Diese Arbeit wurde vom NIH gewährt DA018109 und MH78823 und NIH Neuroscience Blueprint Core-Stipendium P30NS057105 zu Washington University unterstützt.

Kultur Vorbereitung <ol><li> Bereiten dissoziierten Zellkulturen von Ratte oder Maus Hippocampus-Zellen aus postnatalen Tag 0 bis 3 Tiere 1. Unsere Neuronen auf eine zugrunde liegende Astrozyten Monoschicht, was wiederum hält sich an ein Kollagen-Schicht auf eine Zahl 0 Dicke Deckglas ausgebreitet halten. Platte der Neuronen bei einer Dichte von etwa 500 Zellen pro Quadratzentimeter. </li><li> Lassen Sie die Kulturen für 10-14 Tage wachsen, um synaptische Entwicklung und Reifung zu ermöglichen. Behandeln Sie die Kulturen am Tag in vitro 4 mit dem antimitotische AraC weiter Gliazellen Wachstum zu verhaften. Füttere sie am Tag in vitro 5 mit einem halben mittleren Austausch mit Neurobasal Medium plus B27 Ergänzung zum Überleben von Nervenzellen fördern. </li><li> Während der Kulturzeit, einführen Behandlungen entwickelt, um die Zahl der funktionell Schweigen Synapsen verändern. Wir finden, dass man bequemer Weg, um einen großen Prozentsatz (~ 80%) der Glutamat-Synapsen Stille einer 4-stündigen Behandlung mit 30 mM Kalium ist. Längere Inkubationen mit schwächeren depolarisierende Stimuli induzieren ähnlich silencing 2. 4-stündige Stimulation mit 50 &amp;mgr; M Forskolin, einem Aktivator der Adenylylcyclasen, können verwendet werden, um die Bevölkerung von stillen Terminals zu Studienbeginn 3 erwecken. </li></ol> Visualizing stillen Synapsen <ol><li> Die Zellen sind eine relativ geringe Dichte mit gut getrennten neuritischen Prozesse am Tag in vitro 10-14 haben, um Visualisierung diskreter synaptischen Terminals mittels Fluoreszenzmikroskopie zu erleichtern. </li><li> Bereiten Sie eine Stammlösung von 5 mM FM1-43FX in destilliertem Wasser. Die Aktie sollte in den dunklen bei 4 ° C gehalten werden Alle Experimente mit FM1-43FX sollte auch in der Dunkelheit durchgeführt werden. </li><li> Fordern Sie die Kulturen für 2 Minuten mit 45 mM Kaliumchlorid in einem HEPES-gepufferte Kochsalzlösung mit 100 mM Natriumchlorid, 2 mM Calciumchlorid, 1 mM Magnesiumchlorid, 10 mM Glukose und 10 uM FM1-43FX. Diese Lösung sollte auch 1 uM NBQX und 25 uM D-APV an postsynaptische Rezeptoren zu blockieren und zu verhindern, wiederkehrende Signalisierung. Diese Herausforderung wird label synaptischen Terminals, die in der Lage Exozytose und Endozytose sind. Die Herausforderung der Zeit und Kraft sind entworfen, um mindestens eine Runde Exozytose aller verfügbaren Vesikel in der Recycling-Pool 4 verursachen. Allerdings ist die Herausforderung nicht aus, um zusätzliche Schalldämpfung von Endgeräten führen. </li><li> Waschen Sie die Kultur für 3 bis 5 Sekunden nur, mit den gleichen HEPES-gepufferte Salzlösung ohne Kaliumchlorid, aber mit 500 uM Advasep-7 zu entfernen unspezifische Farbstoff 5. Beachten Sie, dass das Timing dieser Schritt kritisch, da längere Anwendung von Advasep-7 ist, führt zum Verlust aller FM1-43FX Kennzeichnung führen. Next waschen in HEPES-gepufferte ohne Advasep-7 Kochsalzlösung für fünf 2-Minuten-Takt. </li><li> Fix-Zellen mit 4% Paraformaldehyd und 0,2% Glutaraldehyd in PBS, pH 7,4 für 10 Minuten. Waschen Sie die Zellen einmal kurz mit PBS, dann für 15 Minuten in Blocking-Lösung mit 4% normalem Ziegenserum und 0,04% Triton X-100 in PBS inkubiert. Beachten Sie, dass FM1-43FX ist auch sehr empfindlich auf die Höhe der Triton X-100 verwendet hier und in späteren Schritten. Generell ist FM1-43FX Kennzeichnung am besten in Abwesenheit von Triton X-100, aber etwas Spülmittel ist notwendig, um Zellen für die anschließende Immunfärbung permeabilisieren. Wir haben empirisch gezeigt, dass 0,04% Triton X-100 optimal für die Prüfung der präsynaptisch stillen Synapsen bestimmt wird. </li><li> Verdünnen Sie die primäre vGluT-1-Antikörper in Blockierungslösung bei einer Konzentration von 1:2500. Inkubieren Sie die Zellen unter leichtem Schütteln / Rotation in der verdünnten vGluT-1-Antikörper für 3 Stunden bei Raumtemperatur. Achten Sie darauf, Ihre Kulturschalen mit einer Folie zu verhindern Ausbleichen der FM1-43FX Farbstoff zu decken. </li><li> Waschen Sie die Zellen zweimal in PBS und dann Inkubieren Sie die Zellen mit einem Anti-Meerschweinchen-Antikörper, konjugiert mit einem Fluorophor mit unterschiedlichen spektralen Eigenschaften von FM1-43FX, die beiden Flecken unterscheiden. Für unser Experiment verwenden wir Alexa 555-konjugierten anti-Meerschweinchen-Antikörper bei 1:500 in Blocking-Lösung. Inkubieren Sie die Zellen mit dem sekundären Antikörper unter Schütteln für 30 Minuten bei Raumtemperatur. </li><li> Waschen Sie die Zellen noch viermal in PBS und hängen Sie dann das Deckglas auf einem Objektträger aus Glas mit einem kleinen Tropfen Fluoromount-G. Seien Sie sicher zu orientieren dem Deckglas, so dass die Zellen der Gleitfläche. Lassen Sie die Dias über Nacht trocknen. </li><li> Wir sind nun bereit, um Bilder von der aktiven und inaktiven Synapsen zu erwerben. Bereiten Sie eine 60 x Öl-Objektiv mit 1,4 numerische Apertur auf einem konfokalen Laser-Scanning-Mikroskop. Setzen Sie ein Bild auf der Bühne, dass das Deckglas Seite nach dem Ziel. Nach der Fokussierung der Folie finden Sie ein Feld von Neuriten, die nicht allzu dicht. Es wird auch empfohlen, Bildgebung in der Nähe der Zelle soma zu vermeiden, als Rest-FM1-43FX Farbstoff dort bleiben können, auch nach ausgiebigem Waschen. Für unsere experimentellen Bilder, wir in der Regel in einer Feldgröße von 51 51 Mikrometer zu vergrößern. </li><li> Erwerbenein z-Stapel von einem bestimmten Bereich abdecken zu einer Tiefe von 7,8 Mikrometer mit 27 0,3-Mikrometer-Schritten. Dieser Bereich ist genug, um die Dicke der Neuriten in unserer Kultur zu decken. Für jeden Schritt in der z-Stapel ersten Scan mit dem Laserlicht, dass Immunfluoreszenz der vGluT-1-Antikörper zu allen glutamatergen Synapsen in einem Mikroskop Bereich zu identifizieren anregt. Das gleiche Feld ist bei jedem Schritt für FM1-43FX Fluoreszenz erneut gescannt. Set mit neutraler Dichte Filter und Photomultiplier Gewinne konsequent unter allen Deckgläser für ein bestimmtes Experiment, bei gleichzeitiger Vermeidung Sättigung des Signals. Wir in der Regel erwerben ≥ 5 Felder pro Bedingung für jedes Experiment. </li><li> Mit Analyse-Software, konvertieren Sie die z-Stapel in ein Composite Ein-Ebenen-Bild. Der Prozess verwenden wir für diese Umwandlung erzeugt ein Bild auf die maximale Intensität von jeder Ebene in jedem Pixel basiert. Das zusammengesetzte Bild ist Schwellenwert und vGluT-1-Terminals sind für die Analyse identifiziert, ohne Bezugnahme auf die FM1-43FX Fleck. Verschmutzte Stellen, die so genannte puncta, werden als Gebiete von Interesse versehen. Wir in der Regel wählen Sie 10 puncta pro Feld. Als nächstes wird der FM1-43FX Kanal Schwellenwert und die Regionen von Interesse sind, überlagert. Die Analyse-Software wird verwendet, um die Fluoreszenz-Intensität jedes Signals sowie die Fläche der einzelnen punctum zu quantifizieren. Ein absolutes Kriterium Pixel oder als Prozentsatz Pixel Kriterium zur Unterscheidung von inaktiven Synapsen aktiv werden. </li></ol> Repräsentative Ergebnisse <ol><li> In unseren Kulturen finden wir, dass 70-80% der Glutamat-Synapsen durch vGluT-1 Färbung definiert, nachweisbar FM1-43 Färbung 2, 6-8 aufweisen. In überlagert Bilder von grünen FM1-43 Fluoreszenz und rot vGluT-1 Färbung dies als eine Fülle von gelben puncta widerspiegelt: Synapsen mit co-lokalisiert Marker. Diese werden durch die Pfeile angedeutet. </li><li> Etwa 20-30% der (rot) vGluT-1-positiven puncta scheitern an (grün) FM1-43-Fluoreszenz über dem Ausgangswert zu haben. Dies sind die inaktiven präsynaptischen und ein Beispiel wird durch die Pfeilspitze gekennzeichnet. </li><li> Ein Drittel der Bevölkerung von Synapsen wird auch beobachtet werden. Diese sind für die (grüne) FM1-43-Fluoreszenz aber ohne (rot) vGluT-1-Färbung. Positive Diese sind aktiv GABAergen Synapsen und ein Beispiel wird durch den Stern gekennzeichnet. Diese werden von den meisten unserer Analysen ausgeschlossen, kann aber explizit untersucht werden mit Hilfe eines vesikulären GABA-Transporter-Antikörper an die Stelle der vGluT-1-Antikörper. </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>

Bedeutung <ol><li> Typischerweise Synapsen wird angenommen, dass durch die Freigabe Sender mit einem messbaren Wahrscheinlichkeit zu betreiben. Die Arbeit von uns selbst und andere macht es offensichtlich, dass einige Synapsen refraktär Freisetzung von Neurotransmittern sind, trotz einer vollständigen Ergänzung der vesikulären Neurotransmitter-Transporter und andere wichtige präsynaptischen Marker 2, 6-8. Wir haben bereits, dass die basalen Netzwerk-Aktivität in Neuronen entscheidend für diese Bevöl...

<!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

Synaptische Plastizität wahrscheinlich liegt dem Nervensystem die Fähigkeit zu lernen und zu erinnern und auch stellen eine Anpassungsfähigkeit, die sonst verhindert eine Beschädigung Beleidigungen zu neurotoxischen. Wir haben das Studium eine Form der präsynaptischen Plastizität, die interessante Teil ist, weil es als digitale Ein-und Ausschalten eines präsynaptischen Terminal s Fähigkeit, Vesikel mit dem Neurotransmitter Glutamat-Ausschüttung zum Ausdruck kommt. Hier zeigen wir ein Protokoll für die Visualisierung der Aktivität Status der präsynaptischen in dissoziierten Zellkulturen aus dem Hippocampus Nagetier vorbereitet. Die Methode beruht auf der Erfassung aktiven Synapsen mit Färbung mit einem fixierbaren Form der Styryl-Farbstoff FM1-43, häufig verwendet, um synaptische Vesikel-Label. Diese Färbung Profil ist mit Immunfärbung des gleichen Terminals mit einem Antikörper gegen den vesikulären Glutamat-Transporter 1 (vGluT-1), eine Kennzeichnungspflicht aller Glutamat Synapsen unabhängig von Aktivierungsstatus konzipiert Fleck gerichtet verglichen. Wir finden, dass depolarisierende Stimuli präsynaptischen Schweigen zu induzieren. Die Bevölkerung von Synapsen, die still ist unter Baseline-Bedingungen können bei längerer elektrischen Schweigen oder durch Aktivierung des cAMP-Signalwege aktiviert werden.

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

Presynaptically Silent Synapses Studied with Light Microscopy

Glutamatergen Synapsen kann von einem aktiven Modus, um einen Silent-Modus wechseln. Wir zeigen, dass präsynaptische Aktivität Status in dissoziierten Kultur der Nager Neuronen visualisiert mit einem fixierbaren Form des FM1-43 Farbstoff aktiven Synapsen visualisieren und Immunfärbung mit vGluT-1-Antikörper, um alle Glutamat Synapsen zu visualisieren.

Präsynaptisch Stille Synapsen mit Lichtmikroskopie Studium

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 Aufrufe.  Washington University School of Medicine. Glutamatergen Synapsen kann von einem aktiven Modus, um einen Silent-Modus wechseln. Wir zeigen, dass präsynaptische Aktivität Status in dissoziierten Kultur der Nager Neuronen visualisiert mit einem fixierbaren Form des FM1-43 Farbstoff aktiven Synapsen visualisieren und Immunfärbung mit vGluT-1-Antikörper, um alle Glutamat Synapsen zu visualisieren.

Serie: 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.

Vorbereitung der Drosophila S2-Zellen für Lichtmikroskopie

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 ...

Forschung

Biologie

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Zell Elektroschweißmuffe Visualisierte mit Fluoreszenz-Mikroskopie

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

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.

Live Cell Reaktion auf mechanische Stimulation von Integrated Optical und Atomic Force Microscopy Studium

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.

Direct Imaging von Calcium ER mit Targeted-Esterase Induzierte Dye Loading (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.

Prüfung der<em&gt; Drosophila</em&gt; Larven Tracheal Terminal-Cells durch Lichtmikroskopie

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.

Multilayer-Halterung für Langzeit-Licht-Mikroskopie Blatt der Zebrafisch

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.

In vivo Klonale Verfolgung von hämatopoetischen Stamm-und Vorläuferzellen durch Fünf Fluoreszierende Proteine ​​mit Hilfe der konfokalen und Multiphotonen Mikroskopie Markierte

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.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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.

Analyse der Gap-Junction-abhängigen Übertragung von miRNA mit 3D-FRAP-Mikroskopie

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.