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

ÉTHIQUE DECLARATION: Toutes les expériences in vivo d&#39;animaux décrits ont été réalisés selon un protocole approuvé et selon les directives éthiques de la Memorial Sloan Kettering Cancer Center Institutional Animal Care et l&#39;utilisation Comité (IACUC). 1. Synthèse de Tz-Bn-NOTA <ol><li> Dans un petit récipient de réaction, dissoudre 7 mg NH 2 Bn-NOTA (1,25 x 10 -2 mmol) dans 600 ul NaHCO 3 tampon (0,1 M, pH 8,1). Vérifier le pH de la solution. Si nécessaire, ajuster le pH de la solution à 8,1 en utilisant de petites fractions aliquotes de 0,1 M de Na 2 CO 3. </li><li> Ajouter la solution de NH 2 Bn-NOTA à 0,5 mg Tz-NHS (1,25 x 10 -3 mmol) dans un tube de microcentrifugation de 1,7 ml. NOTE: Le TZ-NHS peut soit être pesé à sec ou ajouté à partir d&#39;une solution mère de DMF sec ou le DMSO (&lt;50 pi). </li><li> Laisser la solution de réaction résultant réagir pendant 30 min à température ambianteavec une légère agitation. </li><li> Après 30 min, purifier le produit en utilisant C Chromatographie 18 HPLC en phase inverse pour enlever ne ayant pas réagi NH 2 Bn-NOTA. Le NH 2 Bn-NOTA peut être contrôlé à une longueur d&#39;onde de 254 nm, tandis que le Tz-NHS et Tz-Bn-NOTA sont mieux suivis à une longueur d&#39;onde de 525 nm. NOTE: Les temps de rétention sont évidemment fortement tributaire de la configuration de l&#39;équipement de HPLC de chaque laboratoire (pompes, colonnes, tubes, etc.). Toutefois, pour présenter un exemple, si un gradient de 0: 100 MeCN / H 2 O (tous deux avec 0,1% de TFA) à 100: 0 MeCN / H 2 O de plus de 25 min et une 4,6 x 250 mm C 18 colonne d&#39;analyse est utilisé Les temps de rétention des Tz-Bn-NOTA, Tz-NHS, et NH 2 Bn-NOTA, à environ 15 min, 16,5 min et 10 min, respectivement. Le produit peut être purifié à partir des autres composants de la réaction, soit dans un seul passage ou plusieurs passages en utilisant une colonne semi-préparative HPLC preparative ou C 18. 1 H-RMN, analytical HPLC et ESI-MS sont toutes des méthodes qui peuvent être utilisées pour vérifier la pureté du précurseur Tz-Bn-NOTA terminé. 11 </li><li> Geler l&#39;éluant HPLC recueillies à l&#39;aide de l&#39;azote liquide. </li><li> Enroulez le tube de prélèvement congelé dans de l&#39;aluminium opaque. </li><li> Placer le tube de collecte congelé dans un récipient de lyophilisation O / N pour supprimer la phase mobile de CLHP. </li><li> Stocker le produit purifié (un rose vif solide) dans l&#39;obscurité à -80 ° C. NOTE: Ce est un point d&#39;arrêt acceptable dans la procédure. Le précurseur Tz-Bn-NOTA complété est stable pendant au moins 1 an dans ces conditions. </li></ol> 2. Préparation de huA33-TCO Immunoconjugué <ol><li> Dans un tube de 1,7 ml à centrifuger, préparer un 1 mg / ml (2,7 mM) solution de TCO-NHS dans du DMF sec. </li><li> Dans un tube de microcentrifugeuse de 1,7 ml, préparer une 2 mg / ml (13,3 uM) de huA33 solution dans 1 ml de solution saline tamponnée au phosphate, pH 7,4 (0,01 M PO 4 3-, </sub&gt; 0,154 M de NaCl). </li><li> En utilisant de petites fractions aliquotes (&lt;5 pi) de 0,1 M de Na 2 CO 3, ajuster le pH de la solution d&#39;anticorps à 8,8 à 9,0. Utilisez soit papier pH ou un pH-mètre avec une microélectrode de contrôler le pH, et faire attention de ne pas laisser le pH à élever au-dessus de pH 9,0. </li><li> Une fois la solution d&#39;anticorps est à la bonne pH, ajouter un volume de la solution NHS-TCO correspondant à huit équivalents molaires de l&#39;ester activé. Par exemple, ajouter 7,9 pi de la solution à 1 mg / ml TCO-NHS (1,07 x 10 -7 mol TCO-NHS) pour les 1 ml de solution à 2 mg / ml d&#39;anticorps huA33 (1,33 x 10 -8 mol huA33). Ne pas dépasser 5% de DMF en volume dans la solution. </li><li> Mélanger doucement la solution en inversant le tube à centrifuger à plusieurs reprises. </li><li> Enroulez le microtube dans une feuille d&#39;aluminium opaque. </li><li> Laisser la solution à incuber pendant 1 heure à température ambiante avec une légère agitation. </li><li> Après 1 heure à température ambiante, purifier l&#39;immunoconjugué résultant en utilisant un siz jetable pré-emballése exclusion sur une colonne de dessalage. Premièrement, rincer la colonne d&#39;exclusion de taille comme décrit par le fournisseur pour enlever les conservateurs présents sur la colonne pendant le stockage. Ensuite, ajouter le mélange réactionnel à la colonne d&#39;exclusion de taille, rincer la colonne avec 1,5 ml de solution saline stérile à 0,9%, et ensuite récupérer le produit en utilisant 2 ml de solution saline stérile à 0,9% comme éluant. NOTE: Cette étape donnera l&#39;huA33-TCO complété comme une solution 2 ml. </li><li> Mesurer la concentration de la résultante huA33-TCO sur un spectrophotomètre UV-Vis. </li><li> Si une concentration plus élevée est souhaitée, concentrer la solution huA33-TCO l&#39;aide d&#39;une unité de filtration centrifuge avec un poids moléculaire 50.000 cut-off. NOTE: Il est important de noter que tandis que huA33 et une variété d&#39;autres anticorps bien connus (par exemple, le bevacizumab, le trastuzumab, le cetuximab, et J591) sont très tolérants d&#39;être concentrée, agrégation et la précipitation peuvent se produire lors de la concentration dans les autres cas. Les chercheurs de tenter cette Procedure avec un nouvel anticorps doit faire confiance à la littérature ou leur propre connaissance de l&#39;anticorps en question quant à savoir si ou non de concentrer l&#39;anticorps. </li><li> Rangez la immunoconjugué huA33-TCO complété à 4 ° C dans l&#39;obscurité. NOTE: Ce est un point d&#39;arrêt acceptable dans la procédure. Le conjugué mAb-TCO complété doit être stable pendant au moins trois mois dans ces conditions de stockage. </li></ol> 3. 64 Cu Radiomarquage de Tz-Bn-NOTA Remarque: cette étape du protocole impliquant le traitement et la manipulation de radioactivité. Avant d&#39;effectuer ces étapes - ou effectuer d&#39;autres travaux par la radioactivité - les chercheurs devraient consulter avec le Département de la radioprotection de leur établissement d&#39;origine. Prendre toutes les mesures possibles pour minimiser l&#39;exposition aux rayonnements ionisants. <ol><li> Dans un tube de 1,7 ml à centrifuger, préparer un 0,5 mg / ml (723 uM) solution de Tz-Bn-NOTA. </li><li> Dans une 1,7 ml MicroCTube entrifuge, ajouter 10 ul de la solution Tz-Bn-NOTA (5 ug) à 400 ul de 0,2 M de NH 4 OAc tampon pH 5,5. </li><li> Dans l&#39;intérêt de la note tenue radiochimique appropriée, mesurer et enregistrer la quantité de radioactivité dans l&#39;échantillon en utilisant un calibrateur de dose avant et après les étapes qui ont suivi dans le protocole ci-dessous (03.04 à 03.08). Cela aidera à la détermination précise des rendements radiochimiques. </li><li> Ajouter 2,000 uCi (74 MBq) de 64 Cu à la solution Tz-Bn-NOTA. NOTE: En règle générale, [64 Cu] CuCl 2 est fourni dans un petit volume (&lt;30 pi) de 0,1 N HCl, et donc que de petits volumes (&lt;10 pi) de cette solution d&#39;achat d&#39;actions sont nécessaires pour la réaction de radiomarquage. Si de plus grands volumes de la [64 Cu] CuCl 2 actions sont nécessaires, la réaction de radiomarquage est tolérant d&#39;augmenter le volume global de la réaction. Cependant, le pH de la solution de réaction de radiomarquage doit être surveillée avec soin pour garantirqu&#39;il ne tombe pas en dessous de pH 4,0. </li><li> Laisser la solution à incuber pendant 10 min à température ambiante avec une légère agitation. </li><li> Après 10 min d&#39;incubation, purifier le produit en utilisant 18 C chromatographie HPLC en phase inverse. Les temps de rétention sont évidemment fortement dépendant de la configuration de l&#39;équipement de HPLC de chaque laboratoire (pompes, colonnes, tubes, etc.). Toutefois, pour présenter un exemple, si un gradient de 5:95 MeCN / H 2 O (tous deux avec 0,1% de TFA) à 95: 5 MeCN / H 2 O de plus de 15 min est utilisé, le temps de rétention de 64 Cu-Tz- Bn-NOTA devrait être autour de 9,8 min alors libre, non complexée 64 Cu sera élue avec le front du solvant à environ 2-4 min. </li><li> En utilisant un évaporateur rotatif, retirer l&#39;éluant HPLC. </li><li> Reprendre le produit 64 Cu-Tz-Bn-NOTA 0,9% dans une solution saline stérile. NOTE: Compte tenu de l&#39;12,7 h demi-vie physique de 64 Cu, ce ne est pas un point d&#39;arrêt acceptable dans la procédure. Effectuer la synthèse de 64 Cu-Tz-Bn-PASA immédiatement avant l&#39;injection du radioligand, et suivez l&#39;étape par étape immédiatement 3,7 4,5. </li></ol> 4. En Vivo préalablement visées PET Imaging Remarque: Comme dans la section Protocole 3, cette étape du protocole impliquant le traitement et la manipulation de radioactivité. Avant d&#39;effectuer ces étapes chercheurs devraient consulter avec le Département de la radioprotection de leur établissement d&#39;origine. Prendre toutes les mesures possibles pour minimiser l&#39;exposition aux rayonnements ionisants. <ol><li> Dans une souris nude athymiques femelles, implant sous-cutanée 1 x 10 cellules du cancer du côlon SW1222 de 6 et de permettre à ceux-ci se développent dans une xénogreffe de 100 à 150 mm 3 (9 à 12 jours après l&#39;inoculation). 11 </li><li> Diluer une partie aliquote de la solution huA33-TCO de service du protocole 2 à une concentration de 0,5 mg / ml dans 0,9% de solution saline stérile. </li><li> Injecter 200 ul de la solution huA33-TCO (100 pg) dans la veine caudale de la souris portant une xénogreffe. </li><li> Autoriser 24 h pour l&#39;accumulation de la huA33-TCO dans la tumeur de la souris. </li><li> On dilue le 64 Cu-Tz-Bn-NOTA radioligand à une concentration de 1,5 mCi / ml dans 0,9% de solution saline stérile. </li><li> Injecter 200 ul de la solution de ligand radioactif Cu-Tz-Bn-NOTA 64 (300 uCi; 11,1 MBq; 1,6 nmol de 64 Cu-Tz-Bn-NOTA, en supposant une activité spécifique de 6,7 MBq / nmol) dans la veine caudale de la xénogreffe-souris porteuses. </li><li> Au point de temps d&#39;imagerie désiré (par exemple, 2, 6, 12, ou 24 heures après l&#39;injection), Anesthésier la souris avec un isoflurane 2%: mélange de gaz d&#39;oxygène. </li><li> Placer la souris sur le lit de la petite scanner TEP animale. Maintenir l&#39;anesthésie pendant l&#39;analyse en utilisant un 1% d&#39;isoflurane: mélange de gaz d&#39;oxygène. Avant de placer l&#39;animal sur la vitre du scanner, vérifiez l&#39;anesthésie selon la méthode toe-pincement et appliquer une pommade vétérinaire aux yeux de la souris pour éviter le dessèchement pendant l&#39;anesthésie. </li><li> Acquérir les données PET pour la souris via un statiquebalayer avec un minimum de 20 millions d&#39;événements coïncidents à l&#39;aide d&#39;une fenêtre d&#39;énergie de 350-700 keV et une fenêtre de cadencement de coïncidence de 6 nanosecondes. Après avoir complété l&#39;acquisition de l&#39;image, ne laissez pas la souris sans surveillance et ne pas le placer dans une cage avec les autres souris jusqu&#39;à ce qu&#39;il a repris conscience. </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.

Le principal avantage de cette stratégie d&#39;imagerie par PET préalablement visées, ce est qu&#39;il est capable de délimiter les tumeurs avec la cible et le contraste de l&#39;image en arrière-plan seulement une fraction de la dose de rayonnement d&#39;arrière-plan produit par les anticorps directement marqués. Par exemple, dans le système d&#39;imagerie du cancer colorectal décrit ici, les données provenant des expériences de biodistribution aiguë ont été utilisées pour effectuer des calculs de dosim?...

Au cours des trente dernières années, la tomographie par émission de positons (TEP) est devenu un outil clinique indispensable dans le diagnostic et le traitement du cancer. Des anticorps ont longtemps été considérés comme des vecteurs prometteurs pour la livraison de radio-isotopes émettant des positrons à des tumeurs en raison de leur affinité exquise et la spécificité de biomarqueurs du cancer. 1,2 Toutefois, le relativement lents pharmacocinétique in vivo d&#39;anticorps obligatoire l&#39;utilisation de radio-isotopes avec multi-jours demi-vies physiques. Cette combinaison peut donner de fortes doses de rayonnement aux organes non-cibles de patients, une complication importante qui est d&#39;une importance particulière clinique depuis radioimmunoconjugués sont injectés par voie intraveineuse et donc - contrairement partielles scans corps CT - résultat dans doses absorbées dans chaque partie du corps, quels que soient les tissus interrogés. Afin de contourner ce problème, des efforts importants ont été consacrés à l&#39;dévepement de stratégies d&#39;imagerie PET dissociant le radio-isotope et la fraction de ciblage, se appuyant ainsi les propriétés avantageuses des anticorps en longeant simultanément leurs limites pharmacocinétiques intrinsèques. Ces stratégies - le plus souvent appelés préciblage ou ciblage en plusieurs étapes - utiliser habituellement quatre étapes: (1) l&#39;administration d&#39;un anticorps capable de se lier à la fois un antigène et un radioligand; (2) l&#39;accumulation de l&#39;anticorps dans le tissu cible et de son dégagement à partir du sang; (3) l&#39;administration d&#39;une petite radioligand molécule; et (4) la ligature in vivo du ligand radioactif à l&#39;anticorps suivie de la clairance rapide de l&#39;excès de radioligand. 8.3 Dans certains cas, un agent de compensation supplémentaire est injecté entre les étapes 2 et 3 de façon à accélérer l&#39;excrétion de tout anticorps qui n&#39;a pas encore d&#39;engager la tumeur et reste dans le sang. 5 D&#39;une manière générale, two préciblage types de stratégies sont les plus répandus dans la littérature. Bien que les deux ont fait leurs preuves dans des modèles précliniques, ils possèdent aussi des limites clés qui ont empêché leur applicabilité clinique. La première stratégie se appuie sur la forte affinité entre les anticorps de streptavidine conjugué et radiomarqueurs de biotine modifiée; cependant, l&#39;immunogénicité des anticorps de streptavidine modifiée se est avérée être un problème préoccupant en ce qui concerne la traduction. 5,6,9,10 La seconde stratégie, au contraire, utilise des anticorps bispécifiques qui ont été modifiés par génie génétique pour se lier à la fois un cancer antigène de biomarqueurs et un petit haptène radiomarqué molécule. 3,11-14 Bien que cette dernière voie est certainement créatif, sa large applicabilité est limitée par la complexité, le coût et le manque de modularité du système. Récemment, nous avons élaboré et publié une méthode d&#39;imagerie PET préalablement visées sur la base de la demande d&#39;électrons inverse de Diels-Alder (IEDDA) de réaction de cycloaddition entre -cyclooctene trans (TCO) et tétrazine (Tz;. Figure 1) 11 Bien que la réaction se est connu depuis des décennies, IEDDA chimie a connu une renaissance au cours des dernières années comme une technique de bioconjugaison click chemistry, comme illustré par le travail fascinant des groupes de Ralph Weissleder, Joseph Fox et Peter Conti entre autres. 12-15 Le IEDDA cycloaddition a été appliquée dans un large éventail de paramètres, y compris l&#39;imagerie de fluorescence avec des peptides, des anticorps, et des nanoparticules ainsi que l&#39;imagerie nucléaire . avec les deux radiohalogens et radiométaux 16-26 La ligature est un rendement élevé, propre, rapide (k 1&gt; 30000 M -1 sec -1), sélective et - critique -. bioorthogonal 27 Et si un certain nombre de types de chimie clic - y compris azoture alcyne cycloadditions de Cu-catalysée, cycloadditions azoture-alcyne contrainte promu, et Staudinger ligations -. bioorthogonal sont ainsi, ce est la combinaison unique de la cinétique de réaction rapides et bioorthogonality qui rend IEDDA chimie si bien adapté à préciblage applications dans des organismes entiers 28,29 long de ces lignes, il est important de noter que le récent rapport de notre laboratoires ne était pas le premier à appliquer IEDDA chimie pour préciblage: le premier rapport de l&#39;imagerie préalablement visées avec IEDDA née du travail de Rossin, et al et a présenté une méthodologie SPECT utilisant un 111 tétrazine Dans marqué 30.. Comme nous avons discuté ci-dessus, la méthodologie préciblage comporte quatre étapes assez simples (figure 2). Dans le protocole à portée de main, une stratégie préalablement visées pour l&#39;imagerie PET du cancer colorectal qui emploie une Cu-NOTA radioligand marqué 64 de tétrazine et un conjugué TCO modifiée de l&#39;anticorps huA33 va être décrit. Cependant, en fin de compte la modularité de cette méthodologie est l&#39;un de ses gractifs en mangeras, que la fraction -cyclooctene trans peuvent être ajoutés à tout anticorps non-internalisation et l&#39;tétrazine peuvent être attachés à une grande variété de journalistes radioactives.

<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.

Les trois premières étapes de l&#39;expérience - la synthèse de Tz-Bn-NOTA, la conjugaison du TCO huA33 et le radiomarquage de la Tz-Bn-NOTA construire (figures 3 et 4) - sont très fiables. Dans le cas de la procédure ci-dessus, le produit d&#39;assemblage Tz-Bn-NOTA a été synthétisé avec un rendement élevé et de pureté. L&#39;anticorps huA33 a été modifié avec 4,2 ± 0,6 TCO / mAb et Tz-Bn-NOTA était marqué à 64 Cu pour donner le radioligand purifié dans&...

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.

Exploiter la demande Bioorthogonal inverse Electron Diels-Alder Cycloaddition pour préalablement visées PET Imaging

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

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JoVE Journal

Bioengineering

8.7K vues.  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. 

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

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&#039; 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.

Électrotaxie microfluidique à base d&#39;analyse quantitative à la demande de<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 ...

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Bio-ingénierie

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.

Création de dégradés et adhésifs solubles pour la migration des cellules d&#39;imagerie par microscopie à fluorescence

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.

Fluorescence-trempe d&#39;un proche infrarouge fluorophore encapsulée dans des liposomes comme outil de<em&gt; In Vivo</em&gt; Imagerie optique

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 ...

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.

Numérisation rapide résonance paramagnétique électronique ouvre de nouvelles voies pour l&#39;imagerie physiologiquement Paramètres importants<em&gt; In 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.

Corrélatives microscopie électronique léger (CLEM) de suivi et d’imagerie protéine virale associés Structures des cellules immobilisées Cryo

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.

Mesurer la dépendance au champ magnétique de relaxation spin-lattice de l'hyperpolarisé [1-13C]pyruvate

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

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).

Imagerie du flux sanguin avec Ultrafast Doppler

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

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Établir un écosystème Octopus pour la recherche biomédicale et la bio-ingénierie

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Imagerie chimique bioorthogonale du métabolisme cellulaire régulé par les acides aminés aromatiques

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Analyse de la contraction cardiaque du poisson-zèbre avec interaction en réalité virtuelle

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Pleins feux sur l’auteur : Imagerie 4D haute résolution et réalité virtuelle chez le poisson-zèbre pour l’analyse unicellulaire de la fonction cardiaque

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Imagerie 4D de la contraction cardiaque du poisson-zèbre

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...