Bu çalışma Washington Üniversitesi NIH hibe DA018109 ve MH78823 ve NIH Nörobilim Blueprint Core Hibe P30NS057105 tarafından desteklenmiştir.

Kültür hazırlık <ol><li> Postnatal 0 ila 3 gün hayvanlar 1 sıçan veya fare hipokampal hücrelerinin ayrışmış hücre kültürleri hazırlayın. Bizim nöron sayısı 0 kalınlığı lamel üzerine yayılmış bir kollajen tabakası yapışır açmak altta yatan bir astrosit tek tabaka, uymak. Santimetre kare başına yaklaşık 500 hücre yoğunluğu nöron Plaka. </li><li> Kültürlerin sinaptik gelişme ve olgunlaşma izin 10-14 gün boyunca büyümeye izin verin. Daha glial büyüme tutuklama antimitotic AraC ile in vitro 4 gün kültürleri davranın. Nöronal yaşam geliştirmek için Neurobasal orta artı B27 takviyesi ile bir buçuk orta alışverişi ile in vitro 5 gün onları besleyin. </li><li> Kültür dönemde, işlevsel susturuldu sinaps sayısı değiştirmek için tasarlanmış tedaviler tanıtmak. Biz 30 mM potasyum ile 4 saatlik bir tedavi glutamat sinaps (~% 80) büyük bir yüzdesini susturmak için tek uygun yol olduğunu bulabilirsiniz. Zayıf depolarizan uyaranlara ile uzun inkubasyon benzer susturulması 2 neden olur. 50 mcM forskolin, adenilat cyclases bir aktivatör, 4 saat stimülasyon bazal 3 sessiz terminalleri nüfus uyandırmak için kullanılabilir. </li></ol> Sessiz sinaps görselleştirme <ol><li> Hücreler floresan mikroskopi kullanılarak kesikli sinaptik terminalleri görselleştirme kolaylaştırmak amacıyla in vitro 10-14 gün iyi ayrılmış nöritik süreçleri ile nispeten düşük yoğunluklu olmalıdır. </li><li> Distile su ile 5 mM FM1-43FX bir stok solüsyonu hazırlayın. Hisse senedi, 4 karanlıkta muhafaza edilmelidir ° C FM1-43FX kullanan tüm deneyleri de karanlıkta yapılmalıdır. </li><li> 45 mM potasyum klorür, 100 mM sodyum klorür, 2 mM kalsiyum klorür, 1 mM magnezyum klorür, 10 mM glukoz ve 10 mcM FM1-43FX içeren bir Hepes-tamponlu salin solüsyonu ile 2 dakika boyunca kültürlerin meydan. Bu çözüm aynı zamanda, postsinaptik reseptörleri bloke ve tekrarlayan sinyal önlemek için 1 mcM NBQX ve 25 mcM D-APV içermelidir. Bu meydan okuma, endositoz ekzositoz ve yeteneğine sahiptir sinaptik terminalleri etiket. Meydan okuma zamanı ve gücü, mevcut tüm geri dönüşüm havuz 4 veziküllerin ekzositoz en azından bir tur neden tasarlanmıştır . Ancak, meydan okuma terminalleri ek susturulması için yeterli bir sebep değildir. </li><li> Potasyum klorür olmadan tuzlu Hepes-tamponlu, ancak 500 mcM ile Advasep-7 nonspesifik boya 5 kaldırmak için aynı yöntem kullanılarak, sadece 3 - 5 saniye boyunca kültür yıkayın . Bu adımın zamanlaması tüm FM1-43FX etiketleme kaybına neden olacaktır Advasep-7 uzun süreli uygulama gibi kritik olduğunu unutmayın. Sonraki yıkama beş, 2 dakikalık aralıklarla Advasep-7 olmadan tuzlu Hepes tamponlu. </li><li> 10 dakika PBS, pH 7.4,% 4 paraformaldehid ve% 0.2 glutaraldehid hücreleri saptamak. Bir kez kısaca hücreler PBS ile yıkayın, daha sonra, PBS içinde% 4 normal keçi serumu ve% 0.04 Triton X-100 içeren engelleme çözümü 15 dakika süreyle kuluçkaya yatmaktadır. FM1-43FX Triton X-100 ve sonraki adımları kullanılan miktarı çok hassas olduğunu unutmayın. Genel olarak konuşursak, FM1-43FX etiketleme herhangi bir Triton X-100 yokluğunda iyi, ancak bazı deterjan sonraki immün hücreler permeabilize için gereklidir. Biz 0.04% Triton X-100 presynaptically sessiz sinaps muayene için en uygun olduğunu ampirik olarak tespit ettik. </li><li> 1:2500 &#39;lik bir konsantrasyon çözüm engelleme birincil vGluT-1 antikor sulandırınız. Hücreleri, oda sıcaklığında 3 saat süreyle seyreltilmiş vGluT-1 antikor hafif sallayarak / devir ile inkübe edin. FM1-43FX boya kasar önlemek için folyo ile kültür kaplarına karşılamak için emin olun. </li><li> Hücreler PBS içinde iki kez yıkayın ve sonra hücrelerin iki lekeleri ayırt etmek için bir fluorofor FM1-43FX farklı spektral özelliklere sahip bir anti-kobay konjuge antikor ile inkübe edin. Deneme için, biz çözüm engelleme 1:500 555-konjuge anti-kobay antikor Alexa kullanacaktır. Oda sıcaklığında 30 dakika sallayarak hücreleri ikincil antikor ile inkübe edin. </li><li> Hücreler PBS içinde dört kez daha yıkayın ve ardından küçük bir damla Fluoromount-G kullanarak bir bardak slayt üzerindeki kapağı kayma monte. Hücreler slayt bakacak şekilde yönlendirmek lamel emin olun. Slaytları gecede kurumasını bekleyin. </li><li> Şu anda aktif ve pasif sinaps görüntüler elde etmek için hazırız. Konfokal lazer tarama mikroskobu 1.4 sayısal diyafram ile 60 x yağlı objektif hazırlayın. Lamel yan amacı ile karşı karşıya olduğunu sağlanması, sahnede bir slayt yerleştirin. Slayt odaklanarak sonra, aşırı yoğun değildir neurites bir alan bulabilirsiniz. Artık FM1 43FX boya geniş yıkamadan sonra bile orada kalırlar Ayrıca, hücre soma yakın görüntüleme önlemek için tavsiye edilir. Deneysel görüntüler için, biz genellikle bir alan boyutu 51 51 mikron yakınlaştırmak. </li><li> Kazanmakbelirli bir alanda, 27 0.3 mikron adımları ile 7.8 mikron derinliğe kapsayan bir z-yığını. Bu aralık, bizim kültürümüzde neurites kalınlığı karşılamak için yeterli olacaktır. Z-yığını her adım için, bir mikroskop alanında tüm glutamaterjik sinaps tanımlamak için vGluT-1 antikoru immünofloresan heyecanlandıran lazer ışığı ile tarayın. FM1-43FX floresan her adım için aynı alanda yeniden taranır. Doygunluk sinyali kaçınarak, belirli bir deney için tüm lamelleri arasında sürekli olarak nötr yoğunluk filtreleme ve photomultiplier kazanımlar ayarlayın. Biz genellikle her bir deney için ≥ durum ortalama 5 alan kazanır. </li><li> Kompozit bir tek düzlem görüntü analiz yazılımı kullanarak, z-yığın dönüştürmek. Bu dönüşüm için kullanılan işlem, her bir piksel herhangi bir uçaktan maksimum yoğunluğuna göre bir görüntü üretir. Kompozit görüntü eşiklenir ve vGluT-1 terminalleri FM1-43FX referans leke olmadan, analiz için tanımlanır. Puncta olarak anılacaktır Vitray alanları, ilgi çekici bölge olarak işaretlenmiştir. Biz genellikle alan başına 10 puncta seçin. Sonraki FM1-43FX kanal eşiklenir ve ilgi bölgeleri üst üste. Analiz yazılımı, her bir sinyalin floresan yoğunluğunun yanı sıra, her punktum alanda ölçmek için kullanılır. Inaktif sinapsların aktif ayırt etmek için, mutlak bir piksel kriter ya da bir yüzdesi piksel kriter kullanılabilir. </li></ol> Temsilcisi Sonuçlar <ol><li> Bizim kültürde, biz vGluT-1 boyanmasının tarafından tanımlanan glutamat sinaps% 70-80, saptanabilir FM1-43 boyama 2, 6-8 sergilediğimiz bulabilirsiniz. Ile kaplanan görüntülerde FM1-43 yeşil floresan ve kırmızı vGluT-1 sarı puncta bir zenginliği olarak yansır boyama co-lokalize işaretleri ile sinaps. Bu oklarla gösterilir. </li><li> Yaklaşık% 20-30 (kırmızı) vGluT-1 pozitif puncta bazal yukarıda (yeşil) FM1-43 floresan başarısız olur. Bu inaktif presinaptik terminaller ve örnek bir ok ucu ile gösterilir. </li><li> Sinaps üçüncü bir nüfusun da görülecektir. Bunlar pozitif (yeşil) FM1-43 (kırmızı) vGluT-1 boyama floresan ama yoksun. Bu aktif GABAerjik sinaps ve örnek bir yıldız işareti ile gösterilir. Bu analizlerin dışında ama açıkça vGluT-1 antikor yer veziküler GABA taşıyıcı antikor kullanılarak incelenebilir. </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>

Önem <ol><li> Tipik olarak ölçülebilir bir olasılık verici serbest sinapsların çalıştırmak için düşünülmektedir. Veziküler nörotransmitter taşıyıcı ve diğer önemli presinaptik belirteçlerinin 2, 6-8, kendimiz ve başkaları tarafından tam rağmen, bazı sinaps nörotransmitter serbest dirençli olduğu aşikar kılar . Biz nöronlar bazal ağ aktivite inaktif sinaps bu nüfusu korumak için kritik olduğunu daha önce göstermiştir. </li></ol> Kritik adımlar...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Comment</th>
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<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>
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presynaptically silent synapses studied with light microscopy

Sinaptik plastisite olasılığı altında yatan sinir sistemi yeteneğini öğrenmek ve hatırlamak ve nörotoksik olma, aksi takdirde zarar hakaret engelleyen bir adaptasyon temsil edebilir. Nörotransmitter glutamat içeren veziküller serbest bırakmak için bir presinaptik terminal yeteneği bir dijital anahtarlama ve kapalı olarak ifade edilir, çünkü kısmen ilginç presinaptik plastisite bir form eğitim görüyor. Burada kemirgen hipokampus hazırlanan ayrışmış hücre kültürlerinde presinaptik terminalleri faaliyet durumu görselleştirmek için bir protokol göstermektedir. Bu yöntem yaygın olarak sinaptik veziküller etiketlemek için kullanılan styryl boya tamir edilebilir formu FM1-43 ile boyanma kullanarak aktif sinaps tespit dayanmaktadır. Bu boyama profili veziküler glutamat taşıyıcısı 1 (vGluT-1), glutamat sinaps aktivasyon durumu ne olursa olsun, etiket için tasarlanmış leke yöneltilen bir antikor ile aynı terminalleri İmmünoboyama ile karşılaştırılır. Biz depolarizan uyaranlara presinaptik susturulması neden olduğunu bulmak. Bazal koşullar altında sessiz sinaps nüfusu uzun süreli elektrik susturulması veya cAMP sinyalizasyon yollarının aktivasyonu ile aktive edilebilir.

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

Presynaptically Silent Synapses Studied with Light Microscopy

Glutamaterjik sinaps sessiz mod aktif bir moda geçiş yapabilirsiniz. Biz kemirgen nöronların ayrışmış kültür bu presinaptik aktivite durumu gösteren aktif sinaps görselleştirmek için FM1-43 boya tamir edilebilir formu kullanarak ve tüm glutamat sinaps görselleştirmek için vGluT-1 antikoru ile immün görüntülenmiştir.

Işık Mikroskop ile İncelenen Presynaptically Sessiz Sinapslar

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

11.4K Görüntüleme.  Washington University School of Medicine. Glutamaterjik sinaps sessiz mod aktif bir moda geçiş yapabilirsiniz. Biz kemirgen nöronların ayrışmış kültür bu presinaptik aktivite durumu gösteren aktif sinaps görselleştirmek için FM1-43 boya tamir edilebilir formu kullanarak ve tüm glutamat sinaps görselleştirmek için vGluT-1 antikoru ile immün görüntülenmiştir.

Video: 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.

Hazırlanması Drosophila Işık Mikroskopi S2 hücreleri

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 ...

Biyoloji

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.

Tümleşik Optik ve Atomik Kuvvet Mikroskobu eğitimi Mekanik Uyarım Canlı Hücre Tepki

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.

Hedefli-esteraz ile ER Kalsiyum Doğrudan Görüntüleme Kaynaklı Boya Yükleme (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.

İncelenmesi<em&gt; Drosophila</emIşık Mikroskopi tarafından&gt; Larva Trakeal Terminal Hücreleri

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.

Çok katmanlı Zebra balığı uzun vadeli Işık Sayfalık Mikroskopi için Montaj

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.

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

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.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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 Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

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.

Zebra Balığı Embriyolarında Hızlı Kardiyak Dinamiklerin Işık Tabakası Mikroskopisi

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.

Canlı Mikroskopi ve Açık Kaynaklı Yazılım Kullanılarak Aspergillus nidulans Büyüme Hızının Nicel Analizi

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