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

ÉTICA DECLARAÇÃO: Todas as experiências com animais in vivo descritos foram realizados de acordo com um protocolo aprovado e sob as diretrizes éticas do Memorial Sloan Kettering Cancer Center, Animal Care Institucional e Comitê de Uso (IACUC). 1. Síntese de Tz-Bn-NOTA <ol><li> Num recipiente de reacção pequeno, dissolver 7 mg NH2-Bn-NOTE (1,25 x 10 ~ 2 mmol) em 600 ul de tampão de NaHCO3 (0,1 M, pH 8,1). Verificar o pH da solução. Se necessário, ajustar o pH da solução para 8,1 utilizando pequenas alíquotas de 0,1 M de Na 2 CO 3. </li><li> Adicionar a solução de NH 2-Bn-NOTE a 0,5 mg Tz-NHS (1,25 x 10 -3 mmol) num tubo de microcentrífuga de 1,7 ml. NOTA: O Tz-NHS pode ser pesadas seco ou adicionado de uma solução de estoque de DMSO ou DMF seco (&lt;50 ul). </li><li> Permitir que a solução de reacção resultante a reagir durante 30 min à TAcom agitação moderada. </li><li> Após 30 min, purificar o produto utilizando cromatografia em C 18 de HPLC de fase reversa, para remover não reagido NH2-Bn-NOTE. O NH 2 -Bn-NOTA pode ser monitorado em um comprimento de onda de 254 nm, enquanto o Tz-NHS e Tz-Bn-NOTA são melhor monitorados em um comprimento de onda de 525 nm. NOTA: Os tempos de retenção são obviamente altamente dependente da configuração do equipamento de HPLC de cada laboratório (bombas, colunas, tubos, etc). No entanto, para apresentar um exemplo, se um gradiente de 0: 100 de MeCN / H 2 O (ambos com 0,1% de TFA) até 100: 0 de MeCN / H2O ao longo de 25 min e uma analítica de 4,6 x 250 mm coluna C 18 é usada , os tempos de retenção de Tz-Bn-NOTA, Tz-NHS, e NH2-Bn-NOTE será de cerca de 15 min, 16,5 min e 10 min, respectivamente. O produto pode ser purificado a partir de outros componentes da reacção em um único ou múltiplos de execução é executado utilizando uma coluna C 18 preparativa ou semi-preparativa de HPLC. 1 H-RMN, analytical HPLC e ESI-MS são todos os métodos que podem ser utilizados para verificar a pureza do precursor de completada Tz-Bn-NOTE 11. </li><li> Congelar o eluente HPLC coletados utilizando nitrogênio líquido. </li><li> Enrole o tubo de coleta congelado em papel de alumínio opaco. </li><li> Colocar o tubo de recolha congelado num recipiente de liofilização S / N para remover a fase móvel de HPLC. </li><li> Armazenar o produto purificado (um sólido cor de rosa claro) no escuro a -80 ° C. NOTA: Este é um ponto de parada aceitável no procedimento. O precursor Tz-Bn-NOTE concluída é estável durante pelo menos 1 ano, sob estas condições. </li></ol> 2. Preparação de huA33-TCO Imunoconjugado <ol><li> Num tubo de microcentrífuga de 1,7 ml, preparar um 1 mg / ml de solução de TCO-NHS em DMF seca (2,7 mM). </li><li> Num tubo de microcentrífuga de 1,7 ml, preparar uma solução de (13,3 mM) de huA33 em 1 ml de solução salina tamponada com fosfato, pH 7,4 (0,01 M de PO 4 3- 2 mg / ml, </sub&gt; 0,154 M de NaCl). </li><li> Usando pequenas alíquotas (&lt;5 ul) de 0,1 M de Na 2 CO 3, ajustar o pH da solução de anticorpo para 8,8-9,0. Utilize um papel de pH ou um medidor de pH com uma microeletrodos para monitorar o pH, e ter cuidado para não permitir que o pH a subir acima de pH 9,0. </li><li> Uma vez que a solução de anticorpo é o pH correcto, adicionar um volume da solução de TCO-NHS correspondentes a 8 equivalentes molares do éster activado. Por exemplo, adicionar 7,9 mL de solução a 1 mg / ml TCO-NHS (1,07 x 10 -7 mol TCO-NHS) para 1 ml de solução de 2 mg / mL de anticorpo huA33 (1,33 x 10 -8 moles huA33). Não ultrapasse 5% em volume em DMF a solução. </li><li> Misturar suavemente a solução invertendo o tubo várias vezes microcentrífuga. </li><li> Enrole o tubo de microcentrífuga em papel de alumínio opaco. </li><li> Permitir que a solução a incubar durante 1 hr à temperatura ambiente com agitação suave. </li><li> Após 1 h à temperatura ambiente, purificar o imunoconjugado resultante utilizando um siz descartável pré-embaladoe exclusão coluna de dessalinização. Em primeiro lugar, lavar a coluna de exclusão de tamanho tal como descrito pelo fornecedor para remover quaisquer conservantes presentes na coluna durante o armazenamento. Em seguida, adicione a mistura de reacção para a coluna de exclusão de tamanho, lavar a coluna com 1,5 ml de 0,9% de solução salina estéril, e em seguida recolher o produto utilizando-se 2 ml de solução salina estéril a 0,9%, como o eluente. NOTA: Esta etapa irá produzir o concluída huA33-TCO como uma solução de 2 ml. </li><li> Medir a concentração da resultante huA33-TCO em um espectrofotómetro de UV-Vis. </li><li> Se uma concentração mais elevada é desejada, concentra-se a solução huA33-TCO utilizando uma unidade de filtração centrífuga com um peso molecular de 50.000 de corte. NOTA: É importante observar que enquanto huA33 e uma variedade de outros anticorpos conhecidos (por exemplo, bevacizumab, trastuzumab, cetuximab, e J591) são muito tolerantes de ser concentrada, agregação e precipitação pode ocorrer após concentração em outros casos. Pesquisadores de tentar esta procedure com um novo anticorpo deve confiar na literatura ou o seu próprio conhecimento do anticorpo em questão diz respeito à possibilidade ou não de se concentrar o anticorpo. </li><li> Guardar o imunoconjugado completado huA33-TCO a 4 ° C no escuro. NOTA: Este é um ponto de parada aceitável no procedimento. O conjugado mAb TCO preenchido deve ser estável durante pelo menos 3 meses sob estas condições de armazenamento. </li></ol> 3. 64 Cu Radiomarcação de Tz-Bn-NOTA NOTA: Este passo do protocolo envolve o manuseamento e manipulação de radioactividade. Antes de executar estes passos - ou realizar qualquer outro trabalho com radioatividade - pesquisadores deve consultar com Radiação Secretaria de Segurança de sua instituição de origem. Tomar todas as medidas possíveis para minimizar a exposição à radiação ionizante. <ol><li> Num tubo de microcentrífuga de 1,7 ml, preparar uma solução de Tz-Bn-NOTE 0,5 mg / ml (723 uM). </li><li> Numa MicroC 1,7 mlentrifuge tubo, adicionar 10 ul da solução Tz-Bn-NOTE (5 ug) a 400 uL de tampão NH4OAc 0,2 M pH 5,5. </li><li> No interesse do bom radioquímica nota-keeping, medir e registrar a quantidade de radioactividade na amostra usando um calibrador de dose antes e depois das etapas que se seguiram no protocolo abaixo (3,4-3,8). Isso vai ajudar com a determinação exata da yields radioquímica. </li><li> Adicionar 2.000 ï¿½i (74 MBq) de 64 Cu à solução Tz-Bn-NOTA. NOTA: Tipicamente, [64 Cu] CuCl 2 é fornecido em um pequeno volume (&lt;30 ul) de HCl 0,1 N, e, por conseguinte, apenas pequenas quantidades (&lt;10 ul) desta solução de estoque são necessários para a reacção de marcação radioactiva. Se forem necessárias maiores volumes do [64 Cu] CuCl2 estoque, a reacção de marcação radioactiva é tolerante de aumentar o volume de reacção total. No entanto, o pH da solução de reacção radiomarcação devem ser cuidadosamente monitorizados para assegurarque ele não caia abaixo de pH 4.0. </li><li> Permitir que a solução a incubar durante 10 min à temperatura ambiente com agitação suave. </li><li> Após 10 min de incubação, purificar o produto usando cromatografia de HPLC de fase reversa C-18. Os tempos de retenção são obviamente altamente dependente da configuração do equipamento de HPLC de cada laboratório (bombas, colunas, tubos, etc). No entanto, para apresentar um exemplo, se um gradiente de 5:95 de MeCN / H 2 O (ambos com 0,1% de TFA) a 95: 5 de MeCN / H2O ao longo de 15 min é usada, o tempo de retenção de 64 Cu-Tz- Bn-NOTA deve ser em torno de 9,8 min, enquanto livre, não complexada 64 Cu eluirá com a frente do solvente em cerca de 2-4 min. </li><li> Usando um evaporador rotativo, remover o eluente de HPLC. </li><li> Recolher o produto de 64 Cu-Tz-Bn-NOTA em 0,9% de solução salina estéril. NOTA: Tendo em conta a 12,7 h meia-vida física de 64 Cu, este não é um ponto de paragem no processo aceitável. Realizar a síntese de 64 Cu-Tz-Bn-NOTUm imediatamente antes da injeção do radioactivo, e siga o passo a passo 3.7 imediatamente 4.5. </li></ol> 4. In Vivo Pretargeted de imagem PET NOTA: Como no Protocolo Secção 3, esta etapa do protocolo envolve o manuseamento e manipulação de radioactividade. Antes de executar estes passos pesquisadores deve consultar com Radiação Secretaria de Segurança de sua instituição de origem. Tomar todas as medidas possíveis para minimizar a exposição à radiação ionizante. <ol><li> Em um rato nu atímica feminino, implante subcutâneo 1 x 10 células de câncer colorretal 6 SW1222 e ​​permitir que estes se transformar em um 100-150 mm 3 heterólogo (9-12 dias após a inoculação) 11. </li><li> Dilui-se uma alíquota da solução-huA33 TCO Protocolo de secção 2 para uma concentração de 0,5 mg / ml em 0,9% de solução salina estéril. </li><li> Injectar 200 ul da solução-huA33 TCO (100 ug) na veia da cauda do rato xenoenxerto de rolamento. </li><li> Permitir que 24 horas para a acumulação do huA33-TCO no tumor do mouse. </li><li> Dilui-se a 64 de Cu-Tz-Bn-NOTE radioligando para uma concentração de 1,5 mCi / ml em 0,9% de solução salina estéril. </li><li> Injectar 200 ul da solução de ligando radioactivo 64 Cu-Tz-Bn-NOTE (300 uCi; 11,1; 1,6 MBq de 64 nmol de Cu-Tz-Bn-NOTA, assumindo uma actividade especifica de 6,7 MBq / nmol) na veia da cauda do camundongos portadores de xenotransplante. </li><li> No ponto de tempo de imagem desejada (por exemplo, 2, 6, 12, ou 24 horas após a injecção), anestesiar o rato com uma isoflurano a 2%: mistura de gás oxigénio. </li><li> Posicione o mouse em cima da cama do pequeno scanner de PET animal. Manter a anestesia durante a varredura usando um isoflurano 1%: mistura de gás oxigênio. Antes de colocar o animal na mesa do scanner, verifique anestesia utilizando o método toe-pitada e aplicar pomada veterinária aos olhos do mouse para evitar o ressecamento durante a anestesia. </li><li> Adquirir os dados de PET para o mouse através de um estáticodigitalizar com um mínimo de 20 milhões de eventos coincidentes, utilizando uma janela de energia de 350-700 keV e uma janela de tempo coincidência de 6 ns. Depois de concluir a aquisição da imagem, não deixe o mouse autônoma e não colocá-lo em uma gaiola com outros ratos até que recuperou a consciência. </li></ol>

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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.

A principal vantagem desta estratégia de imagem PET pretargeted é que ele é capaz de delinear com tumores alvo-fundo-de contraste de imagem em apenas uma fracção da dose de radiação de fundo produzida por anticorpos marcados directamente. Por exemplo, no sistema de imagem do cancro colo-rectal descrito aqui, os dados obtidos em experiências de biodistribuição agudas foram utilizados para realizar os cálculos de dosimetria para a estratégia de pré-direccionamento com base de Cu-64, juntamente com ...

Ao longo dos últimos trinta anos, a tomografia por emissão de pósitrons (PET) tornou-se uma ferramenta indispensável para o diagnóstico e tratamento do câncer. Os anticorpos têm sido considerados promissores vectores para a entrega de radioisótopos emissores de positrões para tumores devido à sua afinidade e especificidade para requintado biomarcadores de cancro. 1,2 farmacocinética No entanto, a relativamente lenta in vivo de anticorpos exige a utilização de radioisótopos com multi-dia semi-vidas físicas. Esta combinação pode render altas doses de radiação para os órgãos não-alvo de doentes, uma importante complicação que é de especial importância clínica desde radioimunoconjugados são injetados por via intravenosa e, portanto, - ao contrário de tomografias corpo parciais - resultado em doses absorvidas em cada parte do corpo, independentemente dos tecidos interrogados. A fim de contornar este problema, esforço significativo foi dedicado ao desenvolvimento de estratégias de imagem PET que dissociar o radioisótopo e do grupo alvo, o que reforça as propriedades vantajosas de anticorpos contornando simultaneamente suas limitações farmacocinéticos intrínsecos. Estas estratégias - mais frequentemente denominado prï¿½marcaï¿½o ou segmentação de várias etapas - empregam tipicamente quatro passos: (1) a administração de um anticorpo capaz de se ligar tanto a um antigénio e um radioligando; (2) a acumulação de anticorpos no tecido-alvo e a sua depuração do sangue; (3) a administração de uma pequena molécula radioactivo; e (4) da ligação in vivo do ligando radioactivo ao anticorpo, seguido pela rápida eliminação do excesso de radioligando 3-8 Em alguns casos., um agente de compensação adicional é injectado entre as etapas 2 e 3, a fim de acelerar a eliminação de qualquer anticorpo que ainda tem que se ligar o tumor e permanece no sangue. 5 Em termos gerais, twO tipos de estratégias pré-direccionamento são mais prevalentes na literatura. Embora ambos provaram ser um sucesso em modelos pré-clínicos, eles também possuem limitações fundamentais que têm impedido a sua aplicabilidade clínica. A primeira estratégia baseia-se na elevada afinidade entre os anticorpos e conjugados estreptavidina-radiomarcadores modificados com biotina; no entanto, a imunogenicidade dos anticorpos modificados com estreptavidina provou ser um problema preocupante no que diz respeito à tradução. 5,6,9,10 A segunda estratégia, em contrapartida, utiliza anticorpos biespecíficos que foram geneticamente modificadas para se ligarem tanto um cancro antigénio biomarcador e um pequeno hapteno marcado radioactivamente molécula. 3,11-14 Embora esta última via é certamente criativa, a sua ampla aplicabilidade é limitada pela complexidade, custo e falta de modularidade do sistema. Recentemente, desenvolveu e publicou uma metodologia de imagem PET pretargeted baseado na procura de elétron inversa Diels-Alder (IEDDA) reação de cicloadição entre -cicloocteno trans (TCO) e tetrazina (Tz;. Figura 1) 11 Embora a própria reação tem sido conhecida há décadas, IEDDA química experimentou um renascimento nos últimos anos como uma técnica bioconjugação clique química, como ilustrado pelo o trabalho fascinante dos grupos de Ralph Weissleder, Joseph Fox, e Peter Conti entre outros. 12-15 A IEDDA cicloadição foi aplicado numa grande variedade de configurações, incluindo imagiologia por fluorescência com péptidos, anticorpos e nanopartículas, bem como imagiologia nuclear . com ambas radiohalogens e radiometais 16-26 A ligação é de alto rendimento, limpo, rápido (k 1&gt; 30.000 M-1 s-1), seletivo, e - criticamente -. bioorthogonal 27 E, enquanto uma série de tipos de clique química - incluindo cicloadições catalisada por Cu azida-alcino, cicloadições azida-alcino promoveu-deformação e Staudinger ligções -. bioorthogonal estão bem, é a combinação única de cinética de reação rápida e bioorthogonality que faz IEDDA química tão bem adaptado para prï¿½marcaï¿½o aplicações em organismos inteiros 28,29 Nesse sentido, é importante notar que o relatório recente da nossa laboratórios não foi o primeiro a aplicar IEDDA química para prï¿½marcaï¿½o: o primeiro relato da imagem pretargeted com IEDDA surgiu a partir do trabalho de Rossin, et al e contou com uma metodologia SPECT empregando um 111 tetrazina Em marcado 30.. Como discutimos acima, a metodologia prï¿½marcaï¿½o tem quatro passos bastante simples (Figura 2). No protocolo a mão, uma estratégia para a pretargeted imagens PET de cancro colo-rectal que emprega uma 64 Cu-NOTE-radioligando marcado tetrazina e um conjugado de TCO-modificada do anticorpo huA33 será descrito. No entanto, em última análise, a modularidade desta metodologia é um dos seus grEatest activos, como a porção -cicloocteno trans pode ser adicionado a qualquer anticorpo não de internalização, e o tetrazina pode ser ligado a uma vasta variedade de repórteres radioactivos.

<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.

Os três primeiros passos da experiência - a síntese de Tz-Bn-NOTA, a conjugação da TCO para huA33, e a marcação radioactiva do Tz-Bn-NOTE construir (Figuras 3 e 4) - são altamente fiáveis. No caso do processo acima descrito, o construto Tz-Bn-NOTE foi sintetizado com elevado rendimento e pureza. O anticorpo foi modificado com huA33 4,2 ± 0,6 TCO / mAb, e Tz-Bn-NOTE foi marcado radioactivamente com 64 Cu, para se obter o radioligando purificada em&gt; 99% de pureza r...

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.

Aproveitando a demanda Bioorthogonal Inverse Electron Diels-Alder para Pretargeted PET Imagem

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

Research

JoVE Journal

Bioengineering

8.7K visualizações.  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. 

Vídeo: 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.

De alta resolução de fibra óptica para Microendoscopy In situ Celular imagem

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

Bioengenharia

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.

Electrotaxis baseados em microfluídica para análise quantitativa On-demand 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 ...

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.

Criando Gradientes adesivas e solúvel para a migração celular Imaging com Microscopia de Fluorescência

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.

Fluorescência-têmpera de um Fluoróforo encapsulado em lipossomas Near-infrared como ferramenta para<em&gt; In Vivo</em&gt; Imageamento óptico

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.

Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

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.

Rápida digitalização ressonância paramagnética eletrônica abre novos caminhos para criação de imagens fisiologicamente parâmetros importantes<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.

Correlativo luz microscopia eletrônica de varredura (CLEM) para rastreamento e Imaging proteína Viral associado estruturas nas células Cryo-imobilizado

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.

Medindo a dependência do campo magnético do abrandamento da girar-estrutura de Hyperpolarizado [1-13C] piruvato

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.

Protocolo de injeção de lipidico para medições de cristalografia serial no Síncrotron Australiano

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).

Imagem de fluxo sanguíneo com Doppler Ultrarrápido

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