C. Vinegoni Ulusal Sağlık Enstitüleri (NIH) hibe 1-RO1-EB006432 desteği kabul eder.

Bu deneyde kullanılan Drosophila melanogaster hisse senetleri aşağıdaki gibidir: <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> Tüm deneyler için, ilgi doku (yani tükürük bezleri veya kanat diskler) eksprese GFP UAS Gal4 sistemi kullanıldı. Daha spesifik olarak, uygun Gal4 transgenik erkek sineklerin UAS EGFP transgenik arkadaşları geçti. Haçlar nemlendirilmiş bir kuvöz, 25 ° C sıcaklıkta yetiştirilen idi. Pupa (çapraz başladıktan sonra yaklaşık 5-6 gün) şişeleri doldurma başlattığınızda, GFP floresan beyaz prepupal sahne (puparium oluşumundan sonra 0-1 saat) seçilmiş ve tomografi görüntüleme için toplanmıştır. <ol><li> Set up uygun haçlar ve arka 25 ° C </li><li> Çapraz başlandıktan 5-6 gün sonra, görüntüleme için floresan döl seçin. Flakonun taraftan beyaz prepupae yumuşak kaldırılması için ıslak bir fırça kullanın. </li><li> Gıda parçacıkları ve pupa halinde kapsar ve örnek autofluorescent yapar tutkal kaldırmak için bir petri kabına yerleştirilir, su ya da PBS bir damla prepupae koyun. Yavaşça bir fırça ile temizleyin. Pupa yaşlanmasını gerekli ise, yerde, ıslak bir kağıt havlu (nemli odasının) ya da bir sinek gıda ile temiz bir flakon iki petri beyaz prepupae. Bu hayvanın uygun şekilde gelişimi sağlamak için, yeterli nem var olmasını sağlayacaktır. </li></ol> Drosophila Montaj <ol><li> Tutkal, kapiller cam tüp (800 mikron çaplı) içine pupa halinde alt kısmında sinek anterior / posterior eksen tüp paralel odaklı olduğu gibi. </li><li> Dönme aşamasında dikey kılcal tüp sabitleyin. </li><li> Görüntüleme CCD piksel sütunları paralel olarak dönme ekseni için dönme aşamasının devirme ekseni ayarlayın. </li></ol> Belirlenmesi İleri Modeli <ol><li> Kılcal bir cam bir pupa saptamak ve yukarıda açıklandığı gibi monte </li><li> (Cy5.5) floresan boya ile silika kapiler tüp (100 mikron, OD) doldurun </li><li> Anterior / posterior eksenine göre 45 derece sıcaklıkta pupa içindeki kılcal tüp takın. </li><li> Pupa, düşük bir sayısal diyafram ile bir ikaz lazer ışını ile aydınlatın. 360 derecenin üzerinde dikey eksen boyunca pupa dönen floresan transillüminasyon görüntüleri toplayın. </li><li> Uygun bir ileri modeli ile deneysel veriler Fit </li></ol> Görüntüleme Tükrük Bezleri <ol><li> Önce açıklandığı gibi tükürük bezlerinde Mount pupa ifade GFP </li><li> Bir uyarma ışını ile aydınlatın ve transillüminasyon pupa dikey ekseninde 360 ​​derece dönen üzerinden GFP floresan sinyal toplamak. </li><li> Elde edilen veriler ileri modeli kullanılarak yeniden </li></ol> Zaman Aşımı Görüntüleme <ol><li> Kanat hayali diskler Mount beyaz prepupa ifade GFP </li><li> Pupa dehidratasyonu önlemek için görüntüleme ortamı nemli ve oda sıcaklığında tutun. </li><li> Sürekli aydınlatma önlemek için bir deklanşör yerleştirin. </li><li> Bir uyarma ışını ile aydınlatın ve transillüminasyon pupa dikey ekseninde 360 ​​derece dönen üzerinden GFP floresan sinyal toplamak. </li><li> Yeniden ileri bir model kullanılarak elde edilen veriler. </li></ol> Histoloji <ol><li> 1x PBS tükürük bezleri CNS / göz diskleri ayır. </li><li> % 4 formaldehit, oda sıcaklığında 20 dk 1x PEM (100 mM Borular, 1mm EGTA, 1mm MgCl 2) doku sabitleyin. </li><li> DAPI (vektör) ile Vectashield fiksasyonu ve bağlama çıkarın </li><li> Floresan mikroskobu ile görüntüsü. </li></ol> Zaman Aşımı Histoloji <ol><li> Beyaz prepupae toplayın ve nemli bir odasında aşamasında. </li><li> Farklı gelişim aşamalarında pupa toplayın </li><li> Prick fiksasyon (böcek pin 000, FST) önce ince bir iğne ile iki kez pupa halinde (Puparium Oluşumu sonra 0 saat). </li><li> Yavaşça sallanarak oda sıcaklığında 6-8 saat,% 8 Formaldehit, 1x PEM pupa sabitleyin. </li><li> Fiksasyon 1x PBS ile durulama kaldırma ve keskin bir bıçak, iki adet sabit pupa kesip sonra A / P eksene dik; </li><li> Batmak görüntüleme için coverglass kızaklar üzerinde DAPI (Vektör) ve mount Vectashield iki adet (Lab-Tek). </li><li> Bir floresan konfokal mikroskop ile görüntüler elde. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="Şekil 1" border="0" /> Şekil 1: Lütfen <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">buraya tıklayın</a> al .Şekil 1 arger sürümü.

<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 rekonstrüksiyonlar ve pupa halinde D. GFP ifade tükürük bezleri (mavi, DAPI boyanması, yeşil, GFP floresan) ile ilgili histoloji melanogaster prepupa (Ölçek bar, 500 mikron) (a) Şekil 1 de gösterilmiştir. D. Time-lapse serisi görüntüleme melanogaster kanat hayali diskler Şekil 1 (b) &#39;de gösterilmiştir. Görüntüleri dört farklı zaman noktalarında (0,3.0, 3.5, ve 6.5 saat), tek bir canlı örnek elde edilir. 0 derece pupa dorsal görünümüne ilişkin ...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Gelişen organ oluşumu yanı sıra hastalık ve tedavi progession görselleştirme genellikle ağır optik bozulmadan yaşayan organizmaların moleküler ve fonksiyonel değişiklikler sorgulamak için yeteneği üzerine dayanır. En mevcut optik görüntüleme yöntemleri, modern optik mikroskop penetrasyon sınırları (0.5-1mm) ve optik makroskopi difüzyon dayatılan sınırları arasında yer alan boyutlarda görüntüleme için yeterli (&gt; 1cm) [1]. Böylece, çok önemli bir model organizmalar, örneğin böcekler, hayvan embriyolar ya da küçük hayvan ekstremitelerde, in vivo optik görüntüleme için ulaşılmaz kalır. Nanometre çözünürlüklü optik görüntüleme yöntemlerinin geliştirilmesi yönünde ilgi var artmasına rağmen, birçok başarılı görüntüleme penetrasyon derinliği iyileştirilmesi çabaları olmamış. Mikroskopi sınırlarının ötesinde in vivo görüntüleme gerçekleştirmek için yeteneği dokularda bulunan foton saçılması ile ilgili zorluklar ile bir araya geldi aslında. Örneğin görüntü tüm embriyolar son çabaları [2,3] saçılma onları temizlemek için numunenin özel kimyasal arıtma, sadece post-mortem görüntüleme için uygun bir prosedür gerektirir. Bu yöntemler ancak görüntüleme büyük örnekler için: genellikle iki-foton veya konfokal mikroskopi, özellikle gelişim biyolojisi ve ilaç keşfi tarafından izin olanlardan daha kanıtlar. Biz mezoskopik Floresans Tomografi adlı yeni bir optik görüntüleme tekniği geliştirdi [4], 1mm-5mm boyutlar non-invaziv in vivo görüntüleme için uygun. Penetrasyon derinliği yöntemi değişim çözünürlük, ama eşi görülmemiş bir tomografik görüntüleme performansı sunar ve floresan evrim görüntü yeteneği aktararak gelişimsel biyoloji gözlemler (ve muhtemelen diğer biyolojik araştırma alanlarında) olarak yeni bir boyut eklemek için geliştirilmiştir zamanla yanıtları etiketli. Gibi gen mutasyonları ya da dış uyaranlara morfolojik veya fonksiyonel bağımlılıkları çalışmaları hızlandırmak ve önemlisi, aynı, gelişmekte olan organizmanın uzunlamasına zaman atlamalı görselleştirme izin vererek geliştirme veya doku işlevini tam bir resim çekebilirsiniz. Bu teknik, modifiye edilmiş bir laboratuvarda mikroskop ve 360 ​​derece projeksiyonları veri toplamak için çok projeksiyon aydınlatma kullanır. Mezoskopik aralığı için uygun gerçekçi bir inversiyon şeması oluşturmak amacıyla geometrik optik ilkeleri ile birlikte Fokker-Plank foton transport denkleminin çözümü, Fermi basitleştirilmesi geçerlidir. Bu büyüklüğü birkaç milimetre örneklerinde şeffaf olmayan üç boyutlu yapıları, in-vivo tüm vücut görselleştirme kadar izin verir. In-vivo ve Drosophila dokular, gerçek zamanlı olarak altı saatten fazla üst üste opak Drosophila pupa kanat morfolojilerinden takip ederek gelişmekte olan yapıları görüntüleme üç boyutlu tekniğin in vivo performans göstermiştir.

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

Mezoskopik floresan tomografi doku kesit floresan mikroskopi penetrasyon sınırları ötesinde faaliyet göstermektedir. Tekniği çok projeksiyon aydınlatma ve foton bir taşıma açıklamasına dayanmaktadır. Biz, in vivo morfolojilerinden tüm vücut 3B görselleştirme, GFP ifade kanat hayali diskler göstermektedir<em&gt; Drosophila melanogaster.</em

Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme Drosophila</em

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

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Research

JoVE Journal

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K Görüntüleme.  Massachusetts General Hospital. Mezoskopik floresan tomografi doku kesit floresan mikroskopi penetrasyon sınırları ötesinde faaliyet göstermektedir. Tekniği çok projeksiyon aydınlatma ve foton bir taşıma açıklamasına dayanmaktadır. Biz, in vivo morfolojilerinden tüm vücut 3B görselleştirme, GFP ifade kanat hayali diskler göstermektedir Drosophila melanogaster.

Video: 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.

Tek Hücre Elektroporasyon bozulmamış Gelişmekte olan beyin içindeki in vivo

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

Biyoloji

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.

Tüm Kalp Görüntüleme Floresan Optik Projeksiyon Tomografi tarihi Normalizasyon

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.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

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.

Geliştirilmesi ve Yetişkin hazırlanması Drosophila Brain ve Canlı Görüntüleme için Retinae

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 Drosophila Embriyolar Santrifüj

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.

Alt hücresel Çözünürlük bozulmamış Drosophila Larva in vivo Görüntüleme

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.

Hızlı Analiz ve Floresans Mikroskopi Görüntüler Exploration

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 Hazırlama ve Optik Koherens Mikroskobu Kullanımı In Vivo Kalp Fonksiyonu Boyuna Görüntüleme (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.

68Ga çekirdek katkılı demir oksit nano tanecikleri çift pozitron emisyon tomografi için sentezi / (TRT1) manyetik rezonans görüntüleme

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.