이 작품은 워싱턴 대학에 NIH 보조금 DA018109과 MH78823, 그리고 NIH 신경 과학 청사진 코어 부여 P30NS057105에 의해 지원되었다.

문화 준비 <ol><li> 출생 후의 일 0-3 동물에게 1에서 쥐가 드나 마우스 hippocampal 세포의 세포 dissociated 문화를 준비합니다. 우리의 뉴런은에 숫자 0이 두께 coverslip에 보급 콜라겐 층을 준수 돌려 기본 astrocyte monolayer에 준수. cm2 당 약 500 세포의 밀도에 신경 플레이트. </li><li> 문화가 시냅스 개발과 성숙을 할 수 10~14일위한 성장을 허용합니다. 더 glial 성장을 체포하기 위해 antimitotic의 AraC과 함께 체외 4 일에있는 문화를 처리합니다. 의 연결을 생존을 향상시키기 위해 Neurobasal 중간 플러스 B27 보충과 반 중간 교환과 체외 5 일에서 그들을 먹여 살리지. </li><li> 문화 기간 동안 기능 침묵 시냅스의 숫자를 변경하기위한 트리 트먼트를 소개합니다. 우리는 글루 탐 산염 시냅스의 큰 비율 (~ 80 %) 침묵 한 편리한 방법 30 MM의 칼륨과 함께 4 시간 치료는 것을 발견했습니다. 약한 depolarizing 자극과 함께 더 이상 incubations 유사한 입을 2를 유발. 50 μm의 forskolin, adenylyl의 cyclases의 활성과 4 시간 자극은 기본 3 무성 터미널의 인구를 각성하는 데 사용할 수 있습니다. </li></ol> 자동 시냅스를 떠올리 <ol><li> 세포는 형광 현미경을 사용하여 개별 시냅스 터미널의 시각화를 촉진을 줄 수있는만큼 체외 10-14에서 하루 잘 분리 neuritic 프로세스와 상대적으로 낮은 밀도를 가지고해야합니다. </li><li> 증류수 5 MM FM1 -​​ 43FX의 주식 솔루션을 준비합니다. 주식은 4 어둠 속에서 유지되어야 ° C. FM1 -​​ 43FX를 사용하여 모든 실험도 어둠 속에서 수행되어야합니다. </li><li> 100 MM 나트륨 염화물, 2 MM의 염화칼슘, 1 MM 마그네슘 염화물, 10 MM 포도당 및 10 μm의 FM1 -​​ 43FX를 포함하는 HEPES - 버퍼 생리 식염수 45 MM 염화칼륨 2 분 동안 문화를 도전한다. 이 솔루션은 또한 postsynaptic 수용체를 차단하고 반복 신호를 방지하기 위해 1 μm의 NBQX 25 μm의 D - APV를 포함해야합니다. 이 과제는 exocytosis와 endocytosis 능력이 시냅스 터미널 레이블을 것입니다. 도전의 시간과 강도는 재활용 풀 4 사용 가능한 모든 vesicles의 exocytosis 중 적어도 한 라운드를 일으킬 수 있도록 설계되었습니다. 그러나, 문제는 단말기의 추가 입을를 일으킬 수있는 충분한되지 않습니다. </li><li> 염화칼륨없이 생리 HEPES - 버퍼지만, 500 μm의와 Advasep - 7 특이 현상이 염료, 5를 제거하는 똑같은를 사용하여, 오직 3-5초에 대한 문화를 씻으십시오. 이 단계의 타이밍이 모든 FM1 -​​ 43FX 라벨의 손실됩니다 Advasep - 7의 장기간 응용 프로그램으로 중요합니다. 에서 다음 세척 오 2 분 간격을위한 Advasep - 7 않고 생리 HEPES는 버퍼. </li><li> 10 분 4 % paraformaldehyde와 PBS, pH를 7.4에서 0.2 % 글루 타 알데히드와 세포를 수정. 일단 간단히 PBS로 세포를 씻어 후 PBS에 4% 정상 염소 혈청과 0.04 % 트리톤 X - 100을 포함 차단 솔루션에서 15 분 동안 품어. FM1 -​​ 43FX 또한 트리톤은 X - 100 여기 이상의 단계에서 사용의 양을 매우 민감합니다. 일반적으로, FM1 -​​ 43FX 라벨은 트리톤 Triton X - 100의 부재에서 최고이지만, 일부 세제는 다음 immunostaining에 대한 세포를 permeabilize 필요합니다. 우리는 0.04 % 트리톤 X - 100 presynaptically 침묵 시냅스의 시험에 최적입니다 경험적으로 결정했습니다. </li><li> 1:2500의 농도에 솔루션을 차단의 기본 vGluT - 1 항체를 희석. 상온에서 3 시간 동안 vGluT - 1 항체 희석의 부드러운 흔들림 / 회전 세포를 품어. FM1 -​​ 43FX 색소의 표백 방지하기 위해 호일와 문화 요리를 커버해야합니다. </li><li> 두 번 PBS로 세포를 세척 후 두 얼룩을 구분하는 FM1 -​​ 43FX 다른 스펙트럼 특성 형광단에 복합 방지 실험용 돼지 항체와 세포를 품어. 우리의 실험을 위해, 우리는 솔루션을 차단에 1:500에서 555 - 복합 백신 실험용 돼지 항체 알렉사을 사용합니다. 실온에서 30 분 동안 흔들림 보조 항체와 세포를 품어. </li><li> 세포에게 PBS에서 네번 번 이상 씻으 후 Fluoromount - G의 작은 방울을 사용하여 유리 슬라이드에 커버 슬립을 탑재합니다. 세포 슬라이드를 얼굴 있도록 방향 coverslip해야합니다. 슬라이드가 밤새 건조하도록 허용합니다. </li><li> 우리는 지금 활성 및 비활성 시냅스의 이미지를 얻기위한 준비가되어 있습니다. 공촛점 레이저 스캐닝 현미경에 1.4 조리개 수치와 60 X 오일 목표를 준비합니다. coverslip 측면이 목적에 직면 있도록, 무대에 슬라이드를 삽입합니다. 슬라이드를 집중하면, 지나치게 밀도되지 neurites의 필드를 찾으십시오. 잔여 FM1 -​​ 43FX의 염색도 광범위한 세척 후 남아 수 있으므로 이것은 또한, 셀 소마 근처 이미징을 방지하는 것이 좋습니다. 우리의 실험 이미지의 경우, 우리는 일반적으로 51 51 미크론의 필드 크기에 확대. </li><li> 수집27 0.3 - 마이크론 단계로 7.8 미크론의 깊이에 걸쳐 해당 분야의 Z - 스택. 이 범위는 우리 문화의 neurites의 두께를 충당하기 위해 충분하다. Z - 스택의 각 단계에, 현미경 분야의 모든 glutamatergic 시냅스를 식별하는 vGluT - 1 항체의 immunofluorescence 자극 레이저 빛을 처음에 스캔. 동일한 필드가 FM1 -​​ 43FX의 형광 각 단계에서 다시 스캔합니다. 신호의 채도를 피할 수 있지만, 주어진 실험에 대한 모든 coverslips 중에서 지속적으로 중립적인 밀도 필터링 및 photomultiplier 향상을 설정합니다. 우리는 일반적으로 각 실험 조건마다 ≥ 5 필드를 취득. </li><li> 분석 소프트웨어를 사용하여 복합 단일 평면 이미지로 Z - 스택을 변환합니다. 우리가이 변환에 사용 과정은 각 픽셀에 어떤 비행기에서 최대 강도에 따라 이미지를 생성합니다. 합성 이미지 thresholded하고 vGluT - 1 터미널은 FM1 -​​ 43FX 참조 얼룩없이 분석 식별됩니다. puncta로 불리는 스테인드 영역, 흥미 영역으로 표시됩니다. 우리는 일반적으로 현장 지당 10 puncta을 선택합니다. 다음 FM1 -​​ 43FX 채널이 thresholded하고 관심의 영역은 겹쳐 있습니다. 분석 소프트웨어는 각 신호의 형광 강도뿐만 아니라 각 punctum의 영역을 계량하는 데 사용됩니다. 절대 픽셀 기준 또는 퍼센트 픽셀 기준 비활성 시냅스에서 활성화 구별하는 데 사용할 수 있습니다. </li></ol> 대표 결과 <ol><li> 우리 문화, 우리는 vGluT - 1 염색법에 의해 정의된 글루 탐 산염 시냅스의 70~80%은, 감지 FM1 - 43 얼룩이, 6-8을 전시 것을 발견했습니다. 의 중첩 이미지 녹색 FM1 -​​ 43 형광 붉은 vGluT - 1이이 노란색 puncta 풍부한으로 반영되어 얼룩 : 공동화된 마커와 시냅스. 이들은 화살표로 표시됩니다. </li><li> (적색) vGluT - 1 양성 puncta의 약 20-30%은 기본 위 (녹색) FM1 -​​ 43 형광을 가지고 실패합니다. 이들은 비활성 presynaptic 터미널이며, 예제는 화살촉으로 표시됩니다. </li><li> 시냅스의 3 분의 1 인구도 관찰됩니다. 이들에 대한 (녹색) FM1 -​​ 43 형광하지만 (적색) vGluT - 1 얼룩는 없음. 양수 이들은 활성 GABAergic 시냅스이며, 예제는 별표로 표시됩니다. 이들은 우리의 분석의 대부분에서 제외되지만 명시적으로 vGluT - 1 항체의 자리에 기공을 갖는 GABA의 전송 항체를 사용하여 공부하실 수 있습니다. </li></ol>

<ol>
	<li>Mennerick, S., Que, J., Benz, A., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+and+synaptic+properties+of+hippocampal+neurons+grown+in+microcultures+and+in+mass+cultures.">Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures.</a> J. Neurophysiol. 73, 320-332 (1995).</li><li>Moulder, K. L., Jiang, X., Taylor, A. A., Olney, J. W., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+activity+depresses+synaptic+function+through+an+effect+on+vesicle+priming.">Physiological activity depresses synaptic function through an effect on vesicle priming.</a> J. Neurosci. 26, 6618-6626 (2006).</li><li>Moulder, K. L., Jiang, X., Chang, C., Taylor, A. A., Benz, A. M., Conti, A. C., Muglia, L. J., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+role+for+Ca2+-dependent+adenylyl+cyclases+in+recovery+from+adaptive+presynaptic+silencing.">A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.</a> J. Neurosci. 28, 5159-5168 (2008).</li><li>Sara, Y., Mozhayeva, M. G., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+vesicle+recycling+supports+neurotransmission+during+sustained+stimulation+at+hippocampal+synapses.">Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses.</a> J. Neurosci. 22, 1608-1617 (2002).</li><li>Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 809-817 (1999).</li><li>Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, N., Rhee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+vesicle+priming+and+short-term+plasticity+by+Munc13+isoforms.">Differential control of vesicle priming and short-term plasticity by Munc13 isoforms.</a> Neuron. 33, 411-424 (2002).</li><li>Altrock, W. D., tom Dieck, S., Sokolov, M., Meyer, A. C., Sigler, A., Brakebusch, C., Fassler, R., Richter, K., Boeckers, T. M., Potschka, H., Brandt, C., Loscher, W., Grimberg, D., Dresbach, T., Hempelmann, A., Hassan, H., Balschun, D., Frey, J. U., Brandstatter, J. H., Garner, C. C., Rosenmund, C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+inactivation+of+a+fraction+of+excitatory+synapses+in+mice+deficient+for+the+active+zone+protein+bassoon.">Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.</a> Neuron. 37, 787-800 (2003).</li><li>Ting, J. T., Kelley, B. G., Lambert, T. J., Cook, D. G., Sullivan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+overexpression+depresses+excitatory+transmission+through+both+presynaptic+and+postsynaptic+mechanisms.">Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.</a> Proc. Natl. Acad. Sci. USA. 104, 353-348 (2007).</li></ol>

의미 <ol><li> 일반적으로 시냅스는 측정 확률과 송신기를 출시하여 작동하는 생각되고 있습니다. 자신과 다른 사람이 작업은 기공을 갖는 신경 전달 물질의 수송과 다른 주요 presynaptic 마커 2, 6-8의 전체 보완에도 불구하고, 일부 시냅스가 신경 전달 물질을 방출하는 내화물하는 그것이 분명합니다. 우리는 뉴런의 기초 네트워크 활동이 비활성 시냅스의 인구를 유지하는 것이 중요합니다 ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>FM1-43FX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-35355</td>
<td>A red-shifted FM4-64FX is also available, but has not proven as amenable to assays of presynaptic silencing.</td>
</tr>
<tr>
<td>Advasep-7</td>
<td>&nbsp;</td>
<td>CyDex</td>
<td>AR-0A7-001</td>
<td>&nbsp;</td>
<tr>
<td>vGluT-1</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB5905</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 647-conjugated anti-GP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21450</td>
<td>&nbsp;</td>
<tr>
<td>Fluoromount-G</td>
<td>&nbsp;</td>
<td>Southern Biotechnology Associates</td>
<td>0100-01</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

presynaptically silent synapses studied with light microscopy

시냅스 소성 가능성 underlies 학습과 기억 또한 neurotoxic되는 것을 달리 손상 모욕을 방지 적응력을 나타내는 5 신경계의 능력. 그것이 신경 전달 물질의 글루 탐 산염을 포함한 vesicles 발표 presynaptic 터미널의 능력의 디지털 전환 및 해제로 표현되기 때문에 우리는 부분적으로 흥미로운 presynaptic 소성의 형태를 공부하고 있습니다. 여기서 우리는 설치류의 해마에서 준비 dissociated 셀 문화에 presynaptic 터미널의 활동 상태를 시각화를위한 프로토콜을 보여줍니다. 이 메서드는 일반적으로 시냅스 vesicles 레이블을하는 데 사용되는 styryl 염료의 고칠수 형태 FM1 -​​ 43와 얼룩을 사용하여 활성 시냅스를 감지에 의존합니다. 이 얼룩 프로필은 기공을 갖는 글루 탐 산염 수송 1 (vGluT - 1)에 관계없이 정품 인증 상태의 라벨 모두 글루 탐 산염 시냅스를위한 얼룩에 대한 감독 항체와 같은 단말기의 immunostaining와 비교됩니다. 우리는 depolarizing 자극이 presynaptic 입을를 유도 것을 발견했습니다. 기준 조건 하에서 침묵 시냅스의 인구는 장시간 전기 입을 또는 CAMP 신호 통로의 활성화에 의해 활성화할 수 있습니다.

Watch this Scientific Journal Video about Presynaptically Silent Synapses Studied with Light Microscopy at JoVE.com

Presynaptically Silent Synapses Studied with Light Microscopy

Glutamatergic 시냅스는 자동 모드로 활성 모드에서 전환할 수 있습니다. 우리는 쥐 뉴런의 dissociated 문화에 presynaptic 활동 상태를 보여주는 것은 적극적인 시냅스를 시각화하는 FM1 -​​ 43 염료의 고칠수 양식을 사용하여 모든 글루 탐 산염 시냅스를 시각화하는 vGluT - 1 항체와 immunostaining 시각이다.

라이트 현미경과 연구 Presynaptically 사일런트 시냅스

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

presynaptically-silent-synapses-studied-with-light-microscopy

Research

JoVE Journal

Biology

11.3K 조회수.  Washington University School of Medicine. Glutamatergic 시냅스는 자동 모드로 활성 모드에서 전환할 수 있습니다. 우리는 쥐 뉴런의 dissociated 문화에 presynaptic 활동 상태를 보여주는 것은 적극적인 시냅스를 시각화하는 FM1 -​​ 43 염료의 고칠수 양식을 사용하여 모든 글루 탐 산염 시냅스를 시각화하는 vGluT - 1 항체와 immunostaining 시각이다.

비디오: Presynaptically Silent Synapses Studied with Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

의 준비 Drosophila</em라이트 현미경을 위해> S2 세포

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

연구

생물학

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

의 검사<em&gt; 초파리</em가벼운 현미경으로&gt; 애벌레 기관 내 터미널 셀

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

다층 제브라 피쉬의 장기 라이트 시트 현미경에 장착

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

얼룩말 물고기 배아에서 빠른 심장 역학의 빛 시트 현미경 검사

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

라이브 현미경 및 오픈 소스 소프트웨어를 사용하여 아스퍼길러스 니둘란스 성장률의 정량적 분석

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...