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.

Etik deyimi: onaylanmış bir protokole ve Memorial Sloan Kettering Kanser Merkezi (MSK) Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) etik kuralları uyarınca göre yapıldı tarif edilen in vivo hayvan deneylerinde tümü. Bir püskürtme cihazı 1. İnşaat <ol><li> Iki vidayı (x 25 mm 2 4) kullanılarak kısım B (12.7 x 10.7 x 1.8 cm 3) merkezinde dik ahşap parça A (12.5 x 2.5 x 1.8 cm 3) takın. Ahşap parça C takın (11 × 2.5 × 1.8 cm 3) C bölümü (11 x 2.5 x 1.8 cm 3) hala hareket ettirilebilir şekilde bir vida kullanılarak kısım A (12.5 x 2.5 x 1.8 cm 3) ortasına. Bkz: Şekil 1. </li><li> iki döngüler, ya bir tane oluşturmak yoluyla iki delik plastik 3 oz küçük püskürtücü (D) sprey şişe tetik alt ucundan Matkap ve paslanmaz çelik çubuk itin (1/16 &quot;çelik 10 cm) (E) tetikleyici yan. </li&gt; <li> kaymasını kablo bağları önlemek için koli bandı (F) ile sprey şişesinin alt kısmını sarın. Iki plastik kablo bağları kullanarak ahşap parça C (11 x 2.5 x 1.8 cm 3) (28 cm) (G) sprey şişesi takın. </li><li> 011 servo motor (I) kesiniz ve servo kontrolü (H) gevşek kablolar ile yeniden bağlayın. Ardından, koli bandı kullanarak ahşap kısım A (12.5 x 2.5 x 1.8 cm 3) üstüne servo motora takın. </li><li> ataç (K) kullanarak servo motor koluna bir kalem (J) takın. Sıkıca plastik kaplı büküm telleri (L) kullanılarak çelik çubuk döngüler kalem dış kısımlarını bağlamak ve koli bandı ile kalem üzerinde biter sabitleyin. </li><li> servo motor kontrol ünitesinin manyetik kablo konnektörünü (M) kesiniz ve hoparlör kablosu (N) onu takın. Sonra, odun parçası B (12.7 x 10.7 x 1.8 cm 3) servo motor kontrol ünitesini bantlayın. yarısında manyetik konnektörleri ile bir w1 kablosunu kesin ve bakır s gevşek ucuna bir parçasını takmakPeaker kablosu (1 M). Mevcut w1 kablo ve 9V pil (manyetik) i2 geçiş anahtarı ve p1 gücü bağlayın. </li></ol> Yöntem 2. Hassasiyet Belirlenmesi <ol><li> 1.5 ml mikrosantrifüj tüpü içinde, 260 ug (347 nmol), 52 ug (69 nmol), 26 ug (34 nmol) miktarlarında ters osmoz, su (100 uL) içinde [Ru (bpy) 3] Cl2 çözeltilerini hazırlamak 5 ug (6.9 nmol), 3 ug (3.5 nmol), 520 ng (694 pmol), 260 ng (347 pmol), 52 ng (69 umol), 26 ng (34 pmol), 5 ng (6.9 pmol), 3 ng (3.5 umol). </li><li> Her biri 100 uL karıştırın [Ru (bpy) 3] Cl amonyum seryum nitrat sulu çözeltisi, 100 uL 2 çözeltisi ((NH4) 2 Ce (NO 3) 6) bir mikroskop lamı üzerine, su (25 mM) &#39;de. </li><li> Görüntüleme yazılımı başlatarak biyoparlaklık okuyucuda satın ayarlayın. <ol><li> kullanıcı profiline oturum açın ve Acqui bakmaklan de kontrol paneli. &quot;Başlatma&quot; ve enstrüman hazır olana kadar bekleyin tıklayın. </li><li> denetlenmeyen &quot;Görüntüleme Modu&quot; için bakın ve &quot;Işıklı&quot; ve &quot;Fotoğraf&quot; işaretli olduğundan emin olun ve &quot;Floresan&quot;. </li><li> Değiştir &quot;Temas Süresi&quot; 20 s &quot;Lüminesans&quot; ayarı. &quot;Kutulamanın&quot;: aşağıdaki gibidir: &quot;Lüminesans&quot; için kalan ayarları ayarlayın Orta; &quot;F / durdur&quot;: 1; ve &quot;Emisyon Filtresi&quot;: Açık. NOT: pozlama süreleri enstrümantasyon ve kullanılan deneysel ayarlarına göre adapte edilmesi gerekebilir, sunulan kurulum farklı ise. </li><li> &quot;Temas Süresi&quot; For: &quot;Fotoğraf,&quot; aşağıdaki ayarları kullanın Auto; &quot;Binning&quot;: Orta; ve &quot;F / durdur&quot;: 8. &quot;Görüş Alanı&quot; açılır menüsünden için target.Look görüntüleme göre &quot;Konu Boy&quot; ayarlayın. Başlangıç ​​ayarı &quot;C&quot; dir c arasında &quot;B&quot; olarak değiştirin (14 cm mesafedenamera ve örnek aşaması). </li></ol></li><li> Oksitleyici madde korumak için görüntüleme odasının zemininde siyah inşaat bir kağıda üzerinde mikroskobik slayt koyarak nebulizatör ayarlayın. Bir 100 μLdroplet karıştırın [Ru (bpy) 3] CI2 solüsyonunun sulu bir çözeltisi, 100 uL (NH4) 2 Ce ile (NO 3) 6. yeşil kutu çarpı dikkate alınmalıdır. <ol><li> siyah inşaat kağıt üzerinde görüntüleme konuyu yerleştirin, ilgi alanı yeşil ışık kutusu crosshair merkezinde olacak şekilde numune sahnede sergiledi. ahşap destek plastik sprey şişesinde ayırarak nebülizatör hazırlayın. plastik hazne içine (su / etanol 1: 3) ve ahşap destek yerine tekrar trietilamin ihtiva eden bir çözelti doldurun. </li><li> biyolüminesans okuyucu içinde nebulizatör yerleştirin ve güç kablosu nebulizatör kablosu çıkarılmış olduğundan emin olun. Emin olun güç anahtarıaçık, geçiş anahtarı kapalıdır ve kırmızı LED yanıyor püskürtme memesi kafası tarafından görüntüleme konuya doğru kameradan görünüm tıkanıklığı en aza indirerek sprey akışı, görüntüleme konuyla ilgili ilgi alanına doğru işaret edilmektedir böyle nebülizatör yerleştirin. </li><li> Sprey onları kalkan (mikroskobik slaytlar ya da enjeksiyon sitelerinde örneğin, beyaz işaretleri) herhangi bir potansiyel sıcak noktalar üzerinde inşaat küçük kağıt, siyah parçalarını yerleştirin. Bir yerde görüntüleme konu nebulizer veya manyetik kapı mandalı ile karışmaz şekilde görüntüleme bölmesi içindeki nebulizatör uzaktan kord en az 40 cm. görüntüleme sistemi kapağını kapatın. NOT: Crosshair ayarı &quot;adlı Alanında&quot; dayalı boyutu değişecektir &quot;Görüntü Living;&quot; Bu &quot;B&quot; olarak ayarlanmış olduğundan emin olun. </li></ol></li><li> görüntüleme dizisi başlatarak bir görüntü kazanır. içinde &quot;Acquire&quot; Click &quot;Toplama Kontrol Paneli.&quot; ilk görüntüleme dizisine üzerindeence, istenen (önerilir) ise Otomatik kaydetmeyi etkinleştirmek ve bir veri klasörünü seçin. dizinin sonuna kadar &quot;Edit Görüntüleme Etiketler&quot; iletişim geçiyoruz. NOT: kontrol yazılımı cihazın eylemleri adım-adım, gerçek zamanlı olarak gösterir. ölçüm hazırlanması ve doğru pozisyonda örnek sahne taşındıktan sonra, kamera deklanşörü açılır ve ölçüm zamanını sayar. deklanşör açılış ayrıca makine tarafından oluşturulan bir tık sesi ile duyulabilir. </li><li> kemilüminesansını oluşturmak için geçiş şalteri üç kez geçerek: obtüratör açılır gibi, trietilamin solüsyonu üç patlamaları (su / etanol içinde 3, sprey patlama başına 0.24 ± 0.04 ml 1) püskürtün. NOT: Numune sahne Ölçüm sırasında hareket edecektir. Bu izin enstrümanın içinde yeterli (en az 40 cm) kablo bırakın. Çözelti artan borusu ile talip olabilir nebulizatör ile püskürtülür olduğunu ve borunun hiçbir hava kabarcığı olmadığından emin olun. Birkaç yedek Batterie varihtiyaç halinde hazır nebulizer için s. </li></ol> Vivo Görüntüleme 3. Sistemik İntravenöz Enjeksiyonu Sonrası <ol><li> 1.5 ml mikrosantrifüj tüpü içinde, [Ru (bpy) 3] Cl2 nmol 8 ila 33 ihtiva eden fosfat tamponlu tuz (PBS) çözeltisi, 100 uL hazırlar. Sulu bir çözeltisi (NH4) 2 Ce hazırlama (NO 3), aynı zamanda, su (25 mM) 6. </li><li> İntravenöz, sağlıklı farelerin kuyruk venine 100 uL [Ru (bpy) 3] CI2 enjekte (n = 5). </li><li> CO 2 boğulma yoluyla enjeksiyondan sonra fareler 10 dakika euthanize. <ol><li> Daha sonra kalp ve akciğerleri maruz U-şeklinde kaburga kemer kaldırmak, gövde bir Y-cut ile cildi çıkarın. Sol ventrikül 18 bir 24 gauge iğne yoluyla PBS 20 mL sağ atriyum bir çıkış kesme ve enjekte fareler serpmek. Dikkatle göbek kesticilt ve böbrek ve karaciğer maruz kalmaktadır. görünür bir kesim oluşturmak için organları aracılığıyla uzunlamasına kesin. </li></ol></li><li> Aşağıdaki değişikliklerle, adım 2.3-2.6 anlatıldığı gibi tedarik ayarlayın. <ol><li> İyice nebülizör plastik hazne yıkandıktan sonra, bunun yerine trietilamin su (NH4) içindeki bir 2 Ce (NO 3) 6 (25 mM) ile doldurun. NOT: iyice kristalize beri, her kullanımdan sonra nebulizatör memesini durulanması önemlidir (NH 4) 2 Ce (NO 3) 6 birkaç kullanımdan sonra püskürtme memesini yok edebilir. </li></ol></li><li> Görüntüleme için, bütün hayvanlar veya organ örnekleri kullanın. <ol><li> Bütün karın görüntüleme için, kamera ve cihazın arka işaret kafa bakacak açık karın fare karkas yerleştirin. Organı ortalamak yeşil ışık kutusu crosshair (örneğin, karaciğer veya böbrek) yansıması. </li><li> Fveya organ görüntüleme ve miktar bireysel, görüntüleme cihazdan fare kaldırmak ve onu aşağı pin. Önceden açılmış gövdesinden başlayarak, iç organların (örneğin, böbrek, karaciğer, akciğer, kas, dalak, beyin, kalp) tüketim. kas dokusu çıkarılması için arka bacak deri yoluyla kesti. Dikkatle beyin çıkarılması için bir neşter ile kafatası açın. <ol><li> ilgi organ karaciğer, böbrek veya dalak ise, uzunlamasına ikiye tüm organları kesilmiş bir petri veya siyah inşaat kağıt parçası üzerinde her organı yerleştirin. </li></ol></li><li> Tek organlar için kemilüminesan izleyici nispi emisyon kurmak için adımlar 2,3-2,6 açıklanan prosedürü uygulayın. </li></ol></li></ol> Lenf nodu 4. İn Vivo Görüntüleme <ol><li> [Ru (bpy) 3] Cl2 80 nmol ihtiva eden bir PBS çözeltisinden 10 uL hazırlayın. Sulu bir çözeltisi (NH4) 2 Ce hazırlayın (NO 3) 6 waAynı zamanda ter (25 mM). </li><li> subdermal, sağlıklı farelerin arka bacağına çözeltisinin 10 ul enjekte edilir (n = 5). Bir negatif kontrol olarak, saf PBS 10 uL karşı taraftaki pençenin enjekte edilir. Enjeksiyondan sonra CO 2 boğulma yoluyla 15 dakika fareler Kurban. popliteal lenf düğümlerine kadar lenf kanalları ortaya çıkarmak için her iki arka ayakları üzerinde cilt kaldırmak. </li><li> Adım 3.4 anlatıldığı gibi satın ayarlayın. </li><li> , Her iki arka bacak popliteal lenf düğümleri kaldırmak bolun ve miktar tayini amacıyla, (aşama 3.5.3) daha önce açıklandığı gibi, bir Petri tabağına oksidan ile püskürtülür. </li></ol>

<ol>
	<li>Fong, Y., Giulianotti, P. C., Lewis, J., Koerkamp, B. G., Reiner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging and Visualization in The Modern Operating Room: A Comprehensive Guide for Physicians. , (2015).</li><li>Nguyen, Q. T., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-guided+surgery+with+live+molecular+navigation+-+a+new+cutting+edge.">Fluorescence-guided surgery with live molecular navigation - a new cutting edge.</a> Nat. Rev. Cancer. 13 (9), 653-662 (2013).</li><li>Weissleder, R., Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+the+era+of+molecular+oncology.">Imaging in the era of molecular oncology.</a> Nature. 452, 580-589 (2008).</li><li>Heller, S., Zanzonico, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+probes+and+intraoperative+gamma+cameras.">Nuclear probes and intraoperative gamma cameras.</a> Semin. Nucl. Med. 41 (3), 166-181 (2011).</li><li>van Dam, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+tumor-specific+fluorescence+imaging+in+ovarian+cancer+by+folate+receptor-&#945;+targeting:+first+in-human+results.">Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-&#945; targeting: first in-human results.</a> Nat. Med. 17 (10), 1315-1319 (2011).</li><li>Zavaleta, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Raman-based+endoscopic+strategy+for+multiplexed+molecular+imaging.">A Raman-based endoscopic strategy for multiplexed molecular imaging.</a> Proc. Natl. Acad. Sci. U S A. 110 (25), E2288-E2297 (2013).</li><li>Harmsen, S., Bedics, M. A., Wall, M. A., Huang, R., Detty, M. R., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+chalcogenopyrylium-based+surface-enhanced+resonance+Raman+scattering+nanoprobe+with+attomolar+sensitivity.">Rational design of a chalcogenopyrylium-based surface-enhanced resonance Raman scattering nanoprobe with attomolar sensitivity.</a> Nat. Commun. 6, 1-9 (2015).</li><li>Thorek, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+Lymphography:+Multimodal,+High-Resolution,+Dynamic+Mapping+and+Resection+of+Lymph+Nodes+After+Intradermal+Injection+of+18F-FDG.">Positron Lymphography: Multimodal, High-Resolution, Dynamic Mapping and Resection of Lymph Nodes After Intradermal Injection of 18F-FDG.</a> Nucl. Med. 53 (9), 1438-1445 (2012).</li><li>Thorek, D. L. J., Riedl, C. C., Grimm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Cerenkov+Luminescence+Imaging+of+18F-FDG.">Clinical Cerenkov Luminescence Imaging of 18F-FDG.</a> Nucl. Med. 55 (1), 95-98 (2014).</li><li>Gross, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+myeloperoxidase+activity+in+vivo.">Bioluminescence imaging of myeloperoxidase activity in vivo.</a> Nat. Med. 15 (4), 455-461 (2009).</li><li>Lee, J. -. J., White, A. G., Rice, D. R., Smith, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+using+polymeric+nanoparticles+stained+with+near-infrared+chemiluminescent+and+fluorescent+squaraine+catenane+endoperoxide.">In vivo imaging using polymeric nanoparticles stained with near-infrared chemiluminescent and fluorescent squaraine catenane endoperoxide.</a> Chem. Commun. 49 (29), 3016-3018 (2013).</li><li>Lee, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+hydrogen+peroxide+with+chemiluminescent+nanoparticles.">In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles.</a> Nat. Mater. 6 (10), 765-769 (2007).</li><li>Baumes, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=thermally+activated,+near-infrared+chemiluminescent+dyes+and+dye-stained+microparticles+for+optical+imaging.">thermally activated, near-infrared chemiluminescent dyes and dye-stained microparticles for optical imaging.</a> Nat. Chem. 2 (12), 1025-1030 (2010).</li><li>Siraj, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence,+Phosphorescence,+and+Chemiluminescence.">Fluorescence, Phosphorescence, and Chemiluminescence.</a> Anal. Chem. 88 (1), 170-202 (2016).</li><li>Hercules, D. M., Lytle, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescence+from+Reduction+Reactions.">Chemiluminescence from Reduction Reactions.</a> Am. Chem. Soc. 88 (20), 4745-4746 (1966).</li><li>Kerr, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annihilation+electrogenerated+chemiluminescence+of+mixed+metal+chelates+in+solution:+modulating+emission+colour+by+manipulating+the+energetics.">Annihilation electrogenerated chemiluminescence of mixed metal chelates in solution: modulating emission colour by manipulating the energetics.</a> Chem. Sci. 6, 472-479 (2015).</li><li>Montalti, M., Credi, A., Prodi, L., Gandolfi, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Photochemistry 3rd Ed. , 379-404 (2006).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rhodents.">Whole Animal Perfusion Fixation for Rhodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>B&#252;chel, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+intraoperative+chemiluminescent+imaging.">Near-infrared intraoperative chemiluminescent imaging.</a> ChemMedChem. , (2016).</li><li>Ntziachristos, V., Ripoll, J., Wang, L. V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+Listening+to+Light:+the+Evolution+of+Whole-Body+Photonic+Imaging.">Looking and Listening to Light: the Evolution of Whole-Body Photonic Imaging.</a> Nat. Biotechnol. 23, 313-320 (2005).</li><li>Koch, J. H., Gyarfas, E. C., Dwyer, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Activity+of+Complex+Ions+Mechanism+of+Inhibition+of+Acetylcholinesterase.">Biological Activity of Complex Ions Mechanism of Inhibition of Acetylcholinesterase.</a> Austral. J. Biol. Sci. 9 (3), 371-381 (1956).</li><li>Juris, A., Balzani, V., Barigelletti, F., Campagna, S., Belser, P., Zelewsky, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ru(II)+polypyridine+complexes:+photophysics,+photochemistry,+electrochemistry,+and+chemiluminescence.">Ru(II) polypyridine complexes: photophysics, photochemistry, electrochemistry, and chemiluminescence.</a> Coord. Chem. Rev. 84, 85-277 (1988).</li><li>Knoll, J. D., Turro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+utilization+of+ruthenium+and+rhodium+metal+complex+excited+states+for+photoactivated+cancer+therapy.">Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy.</a> Coord. Chem. Rev. 282, 110-126 (2015).</li><li>Haley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+and+toxicology+of+the+rare+earth+elements.">Pharmacology and toxicology of the rare earth elements.</a> J. Pharm. Sci. 54 (5), 663-670 (1965).</li><li>Siekierski, S., Mioduski, T., Salomon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IUPAC Commission on Solubility Data. Solubility Data Series. Vol 13. Scandium, Yttrium, Lanthanum, and Lanthanide Nitrates. , (1983).</li><li>Reiner, T., Jantke, D., Marziale, A. N., Raba, A., Eppinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-Conjugated+Affinity+Labels:+A+New+Concept+to+Create+Enantioselective+Artificial+Metalloenzymes.">Metal-Conjugated Affinity Labels: A New Concept to Create Enantioselective Artificial Metalloenzymes.</a> ChemistryOpen. 2, 50-54 (2013).</li><li>Zanarini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Electrochemiluminescence+of+a+Ru(bpy)3-Labeled+Coupling+Adduct+Produced+on+a+Self-Assembled+Monolayer.">Synthesis and Electrochemiluminescence of a Ru(bpy)3-Labeled Coupling Adduct Produced on a Self-Assembled Monolayer.</a> J. Phys. Chem. C. 112 (8), 2949-2957 (2008).</li><li>Liu, R., Lv, Y., Hou, X., Yang, L., Mester, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Quantitation+Using+Ru-NHS+Ester+Tagging+and+Isotope+Dilution+High-Pressure+Liquid+Chromatography-Inductively+Coupled+Plasma+Mass+Spectrometry+Determination.">Protein Quantitation Using Ru-NHS Ester Tagging and Isotope Dilution High-Pressure Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry Determination.</a> Anal. Chem. 84 (6), 2769-2775 (2012).</li><li>Jantke, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+efficient+conjugation+of+organometallic+complexes+with+pendant+protein+reactive+markers.">Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers.</a> J. Organomet. Chem. 744, 82-91 (2013).</li><li>Aoki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+xenograft+mouse+model+of+diffuse+pontine+glioma+designed+for+therapeutic+testing.">An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing.</a> J Neurooncol. 108 (1), 29-35 (2012).</li><li>Forster, R. J., Bertoncello, P., Keyes, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenerated+Chemiluminescence.">Electrogenerated Chemiluminescence.</a> Annual Rev. Anal. Chem. 2, 359-385 (2009).</li><li>Connell, T. U., James, J. L., White, A. R., Donnelly, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Labelling+with+Versatile+Phosphorescent+Metal+Complexes+for+Live+Cell+Luminescence+Imaging.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+labeling+and+bioanalytical+applications+of+a+tris(2,2&#8242;-bipyridyl)ruthenium(II)-based+electrochemiluminescence+probe.">Synthesis, labeling and bioanalytical applications of a tris(2,2&#8242;-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.</a> Nat. Protoc. 9 (5), 1146-1159 (2014).</li><li>B&#339;uf, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+Ruthenium(II)+Complexes+in+Biocompatible+Poly(d,l-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+its+applications+in+surgery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+for+Metals+in+Medicine:+How+Nanotechnology+May+Help+To+Shape+the+Future.">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., Rold&#225;n-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Emission+of+Cationic+Iridium+(III)+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescence+detection+with+water-soluble+iridium(III)+complexes+containing+a+sulfonate-functionalised+ancillary+ligand.">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.

Burada, optik kemilüminesan muhabir tarafından oluşturulan fotonların emisyon yoluyla doku ortaya koymaya yeteneğine sahip bir teknoloji sundu. Diğer aksine, daha bu kemilüminesans muhabir sistemi radyoaktif olmayan ve çok yüksek hassasiyet seviyelerinde tespitini kolaylaştırır bir görüntüleme probu istihdam, teknoloji 4, 5, 6, 7, 8,...

Son yıllarda, görüntüleme teknolojileri doktorlar hastalığı teşhis ve monitör yol devrim var. Bu görüntüleme teknolojileri, ancak bu tür pozitron emisyon tomografisi (PET), tek foton emisyon bilgisayarlı tomografi (SPECT), bilgisayarlı tomografi (BT) ve manyetik rezonans görüntüleme (MRI) gibi tüm vücut görüntüleme sistemleri, büyük ölçüde sınırlı olmuştur. Özellikle dikkat kanseri ödenmiştir ve teknolojik görüntüleme devrimler büyük ölçüde bu hastalığın teşhis ve tedavi edilir şekilde düzeldi. ameliyathane: Bu ilerlemelere rağmen, bu görüntüleme teknolojileri sadece uymaz bir yer var. Tüm vücut görüntüleme teknikleri, cerrahi planlamada yardımcı olabilir, bunlar genellikle tümör dokusunun tüm kaldırıldı veya artık tümör dokusu cerrahi sınırların 1&#39;de gizli kalır olsun hekimler gerçek zamanlı olarak belirlemeye yardımcı olmak için yeterince yüksek uzaysal çözünürlük yoksundur. Hiçbir infiltratif emin yapmaTümör marjları geride en önemli cerrahi hedeflerinden biridir ve cerrahlar titiz ve tedbirli doku rezeksiyonu arasında sıkı-ip yürümek gerekir edilir. Çok fazla çıkarılırsa, hastada istenmeyen yan etkiler arttırılmış olur çok az kaldırılan ise, nüks oranları, 3 2 artmıştır. Nedenle, doğru tümör marjları tasvir için çok önemlidir ve biz kemilüminesan intraoperatif görüntüleme aksi kurulan teknikleri ile tespit edilmemiş kalabileceği malign doku görselleştirmek için cerrahlara yardımcı olarak tümör marjlarının belirlenmesi doğruluğunu geliştirmek için yardımcı olabilir inanıyoruz. Şu anda intraoperatif görüntüleme sistemleri gibi olası yarar için araştırılan birçok görüntüleme teknolojileri vardır. Bu β- ve γ-radyasyon yayan sondalar 4, optik floresan 5, Raman spektroskopisi 6 dahil </sup&gt;, 7 ve Cherenkov ışıma 8, 9. Ancak bugüne kadar, bunların hiçbiri, standart klinik araçlar olarak kullanılır hale gelmiştir. Optik floresan görüntüleme şimdiye kadar bu tekniklerin en umut verici olduğu kanıtlanmıştır ve en çok araştırdı nedenle etti. Zaten birçok uygulama için değerli bir araç olarak gösterilmiş olsa da, bu sınırlamalar olmadan değildir. Gerçekten de, asıl dezavantajı doğal autofluorescent biyolojik doku tarafından üretilen arka plan floresan olduğunu. Bu arka plan otofloresan sinyali, floresan sinyalinin üretimi için gerekli olan, harici bir ışık kaynağı tarafından, florofor ilave olarak, çevre doku uyarılmasının bir ürünüdür. Pratik açıdan bakıldığında, bu otofloresans potansiyel ameliyathanede bu teknolojinin faydasını sınırlayabilir düşük sinyal-gürültü oranları, yol açabilir. Müdürfloresan görüntüleme üzerinden Kemiluminesan görüntüleme avantajı uyarma ışık gerekli olmasıdır. Bunun bir sonucu olarak, herhangi bir arka plan otofloresansı yoktur. Kemiluminesan görüntülemede, uyarma enerjisi yerine kimyasal oluşturulur. Bu işlem, daha yüksek bir sinyal-ses oranlarına neden olabilir, bu nedenle herhangi bir istenmeyen arka plan sinyali üretir ve. Bu sonuçta cerrahi sınırların daha hassas ve doğru algılama neden olabilir. Biraz şaşırtıcı bir ameliyat görüntüleme tekniği olarak bu yaklaşımın yarar 10 keşfedilmemiş kalmıştır. Gerçekten de, bu tekniğin en yakın örnek, farelerde 11, 12, 13, miyeloperoksidaz tarafından luminol oksitlenmesidir. merhaba ile düşük arka plan sinyali sonuçlanan (1) en az otofloresans: kemilüminesans biyomedikal görüntüleme aşağıdaki avantajları sunabilir araştırma oldukça keşfedilmemiş alan nedenleGher sinyal-gürültü oranları; (2) görünür gelen yakın-kızılötesi değişen kemilüminesan emisyonların ayarlanabilir dalga boyları; ve (3), bir bağlayıcı teknolojileri ile bir araya getirilmiş ve zaten mevcut biyomoleküllerin hedef olduğunda, hedef moleküler görüntüleme sondaları 14 tüm kütüphanelerin erişim sağlayan fonksiyonalitesine sahip kimyasal olarak ışık veren kompleksleri. Bu proof-of-prensibi çalışma rutenyum tabanlı görüntüleme maddesi kullanarak biyomedikal ortamda kemilüminesan görüntülemenin potansiyel yarar göstermektedir. Bu bileşiğin kemilüminesan özellikleri iyi 1960&#39;ların ortalarında 15 kadar uzanan araştırmaları ile incelenmiştir. Kimyasal aktivasyonu üzerine, madde, tıbbi görüntüleme amacıyla uygundur 600 nm 16 ° ışık üretir. Aktivasyon enerjisi uyarılmış durumda su 17 -foll 650 ns ömrü vardır yol açan bir redoks reaksiyonu sonucu elde edilenBu heyecanlı devletin gevşeme üzerine foton kuşağının borçlu. Bir özel tasarlanmış uzaktan nebulizatör kullanımı sayesinde, biz bileşik hem ex vivo ve in vivo tespit başardık. İlk deneylerin sonuçları bu teknolojinin daha fazla araştırılması düşündüren, oldukça ümit vericidir.

<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.

Protokol 1. bölümde tarif edilen vaporizatör, sistemin düşük bir maliyetle kolayca mevcut malzemelerden yapılabilir. Uzak tetikli bir biyolojik olarak ışık veren okuyucu içinde indirgeme / oksitleme maddesinin püskürtülmesi (Şekil 1) için bir ilave olacak şekilde tasarlanmıştır. Tasarım lensten 14 cm mesafeden biyoparlaklık okuyucunun içinde nebulizer güvenli çalışması için izin verir. lensin Hiçbir sisleme veya bulanıklaştırma işlemi s?...

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.

Biyolojik ortamda oluşturuluyor ve Gözlem Kemilüminesans için Yeni Bir Teknik

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.4K Görüntüleme.  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.

Video: 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.

Farelerde Görüntü Bakteriyel Enfeksiyonlar Lusiferaz Kullanımı

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

İmmünoloji ve Enfeksiyon

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.

Bir Optogenetic kontrol etmek ve hücre-hücre etkileşimleri gen ifade desenleri analiz yöntemi

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.

Vivo Çift Yüzey Bioluminescent Imaging&#39;de

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

Tıp

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.

Situ Ölçüm ve korelasyon hücre yoğunluğu ve kollarındaki bakterilerin ışık emisyon

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 ...

Biyokimya

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 Bakteriyel Görüntüleme<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.

BRET Ötesinde Bir Adım: Sınırsız Uyarma tarafından Flüoresan Lüminesans dan (YAKIT)

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

Biyomühendislik

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.

Biyolüminesan Optogenetik 2.0: Fotosensör Proteinleri In Vitro ve In Vivo'yu Aktive Etmek için Biyolüminesanstan Yararlanma

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.

Lusiferaz Biyoparlaklık Gazeteciler kullanma Gen Ekspresyonunun Hücre-özerk Sirkadiyen Saat Ritimleri İzlenmesi

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

Biyoloji

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.

Sitotoksisite Sürekli ve gerçek zamanlı izlenmesi için özerk Bioluminescent Memeli Hücre

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.

Biyolojik ortamda oluşturuluyor ve Gözlem Kemilüminesans için Yeni Bir Teknik

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