NIH concede R01 HL085369.

1. Preparação prévia das soluções de reserva <ol><li> Prepare duas soluções estoque de solução de Tyrode do (16x) de antecedência em água deionizada e armazená-los a 4 ° C: <ol class="lalpha"><li> Ações I (119,872 g / L NaCl, 3,056 g / L CaCl 2 (2H 2 O), 5,6 g / L KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), (Fisher Lawn, científico Fair, NJ)); </li><li> Estoque II (26,88 g / L NaHCO3, (Fisher Scientific, Fair Lawn, NJ)). </li></ol></li><li> Preparar soluções estoque de corantes fluorescentes. Para evitar congelamento e descongelamento repetidos, nós armazenamos 30 mL alíquotas de ambos os corantes a -20 ° C, o que é suficiente para uma experiência: <ol class="lalpha"><li> Sensíveis à voltagem solução estoque de corante RH237 (Invitrogen, Carlsbad, CA), 1,25 mg / mL em dimetil sulfóxido (DMSO, Sigma, St. Louis, MO); </li><li> Cálcio indicador Rhod-2:00 solução-mãe (Invitrogen, Carlsbad, CA), 1 mg / ml de solução em DMSO. </li></ol></li><li> Prepare excitação-contração desacoplador solução estoque blebbistatin (Tocris Bioscience, St. Louis, MO, 2 mg / mL em solução de DMSO) de antecedência e guardar o blebbistatin dissolvida a 4 ° C. </li></ol> 2. Preparar soluções de perfusão e configuração experimental 7 <ol start="4"><li> Recém preparar solução Tyrode 2L do (128.2mM NaCl, 1,3 mM CaCl 2 (2H 2 O), 4,7 mm KCl, 1,05 mm MgCl 2 (6H 2 O), 1.19mM NaH 2 PO 4, NaHCO 20mM 3, 11,1 mM de glicose-D em água deionizada, pH = 7,35 ± 0,05). Se a solução de ações está sendo usado para fazer 2L de solução de Tyrode do (suficiente para um experimento) Tomar 1750 mL de água deionizada e misturar em 125 mL de Ações I, 125 mL de Ações II, e 4g de glicose. </li><li> Ligue as duas bombas dos sistemas de perfusão. Definir a bomba peristáltica (Peri-Star, WPI, Sarasota, EUA) que é usado para perfusão retrógrada a 40 mL / min. Definir a bomba peristáltica outros (Cole-Parmer Masterflex Bomba Peristalic L / S, empresa Instrumento Cole-Parmer, Vernon Hills, Illinois) que é usado para superfusão e devolver o perfusato de volta para o reservatório de exploração para 80 mL / min. </li><li> Lavar o sistema de perfusão com etanol 70% por 30 min e depois com água deionizada 2L. </li><li> Uma vez que toda a água deionizada é evacuada da câmara, circula a solução Tyrode e passá-lo através de um filtro 5 mícrons (Millipore, Billerica, MA, EUA). Aqueça o perfusato a 37 ° C com uma jaqueta de água e circulador (ThermoNESLAB EX7, Newtown, EUA) e do perfusato a oxigenar por borbulhamento O 2 / CO 2 (95% / 5%) de gás na solução. Monitorar o pH da solução com um medidor de pH (Oakton Instruments, Vernon Hills, IL) e ajustar a taxa de O 2 / CO 2 borbulhando para manter o pH em 7,35 ± 0,05. Continue pHmetria e temperatura durante o experimento. </li><li> O aparelho de mapeamento de dupla óptica consiste em duas Micam Ultima-L CMOS câmeras (SciMedia, Costa Mesa, CA), que têm alta espacial (100x100 pixels, 230 ± 20 mM por pixel) e resolução (1.000-3.000 frames / seg) temporal. Fixar um filtro passa-banda (590 ± 15 nm, Thorlabs, Newton, NJ) na frente da câmera designada imagem de cálcio, enquanto que, um filtro passa-tempo (&gt; 700 nm, Thorlabs, Newton, NJ) precisa ser posicionado na frente da câmera designada tensão de imagem. As câmeras são dispostos perpendicularmente uns aos outros por um titular, que contém um espelho dicróico (635 nm de corte, Omega Optical, Brattleboro, VT). Imediatamente abaixo do titular câmera dupla, há uma lente (Nikon Nikkor 55 milímetros 1:1.4 235.052), que concentra a emissão de luz que vem do coração para o espelho dicróico. A distância de trabalho é de aproximadamente três centímetros. A luz de excitação é gerada por uma lâmpada halógena (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) e é transmitida através de um filtro de calor, obturador, e filtro passa-banda (520 ± 45 nm). A guia de luz flexível direciona a luz passa-banda filtrada para a preparação, e um obturador é usada para garantir que a preparação é exposto à luz apenas durante a aquisição de imagem para evitar fotodegradação dos corantes. </li><li> Prepare Ag / AgCl 2 eletrodos para estimulação e detecção antecipada e instalá-los na câmara antes de colocar o coração. Certifique-se que amplificadores e filtros são ajustados para níveis apropriados. </li></ol> 3. Colheita do coração mouse, canular e configurar perfusão Langendorff <ol start="10"><li> Anestesiar o rato com cetamina / xilazina (cetamina, 80mg/kg de peso corporal; xilazina, 10 mg / kg) e heparina (100 unidades) por injeção intraperitoneal. Assegurar um nível adequado de anestesia com a falta de reflexo de dor. </li><li> Após uma incisão médio-esternal, remover rapidamente o coração e lave-o em oxigenada (95% O 2, 5% CO 2), a temperatura constante (37 ± 1 ° C) solução Tyrode é. </li><li> Usando um microscópio de dissecação, Identificar rapidamente a aorta e fazer um corte limpo em toda a aorta ascendente abaixo da artéria subclávia direita. Uma pequena secção de aorta é então ligado a uma custom made 21 calibre da cânula com uma ponta queimado. Fio de seda preto trançado 4-0 (Corporação especialidades cirúrgicas, Reading, PA) é usado para corrigir o coração para a cânula. Após canulação, o coração é perfundido retrogradamente e superfused com solução Tyrode é. A taxa de perfusão retrógrada é ajustado na faixa de 2-5 mL / min para manter a pressão aórtica entre 60 e 80 mmHg (transdutor de pressão, instrumentos de precisão Inc Mundial (WPI), Sarasota, EUA; Ponte Amplificador TBM4M, WPI, Sarasota, EUA). </li><li> Depois que o coração é canulada, pulmão, timo, tecido adiposo e são, então, dissecados e removidos. </li><li> O coração isolado é preso (Ferramentas Ciência Fine) no ápice para a parte inferior da câmara de perfusão (revestido Sylgard) para evitar fluxo induzido por movimento. Os apêndices atriais direito e esquerdo também são esticados e presos (Ferramentas Ciência Fine, Inc, Foster City, CA) para o fundo da câmara, que fornece superfície máxima para medidas ópticas dos átrios. Muito importante! Um pequeno tubo de silicone é inserida no ventrículo esquerdo através das veias pulmonares e fixado por fio de seda para o tecido conjuntivo nas proximidades. Isso evita que o congestionamento solução e acidificação do perfusato preso no ventrículo esquerdo, que é especialmente importante após a supressão das contracções ventriculares com um desacoplador excitação-contração (consulte a etapa Parte 4 19). </li><li> Um eletrodo feito sob medida é colocada sobre a superfície do coração para conduzir os estímulos ritmo, que é gerado pelo Mestre-8 (AMP Instruments Ltd, Jerusalém, Israel) ou 26T PowerLab (AD Instruments, Sydney, Austrália). </li><li> A tampa de vidro de pequeno porte é fixa na superfície da solução sobre o coração para reduzir artefatos de movimento a partir da solução de vibração. </li><li> O foco da luz de excitação sobre o coração. Além disso, ajustar a distância entre o aparelho de câmara dupla e do coração para que a resolução máxima será obtido. </li><li> Desligar todas as luzes na sala e iniciar as gravações elétrica usando PowerLab 26T. </li></ol> 4. Tensão de carga de cálcio e corantes sensíveis e excitação-contração desacoplador <ol start="19"><li> Warm-up de 0,6 mL blebbistatin. Misture 0,5 mL da blebbistatin com o perfusato no reservatório de retenção. Diluir a 0,1 mL restantes de blebbistatin em 1 mL de solução de Tyrode e injetá-la lentamente (ao longo de um período de 20 minutos) através de uma porta de drogas localizado perto da cânula. Observe como blebbistatin reduz gradualmente o artefato de movimento. </li><li> Diluir 30 mL da solução estoque de tensão sensíveis corante RH237 em solução 1 mL Tyrode e injectar lentamente ao longo de 5-7 minutos na porta de injeção mesma blebbistatin. </li><li> 30 mL de cálcio indicador Rhod-02:00 01:01 é misturado com PLURONIC F127 (Invitrogen, Carlsbad, CA, solução a 20% em DMSO) e, em seguida, diluídas em 1 ml de solução Tyrode e lentamente aplicado sobre min 10/05, através da porta de injeção mesmo . </li><li> Aguarde 5-10 minutos para blebbistatin e corantes para alcançar a membrana celular e citoplasma. Continue com o protocolo quando o movimento é completamente suprimida. </li><li> Continuamente gravações monitor ECG sobre todo o procedimento para garantir a função elétrica normal do coração. </li><li> Iniciar as gravações usando o sinal fluorescente SciMedia custom software (SciMedia, Costa Mesa, CA). </li></ol> 5. Resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Figura 1. Instalação experimental para a perfusão, gravações elétricas e mapeamento óptico. EM = emissões; Lp = longpass <img alt="Figura 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Figura 2. Preparação Experimental e exemplos sinal gravado durante a estimulação ventricular. Esquerda: Os sinais de ECG são coletados de Ag / AgCl dois eletrodos de disco (Top) e um exemplo de S1S1 protocolo de estimulação é mostrado (Bottom). Center: Langendorff preparação do coração mouse. Direita: Representante potenciais de ação óptica e sinais de cálcio transiente de átrios (Top) e ventrículos (Bottom) são mostrados. A seta amarela (Top) aponta para a dispersão de sinal fluorescente vinda dos ventrículos, que é visto nas gravações atrial. LV = ventrículo esquerdo, VD = ventrículo direito; LA = átrio esquerdo; RA = átrio direito <img alt="Figura 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Figura 3. Representante gravações ópticas de V m e CAT dos ventrículos do coração selvagem do tipo de mouse. A. A preparação experimental com uma série de pontos espaçados marcada por pontos pretos, cuja óptica gravações pode ser visto em (C). Exemplo B. rastreamento de V m eo gato de uma localização central na matriz (ver caixa in (C)). C. V m (azul) e CAT (vermelho) da série de pontos espaçados. Sinais foram binned 3x3. <img alt="Figura 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Figura 4. Mapa de ativação e de condução. A. Um mapa de ativação exemplo de um coração selvagem do tipo de mouse com transversal (T) e (L) longitudinal direções indicadas pelas setas brancas. Sinais B. V m (Top) e dV / dt (Bottom), correspondentes aos três pontos de vista em A (T1, T2, T3, L1, L2, L3). <img alt="Figura 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Figura 5. Potencial de ação e de cálcio análise de duração transitória. A. duração Ação potencial de 80% repolarização (APD80) e duração de cálcio transiente menos 80% de relaxamento (CaD80) mapas são mostrados de um coração em condições controle (esquerda) e depois de 30 nM isoproterenol aplicação (Direito). A cor amarela / verde nos ventrículos (direito) indica isoproterenol encurtado APD80 e CaD80. Exemplo B. traçados de APD80 e CaD80 dos ventrículos do tipo selvagem mouse (Top) e átrios (Bottom).

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Neste experimento nós modificamos o método de perfusão Langendorff adicionando um pequeno tubo de silicone, que é especialmente crucial depois da supressão das contracções ventriculares com um desacoplador excitação-contração. O tubo de silicone é usado para evitar o congestionamento solução, a acidificação da solução de perfusão, e desenvolvimento de isquemia do ventrículo esquerdo. O coração do mouse é muito sensível à hipotermia, assim, variações de temperatura em todo o coração vai causar diferenças artificial em durações potencial de ação. Conseqüentemente, um sistema de aquecimento foi implementado na câmara de perfusão, a fim de manter uma temperatura constante de 37 ° C durante a totalidade do experimento 8. Uma vez que um modelo Langendorff não retém a inervação do coração, é preciso considerar a adição de neurotransmissores para o perfusato, a fim de investigar o tônus ​​simpático e parassimpático fisiológicas 9. Além de perfusão retrógrada, a adição de superfusão do coração ajuda a manter adequados parâmetros ambientais tais como pH e temperatura. Neste método, o coração foi perfundido Langendorff horizontalmente colocado. Uma configuração de perfusão verticais Langendorff também pode ser usado 10, mas pode resultar em um pouco diferente mecânica cardíaca 11. Além de câmeras CMOS, detectores de alternativas também estão disponíveis e podem ser aplicados para mapear V m e CAT simultaneamente 12. Aplicação de câmeras CMOS de resolução espaço-temporal de alta garante a precisão das gravações, no entanto, sinais de mapeamento óptico não são de uma única célula. Ao contrário, cada sinal fluorescente vem de centenas ou milhares de células, dependendo de ampliação ótica. A fluorescência ventricular muito maior pode distorcer os sinais atrial pelo espalhamento óptico e, portanto, a interpretação cuidadosa dos sinais gravados oticamente é necessária. Outra limitação da preparação do mouse é a distorção de sinal e ruído induzido pela curvatura da superfície, devido ao pequeno tamanho do coração 13. Medições de velocidade de condução pode ser alterado não só a partir da curvatura do coração de rato, mas também da polaridade do eletrodo e eletrodos virtual. Para alcançar a precisão de velocidade de condução, anisotropia, ativação e repolarização mapas, correto foco da câmera na superfície do coração é essencial. Neste método, as gravações em tempo real ECG pode complementar a investigação óptica de eletrofisiologia cardíaca. Sensíveis à voltagem corante (RH237) e indicador de cálcio (Rhod-2:00) são utilizados no protocolo por causa de sua rápida resposta, excitação semelhante, e espectros de emissão de 3,7. Existem combinações alternativas de corantes que podem ser usados ​​para medir V m e CAT que não RH237 e-Rhod 02:00 3. Um corante sensíveis à voltagem romance, PGHI, com uma grande mudança do Stoke (&gt; 200 nm) foi encontrada para permitir uma melhor m V e sinais de CaT, devido à maior separação entre os comprimentos de onda de emissão entre PGHI e Rhod-2:00 14. Melhoramentos futuros podem se concentrar em explorar novas sondas fluorescentes, o desenvolvimento de detectores de novas imagens, e software de processamento melhorado a imagem. Maior resolução e novas modalidades de imagem óptico para o mapeamento óptico 3D também são importantes direções futuras de mapeamento óptico 5.

<table border="1"><tbody><tr><td> Químico </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S271-1 </td></tr><tr><td> CaCl 2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> Técnica S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S369-500 </td></tr><tr><td> NaHCO 3 </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S233-3 </td></tr><tr><td> D-Glicose </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> D16-1 </td></tr><tr><td> Blebbistatin </td><td> Tocris Bioscience, Ellisville, MO </td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen, Carlsbad, CA </td><td> S1109 </td></tr><tr><td> Rhod-02:00 </td><td> Invitrogen, Carlsbad, CA </td><td> R1244 </td></tr><tr><td> PLURONIC F127 </td><td> Invitrogen, Carlsbad, CA </td><td> P3000MP </td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Sigma, St. Louis, MO </td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

O coração do rato é um modelo popular para estudos cardiovasculares, devido à existência de tecnologia de baixo custo para a engenharia genética nesta espécie. Fenotipagem fisiológicas cardiovasculares do coração do mouse pode ser feito facilmente usando imagens de fluorescência empregando várias sondas para potenciais transmembrana (V m), transientes de cálcio (CAT), e outros parâmetros. Excitação-contração de acoplamento é caracterizado pela dinâmica do potencial de ação e intracelular de cálcio e, portanto, é extremamente importante para mapear tanto m V e CAT simultaneamente a partir do mesmo local no coração 1-4. Mapeamento óptica simultânea de Langendorff perfundidos corações do rato tem o potencial para elucidar os mecanismos subjacentes insuficiência cardíaca, arritmias, doença metabólica e outras doenças cardíacas. Visualização de ativação, velocidade de condução, a duração do potencial de ação, e outros parâmetros em uma infinidade de sites não pode ser alcançado a partir de investigação nível celular, mas é bem resolvido pelo mapeamento óptico 1,5,6. Neste artigo apresentamos a configuração de instrumentação e das condições experimentais para o mapeamento de óptica simultânea de V m e CAT nos corações do rato com a resolução espaço-temporal de alta usando state-of-the-art tecnologia de imagens CMOS. Consistente gravações ópticas obtidas com este método ilustrar que o mapeamento simultâneas óptico de corações de camundongos perfundidos Langendorff é viável e confiável.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Este artigo detalha o procedimento de dissecação, a configuração instrumental, e as condições experimentais durante o mapeamento óptico de potencial transmembrana (Vm) e transientes de cálcio intracelular (CAT) em Langendorff isolados intactos corações perfundidos mouse.

Mapeamento de potenciais de ação óptica e transientes de cálcio no coração de rato

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

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27.0K visualizações.  Washington University in St. Louis. Este artigo detalha o procedimento de dissecação, a configuração instrumental, e as condições experimentais durante o mapeamento óptico de potencial transmembrana (Vm) e transientes de cálcio intracelular (CAT) em Langendorff isolados intactos corações perfundidos mouse.

Vídeo: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional Optical resolução Microscopia Fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengenharia

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico Mapping Optical do Coração Coelho Langendorff-perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox Abordagem Recombinação P para Detecção de fusão celular In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Em síntese in vitro de Nativo, Espaçamento fibroso Long e Colágeno Espaçamento segmentar Longo

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A detecção óptica de<em&gt; E. coli</em&gt; Bactérias por mesoporosos Silicon Biossensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Microscopia de fluorescência de luz-folha para capturar imagens 4-Dimensional dos efeitos de modulação de tensão de cisalhamento sobre o coração em desenvolvimento de Zebrafish

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia de fluorescência de luz-folha para o estudo do coração murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxial caracterizações mecânicas das válvulas cardíacas Atrioventricular

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...