The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

ETİK TABLOSU: anlatıldığı in vivo hayvan deneyleri Tüm onaylanmış protokole ve Memorial Sloan Kettering Kanser Merkezi Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) etik kurallar altında göre yapıldı. Tz-Bn-Nota 1. Sentezi <ol><li> Küçük bir tepkime kabında, 600 ul NaHCO 3 tamponu (0.1 M, pH 8.1) içinde 7 mg NH2 -Bn-nota (1.25 x 10 -2 mmol) ile çözülür. Çözeltinin pH kontrol edin. Gerekirse, CO 3 0.1 M Na-2 az miktar kullanılarak 8.1 solüsyonun pH derecesinin ayarlanması. </li><li> 1.7 ml mikrosantrifüj tüpü içinde 0.5 mg Tz-NHS (1.25 x 10 -3 mmol) NH2 -Bn-NOTA solüsyonu ekleyin. Not: Tz-NHS, kuru ya tartıldı ve kuru DMF ya da DMSO (&lt;50 ul) içindeki bir stok çözeltisi eklenebilir. </li><li> Elde edilen reaksiyon çözeltisi, oda sıcaklığında 30 dakika boyunca reaksiyona girmesine izinHafif ajitasyon ile. </li><li> 30 dakika sonra, reaksiyona girmeyen NH2 -Bn-nota çıkarmak için ters-fazlı C18 HPLC kromatografisini kullanarak ürünü temizler. Tz-NHS ve Tz-Bn-NOTA en 525 nm dalga boyunda gözlemlenmiştir ise NH2 -Bn-NOTA, 254 nm bir dalga boyunda izlenebilir. NOT: Tutma süreleri açıkça her laboratuvar (pompalar, sütunlar, tüp, vb) HPLC ekipman kurulumu son derece bağlıdır. Bununla birlikte, bir örnek sunmak için ise 0 gradyanı: 100 MeCN / H2O 100 (her ikisi de% 0.1 TFA ile birlikte): 0 MeCN / H2O içinde 25 dakika ve analitik bir 4.6 x 250 mm C18 kolon kullanılmıştır , Tz-Bn-nota, Tz-NHS ve NH2 tutma süreleri -Bn-NOTA sırasıyla yaklaşık 15 dakika, 16.5 dakika ve 10 dakika olacaktır. Ürün tek bir çalışma ya da bir yarı-hazırlayıcı veya hazırlayıcı C18 HPLC kolonu kullanılarak birden çok çalışır, ya da diğer reaksiyon bileşenlerine de saflaştırılabilir. 1H-NMR, analistenen analitik HPLC ve ESI-MS tamamlanmış Tz-Bn-NOTA ön-maddelerin saflığının doğrulanması için kullanılır ve yöntemlerdir. 11 </li><li> Sıvı azot kullanılarak toplanan HPLC yıkama sıvısı dondurun. </li><li> Opak alüminyum folyo dondurulmuş toplama tüpü sarın. </li><li> Bir liyofilize damar O donmuş toplama tüpü yerleştirin / N HPLC mobil faz kaldırmak için. </li><li> -80 ° C&#39;de karanlıkta saflaştırılmış ürünü (bir katı parlak pembe) saklayın. NOT: Bu prosedür kabul edilebilir bir durak noktasıdır. tamamlandıktan Tz-Bn-NOTA ön-madde, bu koşullar altında en az 1 yıl stabildir. </li></ol> HuA33-TCO immünokonjugatın 2. Hazırlık <ol><li> 1.7 ml mikrosantrifüj tüpü içinde, 1 mg / ml kuru DMF TCO-NHS&#39;nin (2,7 mM) çözeltisi hazırlayın. </li><li> 1.7 ml mikrosantrifüj tüpü içinde, bir 2 mg / ml fosfat tamponlu tuzlu su, 1 ml huA33 arasında (13.3 uM) çözeltisi, pH 7.4 (0.01 M PO 4 3- hazırlamak </sub&gt; 0.154 M NaCI). </li><li> Az miktar 0.1 M Na-2 CO 3 (&lt;5 ul) kullanılarak, 8,8-9,0 antikor solüsyonun pH derecesinin ayarlanması. PH değerini izlemek için bir mikroelektrot ile pH kağıdı veya pH metre ya kullanın ve pH, 9.0 üzerinde yükselmeye izin vermemek için dikkatli olun. </li><li> Antikor çözeltisi doğru pH değerinde sonra, aktive edilmiş ester 8 molar eşdeğer tekabül TCO-NHS bir çözelti hacminin ilave edin. Örneğin, 2 mg / ml huA33 antikor çözeltisi (1.33 x 10 -8 mol huA33), 1 ml için 1 mg / ml bir TCO-NHS çözeltisi (1.07 x 10 -7 mol TCO-NHS), 7.9 ul ekleyin. Çözelti içinde hacme göre% 5 DMF aşılmamalıdır. </li><li> Yavaşça mikrosantrifüj tüpü birkaç kez ters yüz edilerek çözelti karıştırılır. </li><li> Opak alüminyum folyo mikrosantrifüj tüp sarın. </li><li> Çözelti, hafif çalkalama ile oda sıcaklığında 1 saat boyunca inkübasyona izin verin. </li><li> Oda sıcaklığında 1 saat sonra, bir önceden paketlenmiş tek SIZ kullanılarak elde edilen immünokonjugat arındırmakE dışlama kullanımlık tuz alıcı sütun. Depolama sırasında sütun üzerinde mevcut olan herhangi bir koruyucu çıkarmak için satıcı tarafından tarif edildiği gibi İlk olarak, boyut dışlama sütununa yıkayın. Daha sonra, boyut dışlama sütununa reaksiyon karışımına ilave edildi, 1.5 ml% 0.9 steril tuzlu su ile sütun durulama ve daha sonra, yıkama sıvısı olarak% 0.9 steril tuzlu su içinde 2 ml kullanılarak ürün toplamak. NOT: Bu adım, bir 2 ml solüsyon olarak tamamlanan huA33-TCO verecektir. </li><li> UV-Vis spektrofotometre üzerinde bileşke huA33-TCO konsantrasyonunu ölçün. </li><li> Daha yüksek bir konsantrasyonu, arzu edildiği takdirde, 50,000 molekül ağırlığı kesmesi olan bir santrifüj filtre birimi kullanılarak huA33-TCO çözelti konsantre edilir. NOT: huA33 ve diğer iyi bilinen antikor (örneğin, bevasizumab, trastuzumab, setuksimab, ve J591) &#39;in bir dizi konsantre edilmeden çok dayanıklı iken, toplama ve çökelme diğer durumlarda konsantrasyonuna bağlı olabileceğine dikkat etmek önemlidir. Araştırmacılar bu proced çalışılıyorYeni bir antikor ile ure edebiyat veya antikor konsantre olsun veya olmasın ile ilgili söz konusu antikorun kendi bilgi güvenmeli. </li><li> Karanlıkta 4 ° C&#39;da tamamlanmıştır huA33-TCO immüno saklayın. NOT: Bu prosedür kabul edilebilir bir durak noktasıdır. tamamlandıktan mAb TCO konjügat, bu saklama koşulları altında en az 3 ay boyunca stabil olmalıdır. </li></ol> Tz-Bn-Nota 3. 64 Cu Radyo-etiketlenmesi NOT: protokolün Bu adım işlenmesini ve radyoaktivite manipülasyonu içerir. Veya radyoaktivite ile başka bir işi yapan - - Bu adımları gerçekleştirmeden önce araştırmacılar kendi kurumun Radyasyon Güvenliği Bölümü ile başvurmalısınız. Iyonize radyasyona maruziyeti en aza indirmek için mümkün olan tüm adımları atın. <ol><li> 1.7 ml mikrosantrifüj tüpü içinde, 0.5 mg / ml (723 uM) t-Bn-nota bir solüsyon hazırlanır. </li><li> 1.7 mi microC olarakentrifuge tüp, 0.2 M NH4OAc, pH 5.5 tampon maddesi, 400 ul Tz-Bn-NOTA çözeltisi (5 ug) ve 10 ul ekle. </li><li> Uygun radyokimyasal not tutma adına, ölçmek ve önce ve aşağıda protokol (3.4-3.8) olarak izleyen adımlardan sonra bir doz kalibratörü kullanılarak numune radyoaktivite miktarı kaydedin. Bu radyokimyasal verimi doğru kararlılıkla yardımcı olacaktır. </li><li> Tz-Bn-NOTA çözeltisine 64 Cu 2,000 uCi (74 MBq) ekleyin. Not: Genellikle, [64 Cu] CuCl 2 0.1 N HCI küçük bir hacimde (&lt;30 ul) temin edilir, ve bu nedenle sadece küçük miktarlarda (&lt;10 | il), bu stok çözeltisi, radyo-etiketleme reaksiyonu için ihtiyaç vardır. [64 Cu] CuCI 2 stokunun büyük hacimler gerekiyorsa, Radyoetiketleme reaksiyonu genel reaksiyon hacmi artan hoşgörülü olduğunu. Bununla birlikte, radyo-etiketleme, reaksiyon çözeltisinin pH&#39;ı sağlamak için dikkatle izlenmelidiro pH 4.0 altına düşmemesi. </li><li> Çözelti, hafif çalkalama ile oda sıcaklığında 10 dakika inkübasyona izin verin. </li><li> Kuluçkadan 10 dakika sonra, ters-fazlı C18 HPLC kromatografisini kullanarak ürünü temizler. Alıkoyma süreleri Açıkçası her laboratuvar (pompalar, sütunlar, tüp, vb) HPLC ekipman kurulumu son derece bağlıdır. Bununla birlikte, bir örnek sunmak için, eğer bir gradyan 5:95 MeCN / H2O 95 (her ikisi de% 0.1 TFA ile birlikte): 5 MeCN / H2O: 15 dak boyunca kullanılan, 64 Cu TZ retansiyon süresi Serbest, kompleks 64 Cu yaklaşık 2-4 dk çözücü ön Zehir olurken Bn-NOTA civarında 9.8 dakika olmalıdır. </li><li> Bir dönen buharlaştıncı kullanarak, HPLC yıkama sıvısı çıkarın. </li><li> % 0.9 steril tuzlu su içinde 64 Cu-Tz-Bn-NOTA ürün içinde yeniden çözülür. NOT: 64 Cu 12.7 saat fiziksel yarılanma ömrü göz önüne alındığında, bu prosedürde kabul edilebilir bir durak noktası değildir. 64 sentezini Cu-Tz-Bn-DEĞİL gerçekleştirinBir radyoligandın enjeksiyonundan hemen önce ve Adım 4.5 hemen 3.7 Adım takip. </li></ol> Vivo Pretargeted PET Görüntüleme 4. NOT: Protokol Bölüm 3 gibi, protokol, bu adımı işlenmesini ve radyoaktivite manipülasyonu içerir. Bu adımları gerçekleştirmeden önce araştırmacılar kendi kurumun Radyasyon Güvenliği Bölümü ile başvurmalısınız. Iyonize radyasyona maruziyeti en aza indirmek için mümkün olan tüm adımları atın. <ol><li> Ve bir kadın atimik çıplak fare, deri altı implantı 1 x 10 6 SW1222 kolorektal kanser hücreleri bu (9-12 gün aşılamadan sonra) 100-150 mm 3 xenograftlarının içine büyümeye izin verin. 11 </li><li> % 0.9 steril tuzlu su içinde 0.5 mg / ml&#39;lik bir konsantrasyona kadar Protokolü Bölüm 2 huA33-TCO çözeltisi bir kısım seyreltin. </li><li> Ksenograft taşıyan fare kuyruk venine huA33-TCO çözeltisi 200 ul (100 ug) enjekte. </li><li> Fare tümör huA33-TCO birikimi için 24 saat izin verin. </li><li> % 0.9 steril tuzlu su içinde, 1.5 mCi / ml &#39;lik bir konsantrasyona kadar 64 Cu-Tz-Bn-NOTA radyoligand seyreltin. </li><li> Kuyruk damarına (6.7 MBq / nmol bir özgül etkinliğe varsayarak, 64 Cu-Tz-Bn-nota 1.6 nmol;, 11.1 MBq 300 uCi) 64 Cu-Tz-Bn-NOTA radyoligand çözeltisi 200 ul enjekte nakil doku taşıyan kobay fareler. </li><li> Istenen görüntüleme zaman noktasında (örneğin, 2, 6, 12 ya da 24 saat sonra enjeksiyon), bir% 2 izofluran ile anestezi fare: oksijen gazı karışımı ile gerçekleştirilmektedir. </li><li> Küçük hayvan PET tarayıcı yatağının üzerinde fare yerleştirin. Oksijen gazı karışımı:% 1 izofluran kullanarak tarama sırasında anestezi koruyun. Tarayıcı yatakta hayvan yerleştirerek önce, ayak-tutam yöntemi kullanılarak anestezi doğrulamak ve anestezi sırasında kurumasını önlemek için fare gözlerine veteriner merhem uygulayın. </li><li> Statik aracılığıyla fare PET veri elde350-700 keV enerji penceresi ve 6 nsaniye bir tesadüf zamanlama penceresini kullanarak 20 milyon tesadüf olaylar minimum tarayın. Görüntü yakalanmasını tamamladıktan sonra, gözetimsiz fare bırakmayın ve bilinci kazanmış kadar diğer fareler ile bir kafese koymayın. </li></ol>

<ol>
	<li>Wu, 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=Antibodies+and+antimatter:+The+resurgence+of+immuno-PET.">Antibodies and antimatter: The resurgence of immuno-PET.</a> Journal of Nuclear Medicine. 50, 2-5 (2009).</li><li>Zeglis, B. M., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+the+construction+of+radiometallated+bioconjugates+for+positron+emission+tomography.">A practical guide to the construction of radiometallated bioconjugates for positron emission tomography.</a> Dalton Transactions. 40, 6168-6195 (2011).</li><li>Hollander, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies+for+cancer+therapy.">Bispecific antibodies for cancer therapy.</a> Immunotherapy. 1, 211-222 (2009).</li><li>Liu, 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=Tumor+pretargeting+in+mice+using+99mTc-labeled+morpholino,+a+DNA+analog.">Tumor pretargeting in mice using 99mTc-labeled morpholino, a DNA analog.</a> Journal of Nuclear Medicine. 43, 384-391 (2002).</li><li>Boerman, O. C., van Schaijk, F. G., Oyen, W. J. G., Corstens, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeted+radioimmunotherapy+of+cancer:+Progress+step+by+step.">Pretargeted radioimmunotherapy of cancer: Progress step by step.</a> Journal of Nuclear Medicine. 44, 400-411 (2003).</li><li>Goldenberg, D. M., Sharkey, R. M., Paganelli, G., Barbet, J., Chatal, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+pretargeting+advances+cancer+radioimmunodetection+and+radioimmunotherapy.">Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.</a> Journal of Clinical Oncology. 24, 823-834 (2006).</li><li>Sharkey, R. M., Chang, C. H., Rossi, E. A., McBride, W. J., Goldenberg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeting:+taking+an+alternate+route+for+localizing+radionuclides.">Pretargeting: taking an alternate route for localizing radionuclides.</a> Tumor Biology. 33, 591-600 (2012).</li><li>Sharkey, R. 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=Improving+the+delivery+of+radionuclides+for+imaging+and+therapy+of+cancer+using+pretargeting+methods.">Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods.</a> Clinical Cancer Research. 11, 7109-7121 (2005).</li><li>Schultz, 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=A+tetravalent+single-chain+antibody-streptavidin+fusion+protein+for+pretargeted+lymphoma+therapy.">A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.</a> Cancer Research. 60, 6663-6669 (2000).</li><li>Lewis, M. R., 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+evaluation+of+pretargeted+64Cu+for+tumor+imaging+and+therapy.">In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.</a> Journal of Nuclear Medicine. 44, 1284-1292 (2003).</li><li>Zeglis, B. 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=A+pretargeted+PET+imaging+strategy+based+on+bioorthgonal+Diels-Alder+click+chemistry.">A pretargeted PET imaging strategy based on bioorthgonal Diels-Alder click chemistry.</a> Journal of Nuclear Medicine. 54, 1389-1396 (2013).</li><li>Blackman, M. L., Royzen, M., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine+ligation:+fast+bioconjugation+based+on+inverse+electron+demand+Diels-Alder+reactivity.">Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity.</a> Journal of the American Chemical Society. 130, 13518-13519 (2008).</li><li>Devaraj, N. K., Upadhyay, R., Hatin, J. B., Hilderbrand, S. A., 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=Fast+and+sensitive+pretargeted+labeling+of+cancer+cells+through+a+tetrazine/trans-cyclooctene+cycloaddition.">Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.</a> Angewandte Chemie-International Edition. 48, 7013-7016 (2009).</li><li>Devaraj, N. K., 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=Biomedical+applications+of+tetrazine+cycloadditions.">Biomedical applications of tetrazine cycloadditions.</a> Accounts of Chemical Research. 44, 816-827 (2011).</li><li>Devaraj, N. K., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-based+cycloadditions:+application+to+pretargeted+live+cell+imaging.">Tetrazine-based cycloadditions: application to pretargeted live cell imaging.</a> Bioconjugate Chemistry. 19, 2297-2299 (2008).</li><li>Keliher, E. J., Reiner, T., Turetsky, A., Hilderbrand, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yielding,+two-step+18F+labeling+strategy+for+18F-PARP1+inhibitors.">High-yielding, two-step 18F labeling strategy for 18F-PARP1 inhibitors.</a> ChemMedChem. 6, 424-427 (2011).</li><li>Reiner, T., Earley, S., Turetsky, A., 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=Bioorthogonal+small-molecule+ligands+for+PARP1+imaging+in+living+cells.">Bioorthogonal small-molecule ligands for PARP1 imaging in living cells.</a> ChemBioChem. 11, 2375-2377 (2010).</li><li>Reiner, T., Keliher, E. J., Earley, S., Marinelli, B., 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=Synthesis+and+in+vivo+imaging+of+a+18F-labeled+PARP1+inhibitor+using+a+chemically+orthogonal+scavenger-assisted+high-performance+method.">Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method.</a> Angewandte Chemie International Edition. 50, 1922-1925 (2011).</li><li>Taylor, M. T., Blackman, M., Dmitrenko, O., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+highly+reactive+dienophiles+for+the+tetrazine-trans-cyclooctene+ligation.">Design and synthesis of highly reactive dienophiles for the tetrazine-trans-cyclooctene ligation.</a> Journal of the American Chemical Society. 133, 9646-9649 (2011).</li><li>Selvaraj, R., 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=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+integrin+alpha(v)beta(3)+targeted+PET+tracer+based+on+a+cyclic+RGD+peptide.">Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin alpha(v)beta(3) targeted PET tracer based on a cyclic RGD peptide.</a> Bioorganic and Medicinal Chemistry Letters. 21 (3), 5011-5014 (2011).</li><li>Liu, 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=Efficient+18F+labeling+of+cysteine-containing+peptides+and+proteins+using+tetrazine-trans-cyclooctene+ligation.">Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.</a> Molecular Imaging. 12, 121-128 (2013).</li><li>Han, H. 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=Development+of+a+bioorthogonal+and+highly+efficient+conjugation+method+for+quantum+dots+using+tetrazine-norbornene+cycloaddition.">Development of a bioorthogonal and highly efficient conjugation method for quantum dots using tetrazine-norbornene cycloaddition.</a> Journal of the American Chemical Society. 132, 7838-7839 (2010).</li><li>Zeglis, B. 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=Modular+strategy+for+the+construction+of+radiometalated+antibodies+for+positron+emission+tomography+based+on+inverse+electron+demand+Diels-Alder+click+chemistry.">Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry.</a> Bioconjugate Chemistry. 22, 2048-2059 (2011).</li><li>Zeng, D., Zeglis, B. M., Lewis, J. S., Anderson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+impact+of+bioorthogonal+click+chemistry+on+the+development+of+radiopharmaceuticals.">The growing impact of bioorthogonal click chemistry on the development of radiopharmaceuticals.</a> Journal of Nuclear Medicine. 54, 829-832 (2013).</li><li>Reiner, T., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inverse+electron+demand+Diels-Alder+reaction+in+radiochemistry.">The inverse electron demand Diels-Alder reaction in radiochemistry.</a> Journal of Labeled Compounds and Radiopharmaceuticals. 57, 285-290 (2014).</li><li>Li, Z., 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=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+18-F+labeled+probes.">Tetrazine-trans-cyclooctene ligation for the rapid construction of 18-F labeled probes.</a> Chemical Communications. 46, 8043-8045 (2010).</li><li>Karver, M. R., Weissleder, R., Hilderbrand, S. A. <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+evaluation+of+a+series+of+1,2,4,5-tetrazines+for+bioorthogonal+conjugation.">Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation.</a> Bioconjugate Chemistry. 22, 2263-2270 (2011).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+chemistry:+fishing+for+selectivity+in+a+sea+of+functionality.">Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.</a> Angewandte Chemie International Edition. 48, 6973-6998 (2009).</li><li>Bosch, S. 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=Evaluation+of+strained+alkynes+for+Cu-free+click+reaction+in+live+mice.">Evaluation of strained alkynes for Cu-free click reaction in live mice.</a> Nuclear Medicine and Biology. 40, 415-423 (2013).</li><li>Rossin, R., 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+chemisry+for+pretargeted+tumor+imaging+in+live+mice.">In vivo chemisry for pretargeted tumor imaging in live mice.</a> Angewandte Chemie International Edition. 49, 3375-3378 (2010).</li><li>Ackerman, M. 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=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. A., 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=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, 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=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., vanden Bosch, S. M., LaForest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+reaction+for+tumor+pretargeting:+In+vivo+chemistry+can+boost+tumor+radiation+dose+compared+with+directly+labeled+antibody.">Diels-Alder reaction for tumor pretargeting: In vivo chemistry can boost tumor radiation dose compared with directly labeled antibody.</a> Journal of Nuclear Medicine. 54, 1989-1995 (2013).</li><li>Rossin, R., 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=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, F., 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=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

The authors have nothing to disclose.

Bu pretargeted PET görüntüleme stratejisinin temel avantajı, doğrudan etiketli antikorlar tarafından üretilen arka plan radyasyon dozunun sadece bir kısmını hedef-arka görüntü kontrast ile tümörlerin ortaya çıkarma yeteneğine sahip olmasıdır. Örneğin, burada açıklanan kolorektal kanser görüntüleme sisteminde, akut biyolojik dağılım deneylerden elde edilen veriler, doğrudan etiketli 64 Cu-NOTA-huA33 ve 89 Zr-DFO-huA33 ile birlikte 64 Cu-tabanlı pretargeting...

Son otuz yılda, pozitron emisyon tomografisi (PET) kanser tanı ve tedavisinde vazgeçilmez bir klinik bir araç haline gelmiştir. Antikorlar uzun kanser biyomarkerların onların zarif yakınlık ve özgüllük nedeniyle tümörlere pozitron yayan radyoizotop teslimi için umut verici vektörleri olarak kabul edilmiştir. Antikorların 1,2 Ancak, nispeten yavaş in vivo farmakokinetiği çoklu-gün radyoizotop kullanılmasını zorunlu kılmaktadır fiziksel yarı-ömürleri. Kısmi vücut BT aksine - - Bu kombinasyon hastalarda hedef olmayan organlar, radioimmunoconjugates intravenöz ve bu nedenle enjekte edilir çünkü özel klinik önemi olan önemli bir komplikasyon yüksek radyasyon dozları verebilir sonucu emilen dozlarda vücudun her yerinde, sorguya dokuların bakılmaksızın. Bu sorunu aşmak için, önemli çaba develo ithaf edilmiştirAynı anda kendi içsel farmakokinetik sınırlamaları süpürgelik ederken böylece antikorların avantajlı özelliklerini yararlanarak, radyoizotop ve hedefleme parçasını ayrıştırarak PET görüntüleme stratejilerinin pment. Bu stratejiler, - genellikle pretargeting ya da birkaç aşamalı hedefleme olarak adlandırılan - tipik haliyle dört adımı kullanır: (1) bir antijen ve bir radyo-iki bağlayabilen bir antikorun uygulanması; (2) hedef doku ve kan onun izni antikorun birikimi; (3) bir küçük moleküllü radyoligandın yönetimi; ve (4), fazla radyoligandın hızlı bir temizlenme ile. Bazı durumlarda, 3-8 ve ardından antikorun Radyoligandın in vivo bağlanması, ek bir temizleme maddesi, herhangi bir antikorun atılımını hızlandırmak amacıyla, aşama 2 ve 3 arasında enjekte Bu tümör bağlamak için henüz ve kanda kalır. 5 Genel olarak, tw konuşmapretargeting stratejilerin o tipleri edebiyatının en yaygındır. Her iki klinik öncesi modellerde başarılı olmuş olsa da, onlar da kendi klinik uygulanabilirliği engellemiştir anahtar sınırlamaları sahip. İlk strateji, streptavidin-konjuge antikor ve biyotin ile modifiye edilmiş radyo etiketlerin arasında yüksek afinite ile dayanır; Bununla birlikte, streptavidin ile modifiye edilmiş antikorların bağışıklık çeviri ilişkin endişe verici bir sorun olduğu görülmüştür. 5,6,9,10 İkinci strateji, aksine, bir kanser, her iki bağlanma genetik olarak inşa edilmiş, bispesifik antikorları kullanır Bu ikinci yol kesinlikle yaratıcı iken biyobelirteç antijen ve küçük molekül radyoaktif işaretli hapten. 3,11-14, geniş uygulanabilirliği sistemi modülerlik karmaşıklığı, gider ve eksikliği ile sınırlıdır. Son zamanlarda, biz geliştirdik ve (ters elektron talebi Diels-Alder dayalı I bir pretargeted PET görüntüleme metodolojisini yayınladıEDDA) trans -cyclooctene (TCO) ve tetrazin (Tz arasındaki sikloadisyon reaksiyonu;. Gösterildiği gibi Şekil 1) 11 reaksiyon kendisi yıllardır bilinen olsa da, IEDDA kimya, bir tıklama kimya bioconjugation tekniği olarak son yıllarda bir rönesans yaşadı Ralph Weissleder, Joseph Fox, ve peptidler, antikorlar ve nanopartiküller ile floresan görüntüleme dahil 12-15 IEDDA Siklokatılma ayarları geniş bir yelpazede uygulanmıştır. diğerleri arasında Peter Conti gruplarının büyüleyici çalışmaları yanı sıra nükleer görüntüleme .. 27 bioorthogonal Ve tıklama kimya türde bir dizi ise - eleştirel - radiohalogens ve radiometals hem 16-26 ligasyon, verimli, yüksek, temiz, hızlı (k 1&gt; 30.000 M -1 sn -1), seçici ve bir - Cu-katalize azid-alkin siklokatılma, gerilme-terfi azid-alkin siklokatılma ve Staudinger lig dahilations -. o bütün organizmalar uygulamaları pretargeting çok uygundur IEDDA kimya yapar hızlı reaksiyon kinetiği ve bioorthogonality eşsiz kombinasyonu, hem de bioorthogonal olan bu doğrultuda 28,29, bu son raporuna dikkat etmek önemlidir bizim laboratuarlar pretargeting için IEDDA kimya uygulamak için ilk değildi:. IEDDA ile pretargeted görüntüleme ilk rapor Rossin çalışmaları, vd ortaya çıktı ve bir 111 yılında etiketli tetrazin kullanan bir SPECT metodolojisi özellikli 30. Yukarıda ele alındığı gibi, pretargeting yöntemi dört oldukça basit adımları içerir (Şekil 2) sahiptir. Eldeki protokolde, 64 Cu NOTA etiketli tetrazin radyoligand ve huA33 antikorun bir TCO modifiye edilmiş konjügat kullanmaktadır kolorektal kanser, PET görüntülemesi için bir pretargeted strateji tarif edilecektir. Ancak, bu yöntemin sonuçta modülerliği kendi gr biridirtrans--cyclooctene kısım olarak eatest varlıklar olmayan içe antikora eklenebilir ve tetrazin radyoaktif muhabir çeşitli eklenebilir.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

Deneyin ilk üç adım - Tz-Bn-Nota, huA33 için TCO konjugasyonun sentezi ve Tz-Bn-Nota Radyoaktif (Şekil 3 ve 4) inşa - son derece güvenilir. Yukarıda tanımlanan prosedüre durumunda, Tz-Bn-NOTA yapısı yüksek verim ve saflıkta sentezlendi. ~ huA33 antikoru 4.2 ± 0.6 TCO / mAb ile değiştirilmiş olan, ve Tz-Bn-NOTA,&gt;% 85 bozulma düzeltilmiş verim, ve bir spesifik aktivite&gt;% 99, radyokimyasal saflık saflaştırılmış radyoligand vermek üzere 64 Cu ile ...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Pretargeted PET Görüntüleme Bioorthogonal Ters Elektron Talep Diels-Alder Katılma Harnessing

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.7K Görüntüleme.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 

Video: Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Biyomühendislik

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

On-talep Kantitatif Analiz için mikroakışkan tabanlı Electrotaxis<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Floresan Mikroskobu ile Görüntüleme Hücre Göç Yapıştırıcı ve Çözünür Degradeler oluşturma

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Aracı Olarak bir lipozomal-kapsüllü Yakın-Kızılötesi floroforun Floresan-söndürme<em&gt; İn Vivo</em&gt; Optik Görüntüleme

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

Hızlı Tarama Elektron Paramagnetik Rezonans Görüntüleme Fizyolojik Önemli Parametreler için Yeni bulvarlar açar<em&gt; İn Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Cryo-immobilize hücreler yapılarda bağdaşık ışık elektron izleme ve Viral Protein Imaging mikroskobu (CLEM) ilişkili

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Hiperpolarize Spin-Kafes Gevşeme manyetik alan bağımlılığının ölçülmesi [1-13C]pirüuvat

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Avustralya Senkrotron'da Seri Kristalografi Ölçümleri için Lipidico Enjeksiyon Protokolü

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Ultra Hızlı Doppler ile Kan Akışı Görüntüleme

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Pretargeted PET Görüntüleme Bioorthogonal Ters Elektron Talep Diels-Alder Katılma Harnessing

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

On-talep Kantitatif Analiz için mikroakışkan tabanlı Electrotaxis

Floresan Mikroskobu ile Görüntüleme Hücre Göç Yapıştırıcı ve Çözünür Degradeler oluşturma

Aracı Olarak bir lipozomal-kapsüllü Yakın-Kızılötesi floroforun Floresan-söndürme

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

Hızlı Tarama Elektron Paramagnetik Rezonans Görüntüleme Fizyolojik Önemli Parametreler için Yeni bulvarlar açar

Cryo-immobilize hücreler yapılarda bağdaşık ışık elektron izleme ve Viral Protein Imaging mikroskobu (CLEM) ilişkili

Hiperpolarize Spin-Kafes Gevşeme manyetik alan bağımlılığının ölçülmesi [1-¹³C]pirüuvat

Avustralya Senkrotron'da Seri Kristalografi Ölçümleri için Lipidico Enjeksiyon Protokolü

Ultra Hızlı Doppler ile Kan Akışı Görüntüleme