C. Vinegoni 건강 (NIH) 부여 1 RO1 - EB006432 국립 연구소의 지원을 인정한다.

본 실험에 사용된 Drosophila melanogaster의 주식은 다음과 같습니다 <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> 모든 실험은 UAS - Gal4 시스템은 관심의 조직 (즉 침샘이나 날개 디스크)에 overexpress GFP로 사용되었습니다. 보다 구체적으로, 적절한 Gal4와 유전자 변형 여성 파리는 UAS - EGFP 형질 전환 메이트로 교차했다. 십자가는 humidified 인큐베이터에서 25 ° C에서 reared했다. pupae가 튜브를 (약 5-6일 십자가의 개시 후) 채우기 시작할 때, 그들은 흰색 prepupal 단계 (번데기 형성 후 0-1 시간)에서 GFP 형광을 위해 선택하고 tomography 이미징에 대한 수집했다. <ol><li> 25 적절한 교차하고 후방을 설정 ° C </li><li> 5~6일 십자가의 개시 후 이미지에 대한 형광 자손을 선택합니다. 유리병의 측면에서 흰색 prepupae의 부드러운 제거 젖은 페인트를 사용하십시오. </li><li> 물 또는 PBS의 드롭 prepupae을 넣어 음식 입자 및 pupal 사건을 커버하고 표본이 autofluorescent 만드는 접착제를 제거하기 위해 페트리 접시에 위치. 페인트 브러쉬로 부드럽게 닦아주십시오. pupae의 고령화가 필요한 경우 장소는 젖은 종이 타월 (습기 챔버) 또는 비행 음식 깨끗한 유리병의 측면에서 배양 접시에있는 흰색 prepupae을 선택했습니다. 이것은 동물의 적절한 개발을 허용하기 위해 충분한 습도가 있는지 확인합니다. </li></ol> Drosophila 장착 <ol><li> 글루 모세관 유리관 (800 미크론 직경)에 pupal 케이스의 하단 부분 파리의 후부 / 앞쪽에 축은 튜브에 평행 방향입니다 같은. </li><li> 회전 무대에서 수직으로 모세관을 수정. </li><li> 이미징 CCD의 픽셀 컬럼에 평행으로 회전 축에 대한 순서로 회전 무대의 틸팅 축을 조정합니다. </li></ol> 결정 전달 모델 <ol><li> 모세관 유리에 번데기를 수정하고 이전에 설명된대로 마운트 </li><li> 형광 염료 (Cy5.5)와 실리카 모세관 (100 미크론, OD)를 작성 </li><li> 후부 / 앞쪽에 축에 대하여 45도에서 번데기 내부의 모세관 튜브를 삽입 </li><li> 낮은 수치 조리개 렌즈 여기 레이저 빔을 번데기를 조명. 의 수직 축을 따라 번데기를 회전 360도 이상의 형광 transillumination 이미지를 수집합니다. </li><li> 적절한 앞​​으로 모델과 실험 데이터를 맞추기 </li></ol> 이미징 침샘 <ol><li> 로 언급한 침샘에서 번데기 표현 GFP 마운트 </li><li> 여기 빔과 함께 조명하고 transillumination 년에 세로 축을 따라 번데기를 회전 360도 이상의 GFP의 형광 신호를 수집합니다. </li><li> 앞으로 모델을 이용하여 획득한 데이터를 재구성 </li></ol> 시간 경과 이미징 <ol><li> 날개 imaginal 디스크에 흰색 prepupa 표현 GFP 마운트 </li><li> 번데기의 탈수를 피하기 위해 이미징 환경을 습기와 실온에서 보관. </li><li> 지속적인 조명을 피하기 위해 셔터를 배치 </li><li> 여기 빔과 함께 조명하고 transillumination 년에 세로 축을 따라 번데기를 회전 360도 이상의 GFP의 형광 신호를 수집합니다. </li><li> 앞으로 모델을 이용하여 획득한 데이터를 재구성. </li></ol> 조직학 <ol><li> 1X PBS의 침샘의 CNS / 눈 디스크를 해부하다. </li><li> 4 % 포름 알데히드, 실온에서 20 분 대한 1X PEM (100mM 파이프, 1mM EGTA, 1mM MgCl 2) 조직을 수정. </li><li> dapi (벡터)로 Vectashield에 고정하고 마운트를 제거 </li><li> 형광 현미경으로 이미지. </li></ol> 시간 경과 조직학 <ol><li> 흰색 prepupae를 수집하고 습기가 실내에서 그들을 무대. </li><li> 다른 발달 단계에서 pupae 수집 </li><li> 비열한 고정하기 전에 좋은 바늘 (곤충 핀 000, FST)를 두 번 pupal 케이스 (번데기 형성 후 0 시간). </li><li> 부드러운 교반과 함께 실온에서 6-8 시간 8 %의 포름 알데히드, 1X PEM에서 pupae을 수정. </li><li> 고정의 제거 및 1X PBS로 rinsing이 두 가지에 날카로운 칼날과 고정 번데기를 잘라 후 A / P의 축에 직각; </li><li> 잠수함 이미징을위한 coverglass 슬라이드에 dapi (벡터)와 마운트와 Vectashield의 ​​두 조각 (랩 - 테크). </li><li> 형광 공촛점 현미경으로 이미지를 획득. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="그림 1" border="0" /> 그림 1. 주시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">여기를 클릭하십시오</a> 알을 볼그림 1의 arger 버전.

<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.

pupal 가지 경우에 - 생체내의 reconstructions 및 D.의 GFP - 표현 침샘 해당 조직학 (블루, dapi의 얼룩, 녹색, GFP 형광)로 melanogaster prepupa (스케일 바, 500 미크론)은 (A) 그림 1에 표시됩니다. D.의 시간 경과 시리즈 이미지 melanogaster 날개 imaginal 디스크는 Fig.1 (B)에 표시됩니다. 이미지는 네 가지 시간 포인트 (0,3.0, 3.5, 및 6.5 시간)에서 한 라이브 표본에서 얻은 수 있습니다. 번데기의...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

개발 기관 형성뿐만 아니라 질병의 progession 및 치료를 떠올리면 자주 많이 광학 그대로 살아있는 생물의 분자 및 기능적 변화를 심문할 수있는 능력에 의존합니다. 대부분의 기존 광학 이미징 방법은 현대적인 광학 현미경의 침투 한계 (0.5 - 1mm)과 광학 macroscopy의 확산 부과 한계 사이의 거짓말 차원에서 영상에 대한 불충 분한 아르 (&gt; 1cm) [1]. 따라서, 많은 중요한 모델 생물은, 예를 들어 곤충, 동물 배아 또는 작은 동물 사지는, - 생체내 광학 이미징을 위해 액세스할 남아있다. 나노미터 해상도 광학 영상 방법의 개발에 대한 관심이 증가하고 있지만, 이미징 침투 깊이를 향상 많은 성공적인 노력이되지 않았습니다. 현미경 제한 이외에 - 생체내 이미징을 수행하는 능력은 조직에있는 광자 산란과 관련된 어려움을 만난 사실이다. 예를 들어, 이미지를 전체 배아 최근 활동 [2,3] 비산에서 선택을 취소 시편의 특수 화학 처리, 오직 사후 영상을 위해 적합하게 절차를 필요로합니다. 이 방법은 그러나 일반적으로 특히 발달 생물학 및 약물 발견의 두 광자 또는 공촛점 현미경에 의해 허용하는 것보다 큰 이미지 표본에 대한 필요성을 증거. 우리는 Mesoscopic 형광 Tomography라는 새로운 광학 이미징 기술을 개발 [4], 1mm - 5mm의 크기에 비침습에서 - 생체내 이미징에 적합한하는. 침투 깊이에 대한 메소드 교환 해상도지만,이 전례없는 단층 이미징 성능을 제공하며, 그것은의 형광 - 진화 이미지 수있는 능력을 imparting하여 발달 생물 관찰에 새로운 차원의 (그리고 생물 학적 연구의 가능성이 다른 영역)로 시간을 추가 개발되었습니다 시간이 지남에 따라 응답을 태그. 따라서 그것은 유전자 변이 또는 외부 자극에 대한 형태학의 또는 기능 종속성의 연구를 가속 수 있으며, 중요한 것은 같은 개발 유기체의 길이 시간 저속 시각화함으로써 개발 또는 조직 기능의 전체 사진을 캡처할 수 있습니다. 기술은 수정된 실험실 현미경 및 멀티 프로젝션은 조명 360도 계획에서 데이터를 수집을 활용합니다. 그것은 mesoscopic 범위에 적합한 현실 반전 체계를 구축하기 위해 기하학적 광학 원칙과 결합 광자 수송 방정식의 Fokker - 플랭크 솔루션에 페르미의 단순화를 적용합니다. 이것은 크기가 몇 mm에 샘플이 아닌 투명한 입체 구조에 - 생체내 전체 - 바디 시각화를 수 있습니다. 우리는 영상 입체적으로 기술의 인 - 생체내 성능을 보여준 - 생체내에서 연속으로 여섯 시간 동안 실시간으로 불투명 Drosophila pupae에있는 날개의 morphogenesis에 따라 Drosophila 조직 개발을 구조.

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

Mesoscopic 형광 tomography는 조직 sectioning 형광 현미경의 침투 제한없는 운영하고 있습니다. 이 기술은 다중 투영 조명 및 광자 수송 설명을 기반으로합니다. 우리의 morphogenesis의 인 - 생체내 전체 - 몸 3D 시각화 GFP - 표현에 날개 imaginal 디스크를 입증<em&gt; Drosophila melanogaster.</em

에 대한 Mesoscopic 형광 Tomography 인 - 생체내</em개발> 이미징 Drosophila</em

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

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

Research

JoVE Journal

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K 조회수.  Massachusetts General Hospital. Mesoscopic 형광 tomography는 조직 sectioning 형광 현미경의 침투 제한없는 운영하고 있습니다. 이 기술은 다중 투영 조명 및 광자 수송 설명을 기반으로합니다. 우리의 morphogenesis의 인 - 생체내 전체 - 몸 3D 시각화 GFP - 표현에 날개 imaginal 디스크를 입증 Drosophila melanogaster.

비디오: 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.

완전한 개발 두뇌 속에 생체내에서 단일 세포 Electroporation

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

연구

생물학

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.

전체 심장 이미징에 대한 형광 광 프로젝션 Tomography에 대한 출생 정규화

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.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

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.

개발 및 성인의 준비 Drosophila 두뇌와 라이브 영상을위한 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.

Drosophila의 배아들 중에서 - 생체내의 원심 분리

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.

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

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.

신속한 분석 및 형광 현미경 이미지의 탐사

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.

초파리 준비 및 빛 간섭 현미경을 사용하여 생체 내에서 심장 기능의 세로 이미징 (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.

이중 양전자 방출 단층 촬영에 대 한 68가 코어 첨가 산화 철 나노 입자의 합성 / (T1) 자기 공명 이미징

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.

에 대한 Mesoscopic 형광 Tomography 인 - 생체내 이미징 Drosophila