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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

연구

신경과학

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

에 대한 간단한 마이크로 유체 장치<em&gt; 생체 내</em의&gt; 이미징<em&gt; C. elegans</em&gt;<em&gt; Drosophila</em&gt;와 Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

생체공학

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

장기의 라이브 영상<em&gt; 초파리</em&gt; 아이 디스크

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

발생학

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

생물학

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

마이크로 컴퓨팅 단층 촬영을 사용하여 드로소필라 멜라노가스터의 전체 동물 이미징

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

초파리 배아에서 지질 함유 소기관의 세포 골격 의존성 밀매 시각화

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

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 ...

Antibody-Mediated Live Imaging을 통한 단백질 시각화 향상

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

저자 스포트라이트: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research (동물 형태 형성 연구에서 기계적 및 생화학적 신호의 역학 공개) 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

형광 항체 주입을 통한 살아있는 초파리 배아의 저농도 단백질 및 번역 후 변형 시각화

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

에 대한 Mesoscopic 형광 Tomography 인 - 생체내 이미징 Drosophila

요약

더 많은 비디오 탐색

이 비디오의 챕터

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

에 대한 간단한 마이크로 유체 장치

장기의 라이브 영상

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

마이크로 컴퓨팅 단층 촬영을 사용하여 드로소필라 멜라노가스터의 전체 동물 이미징

초파리 배아에서 지질 함유 소기관의 세포 골격 의존성 밀매 시각화

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

형광 항체 주입을 통한 살아있는 초파리 배아의 저농도 단백질 및 번역 후 변형 시각화

형광 항체 주입을 통한 살아있는 초파리 배아의 저농도 단백질 및 번역 후 변형 시각화

형광 항체 주입을 통한 살아있는 초파리 배아의 저농도 단백질 및 번역 후 변형 시각화