우리는 안드레아스 Schönle, Biophysical 화학, 독일, 데이비드 J. 샌드 스트롬, 정신 건강의 국립 연구소, 기술적인 조언 건강, 베데스다, MD, 미국의 국립 연구소에 대한 최대 - 플랭크 - 연구소 주셔서 감사합니다. 우리는 프랭크 Kötting, 유럽 신경 과학 연구소, 이미징 챔버 구축을위한 괴팅겐과 anesthetization 장치를 주셔서 감사합니다. 이 작업이 TMRYZ에 알츠하이머 Forschung 이니셔티브에서 교부금에 의해 지원되었고 세포 및 분자 신경 과학, 튀빙겐 대학교에 대한 대학원의 화목에 의해 중국 장학 협의회, SBH의 화목에 의해 지원되었다.

이미지 챔버의) 조립 <ol><li> 선택한 단계 (25에서 약 24 시간의 관찰 간격을두고 ° C를 무대를 방황까지 예 초기 제 3 instar의 애벌레)의 유충을 선택합니다. </li><li> 문화 매체와 지문이 건조의 그것을 깨끗한 물을 가진 유충을 씻어. </li><li> 할로 카본 오일 박막과 챔버의 바닥 요소 유충을 얼굴 영역의 중심 문장. </li><li> 챔버에서 현미경 목표가 직면하고있는 복부 측면과 유충을 넣어 것은 (신경근육학 교차로 (NMJ) 26 27이 몇 군데있을 수 있습니다) (그림 1 AE). </li><li> 그리드 얼굴 위쪽 (그림 1)의 공기 슬롯과 ​​오일 층에 플라스틱 스페이서를 놓습니다. 이 단계에서 확인하시기 바랍니다 스페이서의 높이가 약 절반 애벌레 직경이며, 슬릿의 너비가 유충 약 두 배 직경해야합니다. </li><li> 현미경에 이미지 챔버를 배치 </li></ol> 유충의 B) Anesthetization <ol start="7"><li> 적절한 anesthetization 장치 / 기화기와 챔버의 두 인레츠를 연결합니다. 최대 - 프런트를 확인하십시오 : 룸 공기 desflurane의 효과적인 농도는 안전 규정에 의해 지정된을 초과하지 않습니다! desflurane만이 매우 작은 총 볼륨은 애벌레를 마취하기 위해 필요합니다. desflurane 1 15 %의 응용 프로그램 (V / V)는 실험에 사용되는 이상적인 농도를 결정하기위한 좋은 출발점이 될 입증되었습니다. 안전상의 이유로 우리는 기화기가 생체내 이미징에의 2~4시간에 필요한 것보다 더 desflurane이 포함해서는 안하는 것이 좋습니다. </li><li> 물 한 잔에서 챔버 콘센트에 연결된 튜브의 다른 쪽 끝을 담가 후 약 5 초 동안 챔버에 마취의 흐름을 제어하는​​ 밸브를 엽니다. 이 단계에서 확인하십시오 : 공기 방울이 물속에 올라가나요? 그렇지 않다면 챔버 새는입니다. </li><li> 약 3 초 동안 밸브를 닫습니다. </li><li> 현미경에 잔류 애벌레 움직임을 모니터링합니다. 심장 박동과 근육의 움직임을 모두 확인하십시오. 유충의 anesthetization가 완료될 때까지 필요한 열고 닫는 밸브로 8 단계 9에서 설명한다면, 모든 밸브를 닫고 이미지를 시작합니다. 이 단계에서 확인하십시오 : 잔여 근육의 움직임이나 심장 박동이 유충이 제대로 anesthetized되지 않았음을 나타냅니다. 완료 anesthetization는 고해상도 이미지를 매우 중요합니다. 유충은 일반적으로 주어진 시간에 더 이상보다 15-20분에 대한 anesthetized되지 않습니다. </li></ol> C) 이미징 <ol start="11"><li> 올바른 위치와 이미지를 관심의 구조 (그림 2-4)을 식별합니다. </li></ol> anesthetization에서 D) 복구 <ol start="12"><li> 영상은 챔버에 공기를하게 완료되면. 이 단계에서 확인하십시오 : 전송 할로겐 라이트에 의해 유충의 심장 박동과 근육의 수축을 확인하십시오. 근육이 계약을 시작으로 그것은 이미징 챔버에서 유충을 제거하는 것이 안전합니다. </li><li> anesthetization 장치에서 챔버를 분리하여 현미경에서 제거합니다. 자세히 챔버를 분해하고 으깬 플라이 재배 매체의 유충을 놓으십시오. </li><li> 적절한 온도에서 인큐베이터에있는 요리를 보관합니다. </li></ol> E) 시간 시리즈 <ol start="15"><li> 충분한 시간이 포인트를 얻을 때까지 단계 2-14을 반복합니다. 대체 프로토콜 : 유충은 30 분 간격 내에서 두 번 이상 군데되어야하는 경우는 다음 anesthetization의 시점까지 이미징 챔버에 나가 실질적인 수도 있습니다. 충분한 시간이 포인트를 얻을 때까지 단계 7-14을 반복합니다. 이 단계에서 확인하십시오 : 유충은 한 번에 두 개 이상의 시간 동안 이미징 챔버에 보관하거나 그것이 한 번에 15-20분 이상에 대한 anesthetized되어야하는 수 없습니다. </li></ol><img src="/files/ftp_upload/2249/2249fig1.jpg" alt="그림 1" /> 이미지 챔버 1 조립 그림. (A) 오일 층에 유충과 플라스틱 스페이서를 놓습니다. (B) 공백에 22 X 22mm 커버 슬립을 플레이스와 (C) 금속 고리와 유충의 위치를​​ 수정하고 (D) 챔버를 닫습니다 다음, 챔버에 플렉시 가이드 반지를 삽입합니다. (E) 이제 챔버는 현미경에 장착할 준비가 된 것입니다. <img src="/files/ftp_upload/2249/2249fig2.jpg" alt="그림 2" /> Drosophila의 애벌레 2 바디 - 벽의 근육을 그림. 근육과 NMJs는 CD8 - GFP - sh을 융합 단백질 8 표현으로 시각했다. 유충으로 표피를 통해 복부 측면에 초점을 때 관찰로 근육이 표시됩니다. 년 (AC)가 가장 높은 표면 근육, 27, 표시됩니다 (KL)가 가장 높은 내부 근육, 6과 7에 표시됩니다. 스케일 바 : 100 μm의, 조각 사이 ΔZ 2 μm의 수 있습니다. <img src="/files/ftp_upload/2249/2249fig3.jpg" alt="그림 3" /> Drosophila의 애벌레의 몸 - 벽의 근육 3 ID를 그림. L3 Drosophila의 애벌레, 세그먼트 A3에서 근육의 신분이 표시됩니다. 근육과 NMJs는 CD8 - GFP - sh을 융합 단백질 8 표현으로 시각했다. 애벌레로 표피를 통해 복부 측면에 초점을 때 관찰로 근육이 표시됩니다. AC에서 가장 피상적인 근육, 27, 같습니다, KL 가장 내부 근육, 6과 7에 표시됩니다. 스케일 바 : 100 μm의, ΔZ 조각 사이에 2 μm의 수 있습니다. <img src="/files/ftp_upload/2249/2249fig4.jpg" alt="그림 4" /> 그림 Drosophila의 애벌레의 신경근육학 분기점 4 정체성. (AD) NMJs는 DGluRIIA - mRFP 융합 단백질 1 표현으로 시각했다. 유충으로 표피를 통해 복부 측면에 초점을 때 관찰로 NMJs가 표시됩니다. (AB)가 가장 높은 표면 NMJ, 27에 표시됩니다, CD에서 가장 인테리어 NMJs, 6과 7이 표시됩니다. 스케일 바 : 100 μm의, ΔZ 조각 사이에 5 μm의 수 있습니다. (E)와 (F) 표면 (E)보다 내부 (F) NMJs의 신원을 참조 주어집니다.

<ol>
	<li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+dynamics+organizing+synapse+formation+in+vivo.">Glutamate receptor dynamics organizing synapse formation in vivo.</a> Nat Neurosci. 8, 898-905 (2005).</li><li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vivo imaging of long-term changes in the Drosophila neuromuscular system [dissertation]. , (1988).</li><li>Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+synapse+development+and+measuring+protein+dynamics+using+two-color+fluorescence+recovery+after+photo-bleaching+at+Drosophila+synapses.">Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.</a> Nat Protoc. 2, 3285-3298 (2007).</li><li>Wang, X., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria.">Imaging axonal transport of mitochondria.</a> Methods in enzymology. 457, 319-333 (2009).</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=Mesoscopic+fluorescence+tomography+for+in-vivo+imaging+of+developing+Drosophila.">Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.</a> J Vis Exp. , (2009).</li><li>Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Drosophila+larva+by+multiphoton+autofluorescence+and+second+harmonic+generation+microscopy.">Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy.</a> Journal of biomedical optics. 13, 050502-050502 (2008).</li><li>Levitan, E. S., Lanni, F., Shakiryanova, D. <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+vesicle+motion+and+release+at+the+Drosophila+neuromuscular+junction.">In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction.</a> Nat Protoc. 2, 1117-1125 (2007).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. &. a. m. p. ;. a. m. p., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li></ol>

관심 없음 충돌 선언하지 않습니다.

제출 방법은 처음 Drosophila melanogaster의 애벌레의 본문 벽의 근육에 glutamatergic 시냅스를 공부하기 위해 개발되었습니다. Drosophila 신경근육학 교차로 (NMJ)은 근육과 신경의 틀에 박힌 cyto 아키텍처가 특징이며, 따라서 이상적인 생체내 이미징에 적합합니다. 그러나, 설명 anesthetization 프로토콜은 이미징 NMJ에 국한되지, Drosophila의 애벌레의 투명성은 장기의 개발 연구를 설명하는 프로토콜의 적응, 세포의 마이 그 레이션, 세포 내에서 axonal화물과 하위 세포 조직 개편의 수송을 용이하게합니다.

시약 : <ul><li> Desflurane (Suprane, 백스터, Unterschleißheim, 독일) </li><li> 할로 카본 오일 (예 : Voltalef H10S 오일 / Atofina, Puteaux, 프랑스) </li><li> 문화 매체 플리 </li></ul> 장비 : <ul><li> 거꾸로 공촛점 현미경 </li><li> 쌍안경 현미경 (예 : Stemi 2000, 칼 자이스 혈구, 예나, 독일) </li><li> 정렬하는 데 사용되는 작은 페인트가 날아 </li><li> 보육기 </li><li> 사용자 지정 기본 이미지 챔버 (기계 도면의 경우는 참조를 참조하십시오. 3) </li><li> 기화기 / anesthetization 장치 (자세한 내용은 참조를 참조하십시오. 3) </li></ul>

in vivo imaging of intact drosophila larvae at sub-cellular resolution

원하는 유전자 변형 동물 라인의 효율적인 설립을 허용 광학 이미징, 유전자 인코딩 fluorophores과 유전 도구에 최근 개선은 생물 학적 과정은 생활의 맥락에서 연구되고 활성화하고, 어떤 경우에는 심지어, 유기체 행동했습니다. 이 프로토콜에서는 하위 세포 해상도 1-3에서 시냅스 인구의 개발과 소성에 따라 desflurane 휘발성 마취제를 사용하여 손상 Drosophila의 애벌레를 마취하는 방법을 설명합니다. Drosophila melanogaster의 애벌레를 마취 다른 유용한 방법은 이전에 4,5,6,7,8를 설명했습니다 있지만, 여기에 표시되는 프로토콜은 다음과 같은 조합 주요 기능으로 인해 상당한 개선을 보여줍니다 anesthetization (1) 매우 높은 학위를,도 심장 박동은 일 2 (몇 시간의 기간 동안 개 이상의 시간 점의 녹음을 허락 anesthetization 사이클 당&gt; 90 % (2) 높은 생존율, 최대 150 nm의 1 수평 해상도에 대한 허용 체포 3)이 경우에 우리를 가능하게 높은 민감도는 생리적 수준에서 표현 단백질의 역학을 연구합니다. 세부, 우리는 GluR - IIA 안정 트렌스 제닉 라인에서 내생 모터 1 엑슨 트랩 라인 FasII - GFP 하나를 통해 표현 postsynaptic 글루 탐 산염 수용체 subunit을 시각화 수 있었다. (4) 다른 방법과는 달리 4,7 애벌레가 아니라 살아있는 몇 군데 아니라 손상 수 있습니다 (즉, 비 해부) 관찰은 일 1 숫자를 통해 발생하는 수. 함께 비디오 내용을 생체내 이미징 챔버 2,3에있는 개별 부품의 기능, 애벌레, anesthetization 절차, 방법 유충 내의 특정 위치를 다시 확인하고 애벌레의 안전한 제거 장착 올바른 이미징에서 챔버.

Watch this Scientific Journal Video about In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution at JoVE.com

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

이 프로토콜은 그대로의 anesthetization 및 이미징을위한 신뢰할 수있는 방법을 설명합니다<em&gt; Drosophila melanogaster</em&gt; 애벌레. 우리는 desflurane 휘발성 마취가 몇 일 이내에 대한 구조의 하위 세포 해상도와 다시 인식에서 반복 이미징 수 있도록 활용해야<sup&gt; 1</sup&gt;.

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 ...

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Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

14.5K 조회수.  University of Tübingen. 이 프로토콜은 그대로의 anesthetization 및 이미징을위한 신뢰할 수있는 방법을 설명합니다 Drosophila melanogaster 애벌레. 우리는 desflurane 휘발성 마취가 몇 일 이내에 대한 구조의 하위 세포 해상도와 다시 인식에서 반복 이미징 수 있도록 활용해야 1.

비디오: In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Drosophila의 유충에서 냄새 기반 행동의 고해상도 측정

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

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생물학

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

성인 Drosophila Melanogaster의 로코의 고해상도 비디오 추적

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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.

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 ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Zebrafish 애벌레의 Microgavage

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

설치류기도 상피의 섬모 운동성의 높은 해상도 이미지를위한 생체 방법

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

단일 미토콘드리아 과산화물 온전한 마음에 점멸 또는 공 촛점 이미징<em&gt; 생체</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...