The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

Ethik - Erklärung: Alle in vivo Tierversuchen wurden nach einer genehmigten Protokoll und unter den ethischen Richtlinien des Memorial Sloan Kettering Cancer Center (MSK) Institutional Animal Care und Use Committee (IACUC) ausgeführt beschrieben. 1. Aufbau eines Vernebelungsgerät <ol><li> Bringen Holzteil A (12,5 x 2,5 x 1,8 cm 3) aufrecht im Zentrum von Teil B (12,7 x 10,7 x 1,8 cm 3) mit zwei Schrauben (4 x 25 mm 2). Befestigen Holzteil C (11 × 2,5 × 1,8 cm 3) in der Mitte des Teils A (12,5 x 2,5 x 1,8 cm 3) mit einer Schraube, so dass ein Teil C (11 x 2,5 x 1,8 cm 3) noch bewegt werden. Siehe Abbildung 1. </li><li> Bohren Sie zwei Löcher durch die untere Spitze der Sprühflasche Auslöser einer Kunststoff 3 Unzen Mini Sprayer (D), und drücken Sie eine Edelstahlstange (10 cm von 1/16 &quot;Stahl) (E) durch zwei Schleifen zu bilden, eine auf entweder Seite des Auslösers. </li&gt; <li> Wickeln Sie den unteren Teil der Sprühflasche mit Klebeband (F) die Kabelbinder ein Abrutschen zu verhindern. Bringen Sie die Sprühflasche Holzteil C (11 x 2,5 x 1,8 cm 3) unter Verwendung der beiden Kunststoff - Kabelbinder (28 cm) (G). </li><li> Schneiden Sie das 011-Servomotor (I) aus, und schließen Sie sie mit den losen Kabel der Servosteuerung (H). Befestigen Sie dann den Servomotor an die Spitze der Holzteil A (12,5 x 2,5 x 1,8 cm 3) unter Verwendung von Klebeband. </li><li> Bringen Sie einen Bleistift (J) an den Servomotorhebel mit der Büroklammer (K). Dicht verbinden mit Klebeband die äußersten Teile des Stiftes an den Stahlstab Schleifen Kunststoff abgedeckt Twist Drähte (L) und befestigen Sie die Enden auf dem Bleistift. </li><li> Schneiden Sie die Servomotor-Steuereinheit des Magnetkabelstecker (M) und bringen Sie ihn an die Lautsprecherkabel (N) ab. Dann kleben Sie das Servomotor - Steuereinheit zu Holzteil B (12,7 x 10,7 x 1,8 cm 3). Schneiden Sie ein w1 Kabel mit Magnet Anschlüsse in zwei Hälften und heften sich an das lose Ende des Kupfer s ein Teilpeaker Kabel (1 m). Schließen Sie den (magnetisch) i2 Kippschalter und p1 Leistung der zur Verfügung stehenden w1 Kabel und einer 9V-Batterie. </li></ol> 2. Empfindlichkeit Bestimmung der Methode <ol><li> In einem 1,5 - Mikrozentrifugenröhrchen ml, werden die Lösungen von [Ru (bpy) 3] Cl 2 bei der Umkehrosmose Wasser (100 &amp; mgr; l) in Mengen von 260 &amp; mgr; g (347 nmol), 52 &amp; mgr; g (69 nmol), 26 &amp; mgr; g (34 nmol) , 5 ug (6,9 nmol), 3 &amp; mgr; g (3,5 nmol), 520 ng (694 pmol), 260 ng (347 pmol), 52 ng (69 pmol), 26 ng (34 pmol), 5 ng (6,9 pmol), und 3 ng (3,5 pmol). </li><li> Mischungs 100 ul jeder [Ru (bpy) 3] Cl 2 Lösung mit 100 ul einer wässrigen Lösung von Ammonium - Cernitrat ((NH 4) 2 Ce (NO 3) 6) in Wasser (25 mM) auf einem Mikroskop - Objektträger. </li><li> Stellen Sie den Erwerb in der Biolumineszenz Leser, indem Sie die Imaging - Software zu initialisieren. <ol><li> Melden Sie sich in das Benutzerprofil in und suchen die acquisition Bedienfeld. Klicken Sie auf &quot;Initialisieren&quot; und warten, bis das Gerät bereit ist. </li><li> Suchen Sie nach &quot;Imaging-Modus&quot; und stellen Sie sicher, &quot;Luminescent&quot; und &quot;Photograph&quot; werden überprüft und, dass &quot;Fluorescent&quot; nicht markiert ist. </li><li> Ändern &quot;Belichtungszeit&quot; Einstellung für &quot;Luminescent&quot; bis 20 s. Legen Sie die übrigen Einstellungen für &quot;Luminescent&quot; wie folgt: &quot;Binning&quot;: Medium; &quot;F / Stop&quot;: 1; und &quot;Emission Filter&quot;: Open. HINWEIS: Die Belichtungszeiten benötigen nach der Instrumentierung und experimentellen Einstellung angepasst werden, verwendet werden, falls abweichend von der dargestellten Einrichtung. </li><li> Für &quot;Photograph&quot;, verwenden Sie die folgenden Einstellungen: &quot;Belichtungszeit&quot;: Auto; &quot;Binning&quot;: Medium; und &quot;F / Stop&quot;: 8. Stellen Sie den &quot;Betreff Höhe&quot; gemäß Abbildung target.Look für das &quot;Field of View&quot; Dropdown-Menü. Die Grundeinstellung ist &quot;C&quot; Wechseln Sie zu &quot;B&quot; (14 cm Abstand zwischen der cAmera und Probentisch). </li></ol></li><li> Stellen Sie den Vernebler, indem Sie einen Objektträger auf einem Blatt aus schwarzem Tonpapier auf dem Boden der Imaging - Kammer platziert es aus dem Oxidationsmittel zu schützen. Mischen Sie einen 100 μLdroplet von [Ru (bpy) 3] Cl 2 -Lösung mit 100 &amp; mgr; l einer wässrigen Lösung von (NH 4) 2 Ce (NO 3) 6. Beobachten Sie das grüne Feld Fadenkreuz. <ol><li> Legen Sie die abzubildenden Objekt auf dem schwarzen Tonpapier, so dass der Bereich von Interesse ist in der Mitte des grünen Fadenkreuz-Licht-Box angezeigt auf der Probenbühne. Bereiten Sie den Vernebler durch die Kunststoff-Sprühflasche aus dem Holzbock lösen. Füllen Sie eine Lösung von Triethylamin (1: 3 in Wasser / Ethanol) in seine Kunststoffbehälter und bringen es auf den Holzbock. </li><li> Setzen Sie den Vernebler in der Biolumineszenz Leser und stellen Sie sicher, dass das Netzkabel aus dem Vernebler Kabel ist nicht angeschlossen. Stellen Sie sicher, dass der Netzschaltereingeschaltet ist, ist der Kippschalter ausgeschaltet und die rote LED leuchtet Setzen Sie den Vernebler, so dass der Spritzstrom wird auf den Bereich von Interesse auf dem Imaging-Objekt gerichtet, während die Sicht nach Behinderung von der Kamera auf das Motiv Bildgebung zu minimieren durch die Sprühdüse Kopf. </li><li> Legen Sie kleine, schwarze Stücke Tonpapier über eventuelle Hot - Spots (zB weiße Flecken auf mikroskopische Dias oder Injektionsstellen) , um sie vor Spritzwasser zu schützen. Legen Sie mindestens 40 cm des Zerstäubers Remote-Kabel innerhalb der Imaging-Kammer, so dass es nicht mit dem abzubildenden Objekt, dem Vernebler oder die magnetische Türverriegelung nicht stört. Schließen Sie das Abbildungssystem Tür. HINWEIS: Das Fadenkreuz wird Größe ändern auf der Grundlage der &quot;Field of View&quot; Einstellung in &quot;Bild Wohnen&quot;; stellen Sie sicher, dass diese auf &quot;B&quot; gesetzt. </li></ol></li><li> ein Bild erwerben, indem Sie die Bildsequenz zu initiieren. Klicken Sie auf &quot;Erwerben&quot; im &quot;Acquisition Systemsteuerung&quot;. Auf der ersten Bildgebungs sequrenz, ermöglichen automatisches Speichern, wenn ein Datenordner gewünscht (empfohlen) und wählen. Ignorieren Sie die &quot;Edit Imaging Labels&quot; Dialog bis zum Ende der Sequenz. HINWEIS: Die Steuerungssoftware zeigt das Gerät die Aktionen Schritt-für-Schritt in Echtzeit. Nach der Messung der Vorbereitung und der Probentisch in die rechte Position bewegt, öffnet er den Kameraverschluß und zählt die Messzeit. Die Verschlussöffnung kann auch durch ein Klickgeräusch von der Maschine erzeugten hören. </li><li> Da der Verschluss öffnet, Spray drei Ausbrüche von einer Lösung von Triethylamin (1: 3 in Wasser / Ethanol, 0,24 ± 0,04 ml pro Burst-Spray) durch den Kippschalter dreimal Schalt Chemilumineszenz zu erzeugen. HINWEIS: Die Probenbühne während der Messung bewegt. Lassen Sie genug (mindestens 40 cm) Kabel im Inneren des Gerätes, dies zu ermöglichen. Stellen Sie sicher, dass die Lösung durch den Vernebler versprüht werden kann durch das Steigrohr angestrebt werden, und dass es keine Luftblasen in das Rohr. Haben mehrere Ersatz batteries für den Zerstäuber bereit, falls erforderlich. </li></ol> 3. In - vivo - Bildgebung nach systemischer intravenöser Injektion <ol><li> In einem 1,5 ml Mikrozentrifugenröhrchen, Herstellung von 100 &amp; mgr; l phosphatgepufferter Salzlösung (PBS) Lösung zwischen 8 und 33 nmol [Ru (bpy) 3] Cl 2 enthält. Bereiten einer wäßrigen Lösung von (NH 4) 2 Ce (NO 3) 6 in Wasser (25 mM) zur gleichen Zeit. </li><li> Intravenös injizieren 100 ul [Ru (bpy) 3] Cl 2 in die Schwanzvene von gesunden Mäusen (n = 5). </li><li> Euthanize die Mäuse 10 min nach der Injektion über CO 2 Erstickung. <ol><li> Entfernen Sie die Haut mit einem Y-Schnitt aus dem Rumpf, entfernen Sie dann den Rippenbogen in einer U-Form das Herz und die Lungen zu belichten. Perfundieren die Mäuse durch einen Auslass in dem rechten Vorhof Schneiden und Injizieren 20 ml PBS durch eine 24 Gauge - Nadel in den linken Ventrikel 18. Vorsichtig durch den Bauch geschnittenHaut und der Niere und der Leber aus. Schneiden in Längsrichtung durch die Organe einen sichtbaren Schnitt zu erstellen. </li></ol></li><li> Stellen Sie den Erwerb wie in Schritten von 2,3 bis 2,6, mit den folgenden Änderungen beschrieben. <ol><li> Nach gründlichem den Vernebler Kunststoffbehälter Waschen, füllen Sie es mit einer Lösung von (NH 4) 2 Ce (NO 3) 6 in Wasser (25 mM) anstelle von Triethylamin. HINWEIS: Es ist wichtig , um gründlich die Verneblerdüse nach jedem Gebrauch gründlich, da kristallisieren (NH 4) 2 Ce (NO 3) 6 kann die Sprühdüse nach mehreren Verwendungen zerstören. </li></ol></li><li> Verwenden Sie die ganze Tier oder Organproben für die Bildgebung. <ol><li> Für ganze Bauch Bildgebung, positionieren Sie die Maus Karkasse mit dem offenen Bauch die Kamera und den Kopf nach auf der Rückseite des Geräts zeigt. Das Organ Center abgebildet (zB Leber oder Niere) im grünen Licht - Box Fadenkreuz werden. </li><li> Foder einzelne Organ Bildgebung und Quantifizierung, entfernen Sie die Maus aus dem Abbildungsinstrument und stecken Sie es nach unten. Ausgehend von dem bereits geöffneten Körperhöhle, auszuschneiden die inneren Organe (zB Niere, Leber, Lunge, Muskel, Milz, Gehirn und Herz). Schnitt durch das Hinterbein Haut Muskelgewebe auszuschneiden. Öffnen Sie vorsichtig den Schädel mit einem Skalpell das Gehirn auszuschneiden. <ol><li> Wenn das interessierende Organ Leber, Niere oder Milz, schneiden alle Organe in der Hälfte der Länge nach, legen jedes Organ auf einer Petrischale oder ein Stück schwarzes Tonpapier. </li></ol></li><li> Befolgen Sie die Anweisungen in den Schritten 2,3-2,6 um die relative Emission des chemolumineszenten Tracer für die einzelnen Organe herzustellen. </li></ol></li></ol> 4. In - vivo - Imaging von Lymphknoten <ol><li> Herstellung von 10 &amp; mgr; l einer PBS - Lösung , enthaltend 80 nMol [Ru (bpy) 3] Cl 2. Bereiten einer wäßrigen Lösung von (NH 4) 2 Ce (NO 3) 6 in water (25 mM) zur gleichen Zeit. </li><li> Injizieren Sie 10 ul der Lösung subdermally in die Hinterpfote von gesunden Mäusen (n = 5). Als Negativkontrolle, injizieren die kontralaterale Pfote mit 10 &amp; mgr; l reinem PBS. Opfern , um die Mäuse über CO 2 Ersticken 15 Minuten nach der Injektion. Entfernen Sie die Haut an beiden Hinterbeinen, die Lymphe Kanäle zu belichten bis in die Kniekehlen-Lymphknoten. </li><li> Stellen Sie den Erwerb wie in Schritt 3.4 beschrieben. </li><li> Entfernen Sie die poplitealen Lymphknoten von beiden Hinterbeinen, schneiden sie in zwei Hälften, und sprühen sie mit Oxidationsmittel auf einer Petrischale, wie zuvor beschrieben (Schritt 3.5.3), zum Zwecke der Quantifizierung. </li></ol>

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<li>Connell, T. U., James, J. L., White, A. R., Donnelly, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+Labelling+with+Versatile+Phosphorescent+Metal+Complexes+for+Live+Cell+Luminescence+Imaging%5BTitle%5D)+AND+%22Chem.+Eur.+J%22%5BJournal%5D)">Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.</a> Chem. Eur. J. 21 (40), 14146-14155 (2015).</li>
<li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis%2C+labeling+and+bioanalytical+applications+of+a+tris%282%2C2%E2%80%B2-bipyridyl%29ruthenium%28II%29-based+electrochemiluminescence+probe%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.</a> Nat. Protoc. 9 (5), 1146-1159 (2014).</li>
<li>Bœuf, G., et al. <a target="_blank" href="https://www.researchgate.net/publication/262418715_Encapsulated_RutheniumII_Complexes_in_Biocompatible_PolyDL-lactide-co-glycolide_Nanoparticles_for_Application_in_Photodynamic_Therapy">Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.</a> ChemPlusChem. 79 (1), 171-180 (2014).</li>
<li>Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanotechnology+and+its+applications+in+surgery%5BTitle%5D)+AND+%22Brit.+J.+Surgery%22%5BJournal%5D)">Nanotechnology and its applications in surgery.</a> Brit. J. Surgery. 97 (4), 463-465 (2010).</li>
<li>Barry, N. P. E., Sadler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+for+Metals+in+Medicine%3A+How+Nanotechnology+May+Help+To+Shape+the+Future%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Challenges for Metals in Medicine: How Nanotechnology May Help To Shape the Future.</a> ACS Nano. 7, 5654-5659 (2013).</li>
<li>Hasan, K., Bansal, A. K., Samuel, I. D. W., Roldán-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuning+the+Emission+of+Cationic+Iridium+%28III%29+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand%5BTitle%5D)+AND+%22Nat.Sci.+Rep%22%5BJournal%5D)">Tuning the Emission of Cationic Iridium (III) Complexes Towards the Red Through Methoxy Substitution of the Cyclometalating Ligand.</a> Nat.Sci. Rep. 5, 1-15 (2015).</li>
<li>Truong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemiluminescence+detection+with+water-soluble+iridium%28III%29+complexes+containing+a+sulfonate-functionalised+ancillary+ligand%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Chemiluminescence detection with water-soluble iridium(III) complexes containing a sulfonate-functionalised ancillary ligand.</a> Analyst. 139 (22), 6028-6035 (2014).</li>
</ol>

The authors have nothing to disclose.

Hier haben wir eine Technik vorgestellt, die optisch der Lage ist, durch einen chemolumineszierenden Reporter erstellt Gewebe über die Emission von Photonen abzugrenzen. Im Gegensatz zu anderen, etabliert, Technologien 4, 5, 6, 7, 8, 9, dieses chemilumineszierende Reportersystem verwendet eine Abbildungssonde , die nicht-ra...

In den letzten Jahrzehnten haben die Imaging-Technologien die Art und Weise revolutioniert, die Ärzte diagnostizieren und Überwachung von Krankheiten. Diese bildgebenden Verfahren, wurden jedoch weitgehend auf Ganzkörperbildgebungssysteme, wie Positronenemissionstomographie (PET), Single-Photon-Emissions-Computertomographie (SPECT), Computertomographie (CT) und Magnetresonanztomographie (MRI). Besondere Aufmerksamkeit wurde auf Krebs bezahlt und technologischen Durchbrüche haben Bildgebung stark die Art und Weise verbessert, dass diese Krankheit diagnostiziert und behandelt wird. Trotz dieser Fortschritte gibt es einen Ort, an dem diese Imaging-Technologien einfach nicht passen: der OP. Während Ganzkörperbildgebungstechniken in der chirurgischen Planung helfen können, fehlt ihnen typischerweise räumliche Auflösungen hoch genug Ärzte in Echtzeit , um festzustellen , ob alle Tumorgewebe entfernt wurde oder Gewebe Resttumor bleibt bei den operativen Margen 1 versteckt. Stellt sicher, dass keine infiltrativenTumorränder hinter ist links eine der wichtigsten operativen Ziele und Chirurgen müssen einen Drahtseilakt zwischen strengen und vorsichtigen Gewebsresektion gehen. Wenn zu viel entfernt wird, sind unerwünschte Nebenwirkungen für den Patienten verstärkt; wenn zu wenig entfernt wird, werden die Rezidivrate 2 erhöht, 3. Daher ist es entscheidend, eine genaue Tumorränder zu beschreiben, und wir glauben, dass chemolumineszierende intraoperative Bildgebung kann helfen, die Genauigkeit der Identifikation von Tumorgrenzen zu verbessern, indem Chirurgen sichtbar zu machen bösartigem Gewebe zu helfen, die sonst unentdeckt mit etablierten Techniken bleiben könnte. Es gibt viele Imaging-Technologien derzeit für eine mögliche Verwendung als intraoperativen Bildgebungssysteme untersucht. Dazu gehören β- und γ-Strahlung emittierenden Sonden 4, optische Fluoreszenz 5, Raman - Spektroskopie 6 </sup&gt;, 7 und Cherenkov Lumineszenz 8, 9. Bis heute jedoch keine von diesen als klinische Standardtools etabliert geworden. Optische Fluoreszenz-Bildgebung hat sich bisher die vielversprechendsten dieser Techniken zu sein und ist deshalb die erforscht. Während es bereits ein wertvolles Werkzeug für viele Anwendungen gezeigt worden ist, ist es nicht ohne Einschränkungen. Tatsächlich ist ihr Hauptnachteil der Hintergrundfluoreszenz von Natur aus autofluoreszenten biologischem Gewebe erzeugt. Dieser Hintergrund autofluoreszierenden Signal ist ein Produkt der Anregung von dem umgebenden Gewebe, zusätzlich zu dem Fluorophor, von der externen Lichtquelle zur Erzeugung eines Fluoreszenzsignals erforderlich ist. Aus praktischer Sicht kann diese Autofluoreszenz führen möglicherweise zu niedrigen Signal-zu-Rausch-Verhältnisse, die die Nützlichkeit dieser Technologie im Operationsraum begrenzen. Der DirektorVorteil der Chemilumineszenz-Bildgebung über Fluoreszenz-Bildgebung ist, dass kein Anregungslicht notwendig ist. Als Ergebnis gibt es keine Hintergrundautofluoreszenz. In Chemilumineszenz Bebilderung wird die Anregungsenergie chemisch statt erzeugt. Dieses Verfahren erzeugt keine unbeabsichtigte Signaluntergrund und somit zu höheren Signal-zu-Rausch-Verhältnis führen kann. Dies könnte dazu führen letztlich in die genauere und präzise Erkennung von operativen Margen. Etwas überraschend hat sich der Nutzen dieses Ansatzes als intraoperativen Bildgebungstechnik unerforscht 10 geblieben. Tatsächlich ist das nächste Beispiel dieser Technik die Oxidation von Luminol durch Myeloperoxidase in Mäusen , 11, 12, 13. Chemilumineszenter biomedizinischen Bildgebung ist daher ein ziemlich unerforschtes Gebiet der Forschung, die folgende Vorteile bieten könnte: (1) minimale Autofluoreszenz, was zu einem niedrigen Hintergrundsignal mit hallogher Signal-zu-Rausch-Verhältnisse; (2) abstimmbaren Wellenlängen des chemolumineszenten Emissionen im Bereich vom sichtbaren bis zum nahen Infrarot; und (3) funktionalisierbarer chemolumineszenten Komplexe , die, wenn sie mit Linker - Technologien kombiniert und Biomoleküle gezielt , die bereits existieren, 14 Zugriff auf ganze Bibliotheken von gezielten molekularen Bildgebung Sonden liefern. Diese Proof-of-Principle-Studie zeigt die potentielle Nützlichkeit von Chemilumineszenz-Bildgebung in der biomedizinischen Einstellung eines Ruthenium-basierte Bildgebungsmittel verwendet wird. Die chemolumineszenten Eigenschaften dieser Verbindung sind gut untersucht, mit Untersuchungen aus der Zeit der Mitte der 1960er Jahre 15. Bei chemischer Aktivierung erzeugt das Mittel Licht bei etwa 600 nm 16, die für medizinische Abbildungszwecke gut geeignet ist. Die Aktivierungsenergie wird durch eine Redox - Reaktion zur Verfügung gestellt , die in einen angeregten Zustand-was dazu führt , eine Lebensdauer von 650 ns in Wasser 17 -foll hatdurch die Erzeugung von Photonen bei Entspannung der angeregten Zustand zu verdanken. Durch die Verwendung eines speziell gestalteten Fern Vernebler, konnten wir die Verbindung , die sowohl ex vivo und in vivo zu detektieren. Die Ergebnisse der anfänglichen Experimente sind sehr vielversprechend, was auf eine weitere Untersuchung dieser Technologie.

<table><tbody><tr><td>Wood part A (12.5 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>&amp;nbsp;Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part B (12.7 &amp;times; 10.7 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part C (11 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Screws (4 &amp;times; 25 mm)</td><td>Screwfix</td><td>79939</td><td></td></tr><tr><td>Harmon Face Values 3 oz mini sprayer</td><td>Bed, Bath and Beyond</td><td></td><td></td></tr><tr><td>stainless steel rod (10 cm of 1/16&amp;rdquo; steel)</td><td>Metals Depot Int. Inc.</td><td>2192</td><td></td></tr><tr><td>Pencil Classic HB</td><td>Papermate</td><td>58592</td><td></td></tr><tr><td>Paper clip</td><td>Office Depot</td><td>221720</td><td></td></tr><tr><td>speaker cable</td><td>RCA Inc.</td><td>AH1650SN</td><td></td></tr><tr><td>Energizer 9V alkaline battery</td><td>Energizer Holdings Inc.</td><td>EN22</td><td></td></tr><tr><td>Hitech HS-82MG Micro Servo Motor, 3.4 kg/cm output torque @ 6V</td><td>Hitech RCD USA Inc.</td><td>32082S</td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalog Number</td><td>Comments</td></tr><tr><td>28 cm plastic cable ties</td><td>General Electric Inc.</td><td>50725</td><td></td></tr><tr><td>Duct tape</td><td>3M Inc.</td><td>3939</td><td></td></tr><tr><td>littleBits w1 wire</td><td>littleBits Inc.</td><td>w1 wire</td><td></td></tr><tr><td>littleBits p1 power</td><td>littleBits Inc.</td><td>p1 power</td><td></td></tr><tr><td>littleBits i2 toggle switch</td><td>littleBits Inc.</td><td>i2 toggle switch</td><td></td></tr><tr><td>littleBits 011 servo</td><td>littleBits Inc.</td><td>011 servo</td><td></td></tr><tr><td>20 cm plastic covered wire twist ties</td><td>Four Star Plastics</td><td>71TIE8000</td><td></td></tr><tr><td>Tris(2,2&amp;prime;-bipyridyl)dichlororuthenium(II) hexahydrate</td><td>Sigma-Aldrich Inc.</td><td>224758</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Ammonium cerium(IV) nitrate</td><td>Sigma-Aldrich Inc.</td><td>22249</td><td></td></tr><tr><td>Isofluorane</td><td>Baxter Healthcare</td><td>1001936060</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>PBS1</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich Inc.</td><td>T0886</td><td></td></tr><tr><td>Water</td><td></td><td></td><td>Water was purified using a Milipore Mili-Q (R &amp;ge; 18 M&amp;Omega;)</td></tr><tr><td>Female nude (outbred) mice</td><td>Jackson Laboratories</td><td>1929</td><td>age 5 - 6 weeks</td></tr><tr><td>Strain C57BL/6J&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>NU/J male mice at&amp;nbsp;</td><td>Jackson Laboratories</td><td>2019</td><td>age 6 &amp;ndash; 8 weeks</td></tr><tr><td>IVIS 200 bioluminescence reader</td><td>Caliper Live Science</td><td></td><td></td></tr><tr><td>Live Image 4.2 software</td><td>Perkin-Elmer</td><td>128165</td><td></td></tr><tr><td>Microscope slides</td><td>ThermoScientific</td><td>4951PLUS4</td><td></td></tr></tbody></table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

Der Vernebler in Protokoll Abschnitt 1 kann aus leicht erhältlichen Materialien zu geringen Kosten konstruiert werden. Es ist beabsichtigt , der Reduktions- / Oxidationsmittel in einem Biolumineszenz - Lesegerät (1) ein Einsatz für Fern ausgelöst werden Aufsprühen. Unser Design ermöglicht den sicheren Betrieb des Zerstäubers innerhalb des Biolumineszenz-Lesegerät bei einem 14 cm Abstand von der Linse. Kein Beschlagen oder Verschwimmen der Linse wurde während der...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Eine neue Technik zur Erzeugung und Observing Chemilumineszenz in einem biologischen Rahmen

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

a-novel-technique-for-generating-observing-chemiluminescence

Research

JoVE Journal

Bioengineering

8.4K Aufrufe.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Serie: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Forschung

Immunologie und Infektion

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Eine optogenetische Methode zu kontrollieren und analysieren gen Expressionsmuster in Zell-Zell-Interaktionen

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetik

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Medizin

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Messung und Korrelation der Zelldichte und Lichtemission von Biolumineszenten Bakterien

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Biochemie

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

A Step Beyond BRET: Fluoreszenz von Unbound Anregung von Lumineszenz (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Biotechnik

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

Biolumineszente Optogenetik 2.0: Nutzung der Biolumineszenz zur Aktivierung photosensorischer Proteine in vitro und in vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Überwachung Zell-autonome circadiane Uhr Rhythms der Genexpression mittels Luciferase Biolumineszenz Reporter

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Biologie

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Autonom Bioluminescent Säugerzellen für die kontinuierliche und Echtzeit-Überwachung von Zytotoxizität

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Eine neue Technik zur Erzeugung und Observing Chemilumineszenz in einem biologischen Rahmen

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