptica. De hecho, la presencia de una abertura parada situada en el conjunto de la lente en el punto focal de la lente asegura que los rayos, que hacen que la imagen del diafragma de apertura, se desplazará en paralelo al eje óptico que elimina prácticamente cualquier distorsión de la perspectiva y ofrecer el mismo aumento de varios planos dentro de la profundidad telecéntrico. Preparación de la muestra El siguiente procedimiento con el fin de fijar los tejidos / órganos <ol><li> Muestras se han fijado en PFA durante 8 horas a 4 ° C. </li><li> Muestra se lavó en PBS durante 15 minutos </li><li> La muestra se inserta en un bloque de agarosa 0,8% </li><li> Agarosa bloque se deshidrata a través de una serie de 20% a 100% de las soluciones de etanol. Este procedimiento de deshidratación de etanol serie ayuda a evitar cualquier contracción asimétrica de la muestra. </li><li> Por último, la muestra se incuba 10 horas en etanol al 100% con el fin de eliminar cualquier contenido de agua de la muestra. </li><li> Se coloca la muestra en una solución 1:2 de alcohol bencílico y benzoato de bencilo por el tiempo necesario para que la muestra clara. Tenga en cuenta que el tiempo puede variar considerablemente de una muestra a partir de unas pocas horas hasta varios días. </li><li> De etanol en los rastros mínima puede estar presente al final del proceso de compensación que da lugar a separar los artefactos en las reconstrucciones debido a la agitación térmica del alcohol en la solución de limpieza en sí. Con el fin de evitar este problema, un ciclo de limpieza en segundo recomendado. </li><li> Puntos 4 y 5 se debe realizar en la oscuridad para evitar el blanqueo de agentes de contraste fluorescente o las proteínas. </li></ol> Corazón de preparación de la muestra Es importante eliminar cualquier contenido de sangre de cualquier tejido antes de ser fotografiado con el fin de evitar artefactos de alta absorción en las reconstrucciones. El siguiente procedimiento se puede seguir. <ol><li> Anestesiar a los ratones con una vía intraperitoneal (IP) la inyección de una mezcla de ketamina (90 mg / kg) y xilazina (10mg/kg). Nota: &quot;. De aquí en adelante mantener la muestra protegida de la luz&quot; </li><li> Inyectar 50 U de heparina (IP) y cinco minutos más tarde, realizar una laparotomía longitudinal. </li><li> Abrir la vena renal izquierda (VRI) e infundir 20 ml de solución salina en la vena cava inferior. </li><li> El bloque de agarosa debe estar deshidratado, con cuatro, incubación de una hora, en el 20%. 40%, 60% y el 80% de etanol y, finalmente, las muestras se incuban en el 100% de etanol durante 10 horas (una noche). </li><li> El corazón se fija el procedimiento de preparación de muestras en general Nota: &quot;. De aquí en adelante mantener la muestra protegida de la luz&quot; </li></ol> Reconstrucciones de absorción Reconstrucción de la absorción óptica en la ausencia de dispersión son, en general, análoga a X-CT y se puede obtener mediante un algoritmo común retroproyección filtrada radón. Las imágenes de absorción son recogidas por una cámara CCD en la transiluminación más de 360 ​​proyecciones con un ángulo de 1 grado a lo largo del eje vertical. Es conveniente para alinear el eje vertical de la paralela de la muestra a la columna de píxeles del CCD. <ol><li> Coloque el bloque de agarosa en una cámara llena con una solución de limpieza </li><li> Iluminar la muestra con un haz colimado, tanto para la excitación y las mediciones de transmisión </li><li> Rotar la muestra de más de 360 ​​proyecciones con un ángulo de 1 grado. </li><li> Adquirir imágenes en la transiluminación, tanto en la absorción (intrínseca) y longitudes de onda de fluorescencia. </li><li> Coloque una velocidad de obturación para evitar la iluminación continua y reducir la decoloración. </li><li> Utilice el bloqueo AFalgoritmo de retroproyección iltered radón para reconstruir tomográficamente la muestra </li></ol> Reconstrucciones de fluorescencia El algoritmo utilizado para la reconstrucción de absorción no es válida para la reconstrucción de la distribución de la fluorescencia y, si no se toma en cuenta la contribución de absorción de plomo a los artefactos graves. <ol><li> Proceder como se indica en el &quot;Absoprtion Reconstrucciones&quot; sección. </li><li> Adquirir al mismo tiempo tanto la absorción y fluorescencia </li><li> Combinar los datos en las imágenes de campo normalizado Nacido </li><li> Escriba las ecuaciones del modelo de propagación de la luz hacia longitudes de onda intrínseca y de fluorescencia. </li><li> Calcular el peso de la matriz en una malla de discretización de funciones de Green de la modelo hacia adelante </li><li> Invertir la matriz de pesos y se multiplica por el detectado imágenes normalizado campo Nacido para obtener la reconstrucción de la distribución de la fluorescencia en el 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

Tomografía por proyección óptica es una técnica de imagen tridimensional que se ha introducido recientemente como una herramienta de imagen sobre todo en la biología del desarrollo y estudios de expresión génica. La técnica hace que la muestra biológica ópticamente transparente, en primer lugar a la deshidratación y después colocar en una mezcla de alcohol bencílico y benzoato de bencilo en una proporción de 2:1 (Babb o Murray solución s abierto). La técnica hace que las muestras biológicas ópticamente transparente, en primer lugar a la deshidratación en soluciones de etanol clasificado luego colocarlos en una mezcla de alcohol bencílico y benzoato de bencilo en una proporción de 2:1 (Babb o solución clara Murray s) para borrar. Después de que el proceso de compensación de la contribución de la dispersión en la muestra se puede reducir considerablemente y de hecho casi insignificante, mientras que la contribución de la absorción no puede ser eliminada por completo. Al tratar de reconstruir la distribución de la fluorescencia en la muestra investigada, esta contribución afecta a las reconstrucciones y conduce, inevitablemente, a los artefactos de la imagen y los errores de cuantificación .. Mientras que la absorción podría reducirse aún más con una permanencia de varias semanas o meses en los medios de compensación, esto conducirá a una pérdida progresiva de la fluorescencia y un tiempo de procesamiento de la muestra exageradamente largo. Esto es cierto cuando la reconstrucción de los agentes de contraste exógenos (agentes de contraste molecular), así como el contraste endógenos (por ejemplo, la reconstrucción de la expresión genética 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

Le sugerimos un enfoque normalizado para Nacido Tomografía de Proyección Óptica (BnOPT) que da cuenta de las propiedades de absorción de las muestras de imágenes para obtener reconstrucciones de fluorescencia precisa y cuantitativa tomográfica. Usamos el algoritmo propuesto para reconstruir la distribución de fluorescencia de la sonda molecular dentro de los órganos de pequeños animales.

La normalización nació para tomografía de fluorescencia de proyección óptica de imágenes de todo corazón

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

born-normalization-for-fluorescence-optical-projection-tomography-for

Research

JoVE Journal

Biology

10.3K vistas.  Harvard Medical School. Le sugerimos un enfoque normalizado para Nacido Tomografía de Proyección Óptica (BnOPT) que da cuenta de las propiedades de absorción de las muestras de imágenes para obtener reconstrucciones de fluorescencia precisa y cuantitativa tomográfica. Usamos el algoritmo propuesto para reconstruir la distribución de fluorescencia de la sonda molecular dentro de los órganos de pequeños animales.

Video: 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.

Limpieza y cuidado apropiado del microscopio

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

Investigación

Biología

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.

Los componentes principales del microscopio 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 y de interferencia diferencial microscopía de contraste (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-automatizado de análisis óptico latido de los corazones pequeños

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 fluorescencia Tomografía por En vivo Imágenes de desarrollo 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.

Time-lapse de imágenes de mitosis después de la transfección de 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.

A largo plazo la cultura de muestras humanas del cáncer de pecho y su análisis mediante tomografía óptica de proyección

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.

Imágenes de fluorescencia con un nanómetros Precisión (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 Preparación e Imagen longitudinal de la función cardiaca in vivo utilizando microscopía de coherencia óptica (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.