이 프로젝트는 CIHR와 NSERC에 의해 후원되었다. 우리는 dsRed 표시된 SSR (BJ 선)와 UBC 바이오 이미징 시설을 표현 파리 변종의 생성에 기여를위한 바브 Jusiak을 인정하고 싶습니다.

1 부 : 조직 준비 <ol><li> 우리의 목표는 신​​경 시스템이 그대로 비행 유충의 조직 준비이지만, 본문 벽 근육의 내부 표면은 인공 hemolymph에 노출되며, 가까운 좋은 광학 현미경 커버 슬립에 위치하실 수 있습니다. 즉, 애벌레 준비 밖으로 안으로. 기존의 조직 준비는 절삭 달아 및 신체 벽 근육을 스트레칭하고, 때로는 신경계의 일부를 제거 참여 이후, 우리는 다른 접근이 필요했습니다. 우리가 구조 기능과 버렸네에서 시간이 지남에 따라 구조적인 변화를 잘 보라 얻을 싶어서 안으로 아웃 동물을 원했고, 우리는 신경계를 지키고 싶었어요. 우리는 또한 조직을 스트레칭 방지하려고하고, 근육 트위치 및 이미지를 망치 할거라 스트레치 수용체를 활성화. </li> <li> 첫째, 동물을 무대. 셋째 애벌레 먹이 및 세 번째 애벌레 완더링 것은 우리가 W3 애벌레에서 보여주는 거고 그럼 해부하다에 크고 쉽습니다. 이 동물은 터비 표현형을 가지고 있으며, 다양한이므로 에버트하기 쉽습니다. 우리는 우리가 적극적으로 W3 애벌레를 포함하여 크롤 링 봤다 애벌레를 사용합니다. </li><li> 이중 소주 H2O의 배양 접시에있는 매우 부드러운 페인트 브러쉬와 구더기의 표면을 청소하십시오. 클린 애벌레 더 광학을 가지고 있고 청소 박테리아를 감소시킵니다. </li><li> 3 ML 얼음 추위에 대한 HL - 6의 작은 페트리 접시, 그리고 인공 hemolymph에 동물을 전송합니다. 동물이 이동 중지하고, (5 분) 끝날 때까지 얼음 접시를 넣어. </li><li> 한 손으로 그리고 다른 봄 가위에 아주 좋은 팁 포셉 잡아. 내 지배적인 손으로 가위를 사용합니다. 본문 벽 전체의 압력을 equlibrate하기 위해 가위로 시체 벽에 작은 구멍을 만듭니다. </li><li> 아래 접시의 바닥에 포셉와 함께 부드럽게 동물을 보유하고 뒷부분이 세그먼트를 잘라. 내장 멀리 해부하다 지방 몸은 가능성 뱃속 밖으로 이동합니다. 또한이 조직을 버려야. </li><li> 좋은 포셉 함께 그릇 바닥으로부터 유충을 잡아. 에서 # 0 곤충 핀을 보유 (뭉툭한 끝)와 애벌레의 입 부분에 대해 그것을 밀어. 밖으로 안쪽에 양말 어져 같은 뱃속을 통해 입 부분을 밀어. </li><li> 조직 밖으로 내부는 그림과 같이 표시됩니다 : 아래. 울트라 미세 해부 도구를 사용하여, 신체 벽에서 지방 몸과 trachioles 멀리 해부하다. trachioles 당겨하지 진짜 열심히 노력하거나, 신경 계통을 분리합니다. trachioles을 추출하면 신체 근육 벽에 구멍을 찢어 것입니다. 당신이 할 수만큼 지방 시신을 제거합니다. 이러한 구조 모두는 조직의 광학 품질을 방해. 이 준비를하기 전에 커피를 건너 뛰도록 할 수 있습니다. </li> <li> 작업이 완료되면 근육은 반투명, 불투명 또는 흰색되지 않습니다. 당신은 조미료없이 HL - 6에 조직 준비를 넣으면 CNS의 모터 패턴 발생기가 작동되기 때문에, 적당한 온도에서, 그것은 가능성이 리드미컬 계약 것입니다. 분명히 계약 또는 부정기 계약 신체 벽 근육과 preps를 사용하지 마십시오. 신체 벽 밖으로 그대로 내부는 즉시 지느러미 및 복부 midlines 따라 반으로 접어 경향, 준비는 시각화에 대한 왼쪽이나 오른쪽으로 &#39;헤미 - 동물 &quot;를 제공하므로. </li><li> 1.9에는 워너 챔버와 같은 작은 볼륨 상업 회의소, 또는 다리를 커버 슬립 배열과 현미경 슬라이드 중 하나의 조직을 탑재합니다. 장착 조직에 대한 제안 제 3을 참조하십시오. </li></ol> 2 부 : HRP에 대한 찬란 표시 주요 항체로의 연결을 부통 라벨. <ol><li> 배양 접시에 드롭 HL - 6의 HRP에 대한 찬란 표시 주요 안티 - 본문 50 마이크로 리터 넣어. 염료 목욕의 내부 아웃 바디 벽면 준비 젖어. 당신은 5 분 후에 신경 터미널 라벨을 참조하지만, 완전하고 밝은 라벨 약 10-20 분 품어 수 있습니다. </li><li> 상온에서 HL - 6의 10-30초에 대한 염료의에서 린스. 비 바운드 염료 때문에 세정주기가 적극 필요가 없다, 빨리 떨어져 rinses. </li><li> 염료 농도와 필요에 따라 배양 시간을 다릅니다. </li></ol> 파트 3 : dextran flurophore 공액와 Perisynaptic 공간 라벨링 <ol><li> 희석 찬란 복합 dextran 염료 HL - 6의 희석. 우리의 목적을 위해 잘 작동 집중했습니다 : </li><li> 당신이 당신의 세포외 공간에 색소 접근의 타이밍에 대해 우려하지 않는 경우, &quot;다리를&quot;coverslip (4 부 참조)에 염료의 20 마이크로 리터 드롭을 넣고 색소 목욕탕에서 준비를 입금. </li></ol> 4 부 : 시각화 (공촛점 현미경 포함)에 대한 조직을 장착. 당신이 준비 (단기 관측)를 perfuse 필요는 없다거나 HL - 6 작은 목욕의 볼륨을 유지하려는 경우, 두 다리를 슬라이드 메서드를 사용하여 당신의 준비를 perfuse 싶다면, 재관류 챔버를 사용해보십시오. 우리는 MO를 사용워너 악기에서 dified 상공 회의소. 모두 수행에 대한 세부 정보. 두 다리를 슬라이드에 준비를 장착. <ol><li> 매우 깨끗한 현미경 슬라이드를 사용하십시오. 슬라이드에 Superglue이 광장, 18mm (# 1.5) coverslips. coverslips의 가장자리 사이에 2mm의 간격을 둡니다. </li><li> 접착제가 완전히 마른하자, 또는 그것은 당신의 준비 주위 수성 매체를 통해 이상한 필름을 형성합니다. </li><li> coverslips의 가장자리 사이의 애벌레 밖으로 안으로 위치. 당신은 애벌레가 대형이며 이미지 수집 시스템은 제한된 픽셀 배열이있다면 대각선의 준비를 위치해야 할 수도 있습니다. </li><li> 18mm # 1.5 coverslip, (아주 깨끗한)로 조직을 커버. 슬라이드 최소한의 바셀린과 조직 측면 전표를 붙인 &quot;가기&quot;coverslip을 준수합니다. </li><li> 상단 coverslip에 1.3379의 굴절률 (여러분이 HL - 6을 사용하는 경우)와 카길 사용자 지정 목적 기름 한 방울을 넣고이 어셈블리를 뒤집어. </li><li> 목표쪽으로 당신의 현미경, 석유 측면에서 조직 어셈블리를 위치하고 목표를 중점을두고 있습니다. 수정된 재관류 챔버의 조직을 장착. </li><li> RC 20 챔버의 바닥을 형성 접착제 1.5 coverslip. RC - 20 지침에 따라 같은 챔버에서 조직을 놓습니다. 챔버의 지붕을 형성하는 대신 coverslip의 Nytex 메쉬의 조각을 사용합니다. </li></ol> 제 5 부 : 대표 결과 : <ol><li> 인사이드 - 아웃 조직 준비 해부 순서 : <img src="/files/ftp_upload/1154/fig1.jpg" alt="그림 1" /> W3 애벌레, 세탁 ( &quot;터비&quot;표현형) (5.1.1). W3 애벌레, 해부 후부 두 세그먼트. 당신은 횡단 바디 벽면 압력 (화살표)의 뱃속에서 밀쳐 지방 시신을 볼 수 있습니다. 절개 전에 몸을 벽에 일분 (5.1.2)에 작은 구멍을 만들어 내장 &quot;분화&quot;를 최소화하기 위해보십시오. 내장과 함께 준비 밖으로 안으로 준비 (5.1.3)을 켜기 전에 해부 했어요. 준비 (5.1.4)의 루멘 내부 핀 (화살표)로 대부분 everted 준비,. 근육은 밖에서 지금과 표피 일부 지방 몸과 (5.1.5) 첨부 trachioles 함께 내부에 있습니다. 거의 모든 지방 몸과 해부 trachioles (5.1.6)와 완벽하게 해부 조직 준비. <img src="/files/ftp_upload/1154/fig2a.jpg" alt="그림 2" width="600" height="200" /></li><li> 애벌레 NMJ에서 안티 - HRP를 사용하여 라벨 살고 있습니다. glial 확장 (밖 준비 내부)로 대표 W3 애벌레 신경 근육 버렸네. 모터 신경 부통 터미널은 Cy5에 복합이다 HRP (마젠타), 대한 안티 몸을 기본으로 표시됩니다. glial 프로세스는 GFP (녹색)으로 표시됩니다. <img src="/files/ftp_upload/1154/fig2b.jpg" alt="그림 3" /></li><li> Perisynaptic 공간 찬란 준비 밖으로 안으로 알렉사 680 dextran과 시각. glial 과정 (녹색)가 막 타겟 GFP와 함께 표시됩니다. 근육 표면에 포스트 시냅스 SSR (파란색)은 dsRed와 함께 표시됩니다. 알렉사 dextran (적색) 양식 세포 분야에 중점을두고 있습니다. dextran 염료 perisynaptic 공간에 도넛 모양의 웅덩이를 형성합니다. dextran 라벨 및 라벨 dsRed SSR은 회색으로 표시됩니다. perisynaptic 공간 (화살표)를 강조 여러 도넛 모양의 염료 풀을합니다. </li></ol>

<ol>
	<li>Macleod, G. T., Marin, L., Charlton, M., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicles:+Test+for+a+role+in+presynaptic+Calcium+regulation.">Synaptic Vesicles: Test for a role in presynaptic Calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Macleod, G. T., Hegstro, M., Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast Calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiology. 88, 2659-2663 (2002).</li><li>Morales, M., Ferrus, A., Martinez-Padron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+calcium-channel+currents+in+normal+and+csp+mutant+Drosophila+peptidergic+terminals.">Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals.</a> Eur J Neurosci. 11, 1818-1826 (1999).</li><li>Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J., Kla&#168;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Function+of+the+Blood&#8211;Brain+Barrier+in+Drosophila.">Organization and Function of the Blood&#8211;Brain Barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

이 절차는 라이브 표시된 단백질 및 세포 프로세스의 장기 이미징을 허용합니다. 우리가 설명에서 현장 조직 준비는 손상과 기능 CNS, PNS 및 반사 회로를했다. 이 조직 준비는 애벌레 몸 벽 근육이 늘어는 표준 애벌레 플라이 근육 프로토콜 (그것이 밖으로 고정된 경우) 이상의 장점이 있습니다. 스트레칭하면 시냅스 형태를 왜곡하고 반사 기반의 수축을 일으킬 수있다. 준비 밖으로 우리의 ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.</td>
<td>Reagent</td>
<td>NA</td>
<td>NA</td>
<td>5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).
2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.
References: Macleod et al 2002 and Macleod 2004
</td>
</tr>
<tr>
<td>Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>D34680</td>
<td>Use a small volume perfusion chamber to keep the total volume of dye low</td>
</tr>
<tr>
<td>Anti-HRP-CY5 conjugate (goat)</td>
<td>Reagent</td>
<td>Jackson ImmunoResearc</td>
<td>123-175-021</td>
<td>Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at &#x2013;20C. Dilute one aliquot into 100 microliters of HL-6</td>
</tr>
<tr>
<td>Alexa 647 antibody labeling kit</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>A10475</td>
<td>We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.</td>
</tr>
<tr>
<td>Custom Formulated Objective Oil, refractive index 1.3379</td>
<td>Reagent</td>
<td>Cargill Labs</td>
<td>Custom Formulated</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Fine Forceps</td>
<td>Tool</td>
<td>Fine Science Tolls</td>
<td>11252-23 or 11295-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>91500-09</td>
<td>&nbsp;</td>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion Chamber RC 20 Series</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>64-02222</td>
<td>&nbsp;</td>
<tr>
<td>Spinning Disc confocal</td>
<td>Microscope</td>
<td>Quorum</td>
<td>Quorum Wave FX</td>
<td>Mounted on a Leica DMI6000 Inverted Microscope</td>
</tr>		</tbody>
		</table>
		</div>

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

우리의 프로젝트는 개발 애벌레의 날아 신경근육학의 버렸네에서 GFP 분류 glial 구조를 발견. 라이브 glial - 신경 근육 시냅스의 개발 봐, 우리가 사는 그대로 애벌레의 기능을 가진 애벌레 조직 준비를 개발뿐만 아니라, 좋은 광학 특성을했다. 이 새로운 준비도 버렸네에 perfusates의 액세스 허용. 우리는 파리 애벌레를 사용하여 인공 hemolymph에서 그들을 열중하고, 그들을 재미하여 정상적인 신체 리듬 수축을 완화. 다음 우리는 각 동물의 후부 세그먼트를 해부하고 뭉툭한 곤충 핀으로 뱃속을 통해 뒤로 입 부분을 밀고. 이것은 양말 안에 아웃을 바꾼 것처럼, 애벌레 신체 벽 everted. 우리는 울트라 미세 해부 가위로 절개를 완료하기 때문에 신체 벽 근육의 내장 측면을 노출. NMJ에 glial 구조는 glial 특정 발기인의 통제하에 멤브레인 대상으로 GFP를 표명했다. 근육의 포스트 시냅스 막, SSR (Subsynaptic Reticula)는 synaptically dsRed 대상으로 표시됩니다. 우리는 심하게 모터 신경 터미널 버렸네의 세 번째 부분을 라벨을해야했습니다. 이렇게하려면 우리는 멀리 붉은 발광 flurophore에 복합, HRP에 기본 항체 적용. 모터 신경 터미널과 SSR 사이 perisynaptic 공간으로 염료 확산 속성에 대한 테스트하기 위해, 우리는 멀리 붉은 발광 flurophore 및 회수 이미지를 복합 대형 Dextran 분자의 솔루션을 적용.

Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

우리는 공촛점 현미경과 형광 염료를 사용하여 라이브 파리 애벌레의 소설 내부 아웃 조직 준비에 Glia - 신경근육학 시냅스의 구조 기능을 설명했다. 우리는 HRP에 형광 기본 항체 살 신경 터미널로 표시하고, 또한 형광 Dextrans와 perisynaptic 공간을 시각.

라이브를 떠올리 Drosophila</em형광 염료와 함께> Glial - 신경근육학 융티온

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

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10.7K 조회수.  University of British Columbia - UBC. 우리는 공촛점 현미경과 형광 염료를 사용하여 라이브 파리 애벌레의 소설 내부 아웃 조직 준비에 Glia - 신경근육학 시냅스의 구조 기능을 설명했다. 우리는 HRP에 형광 기본 항체 살 신경 터미널로 표시하고, 또한 형광 Dextrans와 perisynaptic 공간을 시각.

비디오: Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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 ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

의 형광 라벨링 Drosophila 심장 구조

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

에 잔 심장을 떠올리 Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

형광 현미경을 사용하여 Nematodes에 박테리아를 머릿속으로

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

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  ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP 현미경을 이용한 miRNA의 gap junction 의존적 전달 분석

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

라이브를 떠올리 Drosophila Glial - 신경근육학 융티온