우리는 각각 Sciara coprophila 및 Ilyanassa obsoleta에 대한 인 - 생체내의 원심 분리를 테스트하는 자료를 공급 수잔 Gerbi, 하이디 스미스, 데이비드 램버트 감사드립니다. 우리는 기술과 원고에 대한 의견에 대한 Welte 실험실 구성원 감사합니다. 인 - 생체내 원심 분리 분석의 개발 및 개선을 모우에 NIGMS 부여 GM64687에 의해 지원되었다. 이미지가 그림에 표시됩니다. 1 그림. 5E, F는 이전에 다음 ID로 2를 출판 및 권한과 함께 reprinted 있습니다.

<ol>
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F., Heidmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+Drosophila+CID/CENP-A+and+CENP-C+into+centromeres+during+early+embryonic+anaphase.">Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase.</a> Curr Biol. 17, 237-243 (2007).</li><li>Clarkson, M., Saint, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+His2AvDGFP+fusion+gene+complements+a+lethal+His2AvD+mutant+allele+and+provides+an+in+vivo+marker+for+Drosophila+chromosome+behavior.">A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior.</a> DNA Cell Biol. 18, 457-462 (1999).</li><li>Welte, M. A., Gross, S. P., Postner, M., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+vesicle+transport+in+Drosophila+embryos:+forces+and+kinetics.">Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.</a> Cell. 92, 547-557 (1998).</li><li>Tran, S. L., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=unpublished+observations.">unpublished observations.</a> , .</li><li>Rothwell, W. F., Sullivan, W., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+analysis+of+Drosophila+embryos.">Fluorescent analysis of Drosophila embryos.</a> Drosophila Protocols. , 141-157 (2000).</li><li>Guo, Y., Jangi, S., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+Control+of+Intracellular+Transport:+Distinctly+Targeted+Isoforms+of+the+Regulator+Klar.">Organelle-specific Control of Intracellular Transport: Distinctly Targeted Isoforms of the Regulator Klar.</a> Mol Biol Cell. 16, 1406-1416 (2005).</li><li>Meyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+Functions+of+Argonaute+Proteins+in+Patterning+and+Morphogenesis+of+Drosophila+Embryos.">Overlapping Functions of Argonaute Proteins in Patterning and Morphogenesis of Drosophila Embryos.</a> PLoS Genet. 2, 1224-1239 (2006).</li><li>Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lipid-droplet+transport+by+the+Perilipin+homologue+LSD2.">Regulation of lipid-droplet transport by the Perilipin homologue LSD2.</a> Curr. Biol. 15, 1266-1275 (2005).</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+XXII:+The+redistribution+of+the+visible+materials+of+the+egg+by+centrifuging.">Chapter XXII: The redistribution of the visible materials of the egg by centrifuging.</a> Experimental Embryology. , (1927).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+oogenesis+and+embryogenesis+resulting+from+centrifuging+Drosophila+melanogaster+females.">Abnormal oogenesis and embryogenesis resulting from centrifuging Drosophila melanogaster females.</a> J Embryol Exp Morphol. 40, 65-81 (1977).</li></ol>

관심 없음 충돌 선언하지 않습니다.

중요 단계 한천 농도가 충분히 높지되어 있는지 확인합니다. 너무 낮으면, 한천은 원심 분리하는 동안 분해되며, 태아가 파열하거나 임의로 지향합니다. 한천 농도 단지 약간 한천이 (단계 4.3에서 초과하는 TSS의 충분 건조 또는 불충 분한 제거) 젖었거나 너무 낮은 경우 경우 태아는 원심 분리하는 동안 그들의 구멍에서 부동과 부적 절한 중심이 될 것입니다. 한천 너무 얇게 부어있다면, 그것은 또한 축소 또는 원심 분리하는 동안 균열 것입니다. organelles 개별 수천개의 세포 내에 분리된 될 것입니다 때문에 배아는 cellularization 단계 이상의 고급 않았는지 확인합니다. 그렇지 않으면, organelles의 분리 (그림 5C) 작동하지 않습니다. 한 세포 단계에서 젊은 배아에서 organelles 큰 거리를 마이 그 레이션하는 무료이며 자신의 특성 밀도에 따라 축적. 너무 오래 배아를 분석 방지하기 위해, 확실하게 (I)은 암컷은 오래된 달걀 (단계 2.4), (II) (단계 2.5) 3 시간 또는 더 적은을위한 배아를 수집, (III)를하다 수 있도록 미리 컬렉션을 포함 시각 한천에 삽입을위한 cellularization 단계 전에 배아를 선택합니다 (단계 4.2), (IV) cellularization (단계 4.6)의 끝 부분을 통해 개발을 방지하기 위해 짧은 시간을 포함 유지하고, (V) (단계 5.6) 정확하게 레이어 않았 배아를 폐기 . 원심 분리 시간에 날림으로하다하지 마십시오. 배아 중 일부는 심지어 원심 분리의 불과 10 분 후에 좋은 겹겹이 표시되지만, 분리는 배아로부터 배아에 일관성이있다. 30 분 놀이는 매우 일관성있는 결과를 제공합니다. 원심 분리가 끝난 후 또한, 더 이상 원심 분리 시간은 더 이상 레이어는 안정됩니다. 이것은 사용하기 전에 추가로 배아를 처리하는 데 시간이 걸립니다 어떤 이들 응용 프로그램에 중요합니다 (예를 들어, 그들은 이식 실험에 대한 처리 또는 공촛점 분석을 위해 장착해야하는 경우). 가능한 수정 에그 수집 및 chorion 제거 대부분의 다른 프로토콜은 2 단계와 3 단계 1, 3, 9 존재하고, 사용되는 배아는 어릴 같은 것들은 오랫동안 여기에 설명뿐만 아니라 작동합니다, 달걀 수율이 높다, 그리고 분수 잘못 개최 배아가 최소화됩니다. 앞쪽에 - 사후 정렬 프로토콜은 뒷부분 끝이 한천에 묻힌과 앞쪽에 끝이 접속 (그림 3)이다 그러한 모든 배아를 orients. 그것이 원심 분리 (그림 1, 5) 동안 지적 결국 랜드마크로 micropyle을 사용할 수 있습니다 있도록이 방향은 일관성을 위해 좋다. 그러나, 연습, 그것은 밝은 - 필드 현미경에 의한 지질 - 비말와 노른자 레이어의 독특한 외모로 centrifuged 태아의 방향을 선택하기 쉽습니다. 그런 다음 양쪽 끝에와 배아를 탑재 가능됩니다, 이러한 유연성은 설치 과정을 속도. Unoriented 원심 분리 프로토콜의 가장 많은 시간과 기술적으로 도전적인 측면 한천의 배아 (4 단계)를 설치합니다. 그것은 (한 구멍에 삽입하고 한 번 원심 분리 후 복구)을 두 번 각각의 배아를 감동 필요합니다. 대안으로, 하나는 TSS 가득 microcentrifuge 튜브에 4.1 단계에서 배아를 전송할 수 있습니다. 이러한 튜브는 microcentrifuge (13,000 RPM, 10-30 분)에 centrifuged 때, 배아 것입니다 역시 일반적으로 계층 10-13. 이것은 유사 하나가 매우 빠르게 동시에 배아의 수백을 처리하실 수 있습니다. 그러나, 겹겹이은 일관성이 없다는 것입니다, 배아의 일부가 위의 프로토콜에 적용보다 훨씬 높은 원심 분리 세력 (~ 1만6천그램)에도 불구하고 서로 다른 레이어를 개발하지 않습니다. 더 많은 도전 태아의 방향이 변수이고, 하나는 주요 배아 축에 다양한 각도에서 분리를 볼 수있다는 사실입니다. 이 변화는 실험자가 특정 배아가 원심 튜브에 정렬 방식을 원심 분리 후에 정렬해야합니다. 그것이 안정적으로 그것을 할 많은 연습과 더 많은 판단을 필요로하지만 배아의 가장 밀도 부분 마커로 노른자의 autofluorescence의 사용과 함께, 이것은 종종 가능합니다. 샘플에 충분한 배아가있다면, 하나는 배아는 그림처럼 배열하는 일이있는 해당 인스턴스를 정렬하실 수 있습니다. 1. 자신의 알 껍질에서 배아의 원심 분리가 chorion 처음 2를 제거하지 않고 단계 4 한천의 배아를 포함시킬 수도 있습니다. 이 방법에서 수집 접시 배아는 할로 카본 오일 대신 단계 3.2 (할로 카본은 chorion의 투명한 세가)의 50 % 표백제 덮여있다. 해당 단계의 배아가 선택되고 핀셋을 사용하여 새 한천 플레이트로 전송되며, 핀셋, 손상만을 계란 필라멘트를 잡아서배아 적절한는 피할 수 있습니다. 배아는 앞쪽에 계란 필라멘트가 한천에있는 구멍까지 붙어서, 4.8을 통해 단계 4.4로 내장하고 있습니다. 배아가 centrifuged 및 단계 5.4에서와 같이 한천 밖으로 파고되고 난 후에 알 껍질은 50 % 표백 처리에 의해 제거됩니다. 계란 껍질의 존재에 원심 분리는 고정 후 devitellinization의 속도 (단계 6.2) 향상지만, 원심 분리에 대한 올바른 단계의 선택 (단계 4.2) 자세한 도전입니다. 애플 리케이션 특정 단백질이 특정 주요 organelle에 localizes 경우 colocalization 실험, 즉, 테스트를위한 배아의 원심 분리가 특히 유용합니다. 예를 들어, Klar 단백질이 초기 배아 지질 방울에 존재이며, 아직이 지방화는 그대로 태아에서 보여주는 도전이다. Klar puncta 및 지질 방울 모두 열심히 가짜 colocalization은 배제하고, 배아 주변 10 풍부하기 때문에 일부에서는이 있습니다. 그러나, 원심 분리는 별개의 레이어로 지질 방울을 집중하고, 따라서, Klar 신호 colocalization 10 명백한됩니다. 비슷한 분석 unambiguously 특정 histones 같은 놀라운 사례를 포함한 지질 방울 8, 13-15, 2와 단백질 ubiquitously 존재하는 경우에는 다른 단백질의 지방화를 보여주지만, 지질 방울 8 풍부했다. 배아가 발달 단계 (단계 4.2)에 의해 시각적으로 선택할 수 있듯이, 그것은 지질 방울 8, 15 단백질의 발달 규제 채용을 감지도 가능합니다. 지질 방울은 시각적으로 뚜렷한 &quot;모자&quot;에 축적 이후 지질 방울과 colocalization에 대한 테스트하는 것이 특히 쉽게, 아니 더 이상 마커 때문에 또는 얼룩이 레이어를 확인하기 위해 필요합니다. 사실, 때문에 간편하고 잠재적으로 눈에 띄는 시각적인 결과를 (그림 1, 그림 5. D, E), 우리의 실험실은 후보 단백질이 지질 방울로 지역화된 여부를 첫 번째 테스트로 지금은 정기적으로이 기술을 사용합니다. 원칙적으로 유사한 colocalization 연구 그림에 표시된 다른 organelles를 위해 작동합니다. 1, 그의 가능성 유통 centrifuged 배아에서 다른 (세포내 기생충 포함) 문서로 아직 가지고 있죠. 원심 분리가 꽉 밴드로 organelles을 집중하고 있기 때문에 이러한 organelles의 풍부한 소수의 고립을 용이하게합니다. 이 접근법은 이미 기증 배아 이식이에 대한 지질 방울을 복구하는 데 사용되었습니다. 또한 화학적으로 농축 분율 (예, 고정 15 일 이후에 배아에서 레이어를 절단하여) 분석하는 것이 가능해 질 수 있습니다. 원심 분리는 16 일 (개구리, nematodes, tunicates, annelids, 성게, mollusks, 그리고 Cnidarians 포함) 여러 종류의 달걀을 계충화하다하는 데 사용할 수있는 방법은 여기 Drosophila의 배아 이상의 응용 프로그램을 설명했다. 우리 자신의 연구실에서, 우리는 집파리 무스카 domestica 2의 태아에 대한 이상의 프로토콜을 사용하고 곰팡이 냇 Sciara coprophila 17 달팽이 Ilyanassa obsoleta 17 태아의 분석에 unoriented 원심 분리를 고용했습니다. 마지막으로,이 방법은 태아에 제한되지 않습니다 원심 분리 또한 oocytes (예 : Drosophila 2, 18 밀크 위드 버그 Oncopeltus fasciatus 17) 등 다른 대형 세포를 계충화하다에 유용할 수 있습니다. 다른 표본에 대한, 정확한 원심 분리 조건은 최적화할 필요가있다 : 예를 들어, 3,000g 10 분 효율적으로 무스카 domestica 2 배아를 계충화하다 충분한이며, 더 이상 centrifuged 경우, 태아는 고정 및 immunostaining 동안 떨어져 휴식하는 경향이있다.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>60x15 mm Petri dishes</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>#35-1007</td>
<td>From Falcon</td>
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<tr>
<td>Wire mesh</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>CX-0150</td>
<td>stainless steel type 304; mesh size #150 x150, wire diameter: 0.0026 inches</td>
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<td>Tungsten needles</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10130-10</td>
<td>0.25 mm</td>
</tr>
<tr>
<td>Moria Nickel Plated Pin/Needle Holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26016-12</td>
<td>&nbsp;</td>
<tr>
<td>Fly Cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-100 or 59-101</td>
<td>For 65 mm or 100 mm diameter Petri dishes, respectively</td>
</tr>
<tr>
<td>Halocarbon Oil 27</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H8773</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in-vivo centrifugation of drosophila embryos

세포 organelles의 다른 유형의 정화 및 분리를위한 주요 전략은 낙관적인 밀도의 차이에 따라 서로에서 분리하는 것입니다. 그러나, 세포가 이전 원심 분리, 단백질이 아닌 모국어 환경에서 organelles을 방해하는 경우 종종 부적절하게 서로 붙어. 여기 우리는 그대로, 살아있는 Drosophila의 배아에서 밀도에 의해 별도의 organelles하는 방법을 설명합니다. cellularization 전에 초기 배아는 인구 케이지에서 수확하고, 자신의 외부 달걀 껍질은 50 % 표백제와 함께 치료에 의해 제거됩니다. 배아는 다음 한천의 작은 수직 구멍에 먼저 작은 한천 플레이트 및 삽입, 사후 최종으로 전송됩니다. 임베디드 배아가 포함된 플레이트는 3,000그램 30 분 centrifuged 있습니다. 한천은 배아를 지원하며 정의된 방향에서 그들을 유지합니다. 후에, 배아는 뭉툭한 바늘로 한천 밖으로 파고있다. 원심 분리는 밝은 - 필드 현미경으로 충화 쉽게 볼, 층이으로 주요 organelles를 분리합니다. 형광 마커의 숫자는 살아있는 배아의 성공 충화을 확인하실 수 있습니다. 특정 organelles와 연관된 단백질이 colocalization을 보여주는 특정 계층에 풍부한 것입니다. 개별 레이어는 기증자의 난자에 생화 학적 분석 또는 이식을위한 복구할 수 있습니다. 이 기술은 다양한 종류의 계란과 oocytes 등 다른 대형 세포에 organelle 분리에 대해 적용됩니다.

Watch this Scientific Journal Video about In-vivo Centrifugation of Drosophila Embryos at JoVE.com

In-vivo Centrifugation of Drosophila Embryos

우리 삶의 밀도에 의해 별도의 organelles하는 방법을 설명<em&gt; Drosophila</em&gt; 배아. 배아는 한천에 포함된하고 centrifuged 있습니다. 이 기법은 앞쪽에 - 후부 배아 축을 따라 주요 organelles의 재현성 분리 산출. 이 방법은 생화 학적 분석 및 이식 실험에 대한 colocalization 실험 및 수율 organelle의 분수를 용이하게합니다.

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-centrifugation-of-drosophila-embryos

Research

JoVE Journal

Biology

In-vivo Centrifugation of Drosophila Embryos

18.3K 조회수.  University of Rochester. 우리 삶의 밀도에 의해 별도의 organelles하는 방법을 설명 Drosophila 배아. 배아는 한천에 포함된하고 centrifuged 있습니다. 이 기법은 앞쪽에 - 후부 배아 축을 따라 주요 organelles의 재현성 분리 산출. 이 방법은 생화 학적 분석 및 이식 실험에 대한 colocalization 실험 및 수율 organelle의 분수를 용이하게합니다.

비디오: In-vivo Centrifugation of Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrula 스테이지 Drosophila의 태아에서 문화의 연결 준비

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

연구

생물학

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

의 준비 Drosophila</em공중 드롭 프로토콜을 사용하여 라이브 영상을위한> 태아

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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 ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

의 입식 이미징 Drosophila 태아

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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 ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

dsRNA 주입하여 RNAi 간섭에 Drosophila 태아

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin의 분리 및 정제에서 Drosophila 태아

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...