This work was supported in part by the US National Institutes of Health (R01 HL 111600) and the American Heart Association (12SDG9010015).

Todos los procedimientos con animales fueron aprobados por el Comité de Cuidado y Uso de Animales de la Universidad de California, Davis, y se adhirieron a la Guía para el Cuidado y Uso de Animales de Laboratorio publicado por los Institutos Nacionales de Salud. 1. Preparación <ol><li> Prepare dos concentrados (25X) los stocks de solución de Tyrode modificada por adelantado y se almacena a 4 ° C: (1) de la I (en mM: NaCl 3205, CaCl2 32,5, KCl 117.5, NaH 2 PO 4 29.75, MgCl2 26.25) y (2) de la II (en mM: NaHCO3 500). <ol><li> Recién preparar la solución de 2 L Tyrode de la siguiente composición (en mM): NaCl 128,2, CaCl 2 1,3, KCl 4,7, MgCl 2 1,05, NaH 2 PO 4 1,19, NaHCO3 20 y glucosa 11,1 mediante la combinación de 1840 ml de agua desionizada (DI) agua, 80 ml Stock I, 80 ml de Stock II, y 4 g de glucosa. No mezclar directamente de la I y II de la; añadir elm a 1840 ml DI para evitar la precipitación. </li></ol></li><li> Preparar la solución madre del desacoplador blebbistatin excitación-contracción en 10 mg / ml en anhidro di-metilsulfóxido (DMSO) con antelación y almacenar la solución madre a 4 ° C. </li><li> Preparar soluciones madre de colorantes fluorescentes: (1) RH237 sensibles al voltaje colorante (1 mg / ml en DMSO) y (2) indicador de calcio de baja afinidad Fluo-5N AM (2 mg / ml en DMSO) de acuerdo con las recomendaciones del fabricante. Utilice los preparativos de inmediato para evitar la descomposición, con la consiguiente pérdida de capacidad de carga. </li><li> Cebe la recirculación sistema de perfusión Langendorff con oxigenada (95% O 2, 2 5% de CO) solución de Tyrode. Ajustar el flujo de O 2 / CO 2 para mantener el pH de la solución de Tyrode a 7,4 ± 0,05. Utilice un filtro de red en línea de nylon tejido (tamaño de poro: 11 micras) para filtrar continuamente el líquido de perfusión. <ol><li> Cebe el sistema de perfusión a temperatura ambiente, como la posterior loading de Fluo-5N AM se lleva a cabo a temperatura ambiente. Para Fluo-5N AM carga, utilice un pequeño volumen de líquido de perfusión de recirculación (~ 150 ml). Después de colorante de carga, utilizar un volumen mayor de líquido de perfusión (1 - 2 L, véase la Figura 1C). </li></ol></li><li> Encienda el sistema de adquisición de datos y prepararse para la monitorización continua de la presión de ECG y la perfusión. Prepara sistema de presión-vigilancia: Conecte un transductor de presión a la línea de perfusión y monitorear la presión de perfusión con un amplificador transbridge. Ajuste la presión de perfusión basal a 0 mmHg cuando el corazón no está conectado al sistema de perfusión. </li><li> Alinear las cámaras de mapeo óptico para asegurar la alineación espacial entre el V my SR Ca 2 + señales. <ol><li> En primer lugar, se centran las cámaras mediante la colocación de un gobernante o un objeto con el texto en el plato de la perfusión. A su vez las cámaras de mapeo en un modo de visualización en directo y ajustar el enfoque hasta que el texto es nítido y claro. Adquirir una sola imagen Marco de cada cámara. </li><li> Superposiciónestas imágenes y ajustar la transparencia de la imagen de arriba, así que ambas imágenes son visibles. El texto en las imágenes debe superponerse con exactitud. Si no perfectamente alineados, ajustar el ángulo del espejo dicroico o la posición de cada cámara hasta que se alinean las dos imágenes. </li></ol></li></ol> 2. La recolección, perfusión y tinte de carga del corazón de conejo <ol><li> Asegure el conejo en un retenedor de conejo aprobado. Profundamente anestesiar a través de una inyección intravenosa (vena marginal de la oreja) de pentobarbital sódico (50 mg / kg) y heparina (1000 IU). <ol><li> Cuando los conejos pantallas carecen de reflejos nociceptivos, hacer una incisión en la piel en la línea media para exponer el esternón y las costillas. Corte a través del esternón con-contundentes punta tijeras quirúrgicas desde el xifoides al manubrio. Tenga cuidado de no dañar el corazón al abrir el esternón. Difundir las costillas para exponer el corazón. </li><li> Extirpar rápidamente todo el bloque corazón-pulmón cortando rápidamente todos los vasos y tejido conectivo. Immediatamente colocar el bloque corazón-pulmón en solución enfriada con hielo de Tyrode. </li></ol></li><li> Localice la aorta para la perfusión retrógrada. Cortar la aorta ascendente proximal a las tres ramas del cayado aórtico. Canular la aorta a una cánula de 8 G conectado al sistema de perfusión. Use un pedazo de sutura USP 0 seda para asegurar la aorta en la cánula. </li><li> Diseccionar cuidadosamente la tráquea, los pulmones y la grasa epicárdica del corazón. Con un fuerte pinzas y tijeras de disección, localizar la válvula mitral y cuidadosamente dañar o quitar un folleto para evitar la congestión de la solución en el ventrículo izquierdo. </li><li> Sumergir el corazón en la cámara de perfusión con camisa de vidrio horizontalmente con la superficie anterior del corazón hacia arriba para formación de imágenes. Fije la cánula a una pieza de Sylgard con los pernos en forma de U en la parte inferior del plato de silicio para evitar el movimiento del corazón durante el mapeo (Figura 1D). Si lo desea, inserte un alfiler pequeño insecto en el ápice del corazón por stabilización. </li><li> Posición electrocardiograma (ECG) electrodos en el baño en el lado derecho e izquierdo del corazón. Sumergir totalmente los electrodos en el baño y asegurarse de que no están en contacto con la superficie del corazón para proporcionar un ECG volumen realizado análoga a una configuración de derivación I. Verifique un plomo normal, me ECG morfología. Ajuste el flujo (25 - 35 ml / min) para asegurar que la presión aórtica se mantiene a 60 - 70 mmHg. </li><li> Apague las luces de la sala y añadir 0,3 a 0,6 ml de solución madre blebbistatin (paso 1.2) a la perfusión (concentración final 10 a 20 M). <ol><li> Apague las luces de las habitaciones para evitar fotoinactivación de blebbistatin. Si es necesario, un pequeño proyector o faro se pueden usar para proporcionar iluminación de la tarea. Nota: blebbistatin es un inhibidor de la ATPasa de la miosina y un desacoplador excitación-contracción. Debido Fluo-5N carga requiere condiciones prolongados (60 min) RT (hipotermia) (consulte los pasos 2,8 - 2,10), la producción de energía cardiaca podría estar comprometida. Therefore, la adición de blebbistatin en el perfundido antes de colorante de carga puede reducir la demanda de energía 16 y eliminar los artefactos de movimiento durante las grabaciones ópticas posteriores 17. </li></ol></li><li> Cuando la contracción del corazón ha cesado (10 - 15 min, verificado en el registro de la presión aórtica), cambiar al sistema de perfusión de recirculación de pequeño volumen (vea el paso 1.4.1). </li><li> Preparar y cargar solución de carga Fluo-5N AM: Añadir 0,25 ml de Fluo-5N AM solución madre (paso 1.3) a 0,25 ml cálida 20% F127 Pluronic. Añadir 0,5 ml de solución caliente de Tyrode a la mezcla, mezclar bien y agregar al sistema de perfusión de recirculación de pequeño volumen. </li><li> Monitorear continuamente la presión de perfusión y ECG y ajustar el flujo si es necesario. Colorante de carga dura aproximadamente 1 hora a temperatura ambiente. 45 min después de la carga ha comenzado, encienda el baño de agua circulante para comenzar a calentar el sistema de perfusión y la perfusión a 37 ° C. </li><li> Después de 60 minutos de Fluo-5N AM carga, switch la perfusión para el sistema de perfusión recirculante volumen más grande (véase el paso 1.4). </li><li> Diluir 50 l de solución de colorante sensible al voltaje de la RH237 (etapa 1.3) en 1 ml de solución caliente de Tyrode y se añade lentamente (más de ~ 5 min) en un puerto de inyección proximal a la cánula. </li></ol> 3. Mapeo óptico <ol><li> Durante los momentos finales de colorante de carga, coloque el corazón y el enfoque de las cámaras de mapeo óptico para asegurar el campo apropiado de vista para el experimento. </li><li> Si lo desea, coloque un electrodo de estimulación bipolar en la superficie epicárdica del corazón para la estimulación. </li><li> Coloque un plástico o de vidrio cubierta de resbalón en la superficie de la cámara de perfusión para reducir los artefactos de movimiento en la superficie del líquido que puede estar presente debido a la recirculación del líquido de perfusión. Nota: Como alternativa a una hoja de cubierta, utilice una cubierta de plástico de cocina limpio 50 mm Petri. </li><li> Centrarse las guías de luz para iluminar uniformemente la superficie del corazón con el correoluz xcitation. Utilice azul diodo emisor de luz (LED) fuentes de luz y filtrar aún más la luz con un 475 a 495 nm filtro de banda (Figura 1). </li><li> Recoger fluorescencia emitida con un macroscopio y dos de metal-óxido-semiconductor (CMOS) cámaras de mapeo óptico complementarias. Dividir la luz emitida con un espejo dicroico a 545 nm. <ol><li> -Pass largo filtrar la fracción de longitud de onda más larga, que contiene la señal m V, a 700 nm. De paso de banda filtrar la fracción de longitud de onda más corta, que contiene la señal SR Ca 2 +, 502 a 534 nm (Figura 1A). Ajuste la frecuencia de adquisición de datos ópticos de 0,5 a 1 kHz. </li></ol></li><li> Para cada grabación óptica, primero iniciar el protocolo de estimulación deseada, encender la luz de excitación, y recoger 1-4 seg de datos. Si lo desea, sincronizar el protocolos de estimulación y adquisición de datos. Nota: No es posible volver a cargar el Fluo-5N AM, por lo tanto la relación señal a ruido (SNR) se decreaSE lo largo del experimento debido a teñir fuga de la SR. Limite protocolo experimental a 1 - 2 horas para asegurar altos valores de SNR. </li><li> Tras el experimento, lavar el sistema de perfusión en la secuencia de agua DI, 70% de alcohol reactivo, y nuevamente con agua DI. </li><li> Filtra cada conjunto de datos con un filtro gaussiano espacial (radio de 3 píxeles) utilizando paquetes de software disponibles comercialmente de acuerdo con el protocolo del fabricante. </li><li> Si es necesario, espacialmente alinear el V my SR conjuntos de datos Ca 2+. Como en el paso 1.6, superponer imágenes de cada cámara y verificar que las estructuras anatómicas del corazón (es decir., Vasculatura coronaria, bordes de las aurículas o ventrículos) u otros artículos en el campo de visión (es decir, la cánula, el electrodo de estimulación) exactamente solapamiento. Si no, la alineación espacial de los conjuntos de datos es necesario. </li><li> Cambie la imagen transparente superior se logra en las direcciones x e y-dirección hasta que la superposición exacta, tomando nota del número de píxelesla imagen debe ser desplazado en cada dirección. Todo el conjunto de datos correspondiente a la parte superior de la imagen (ya sea V m o SR Ca 2+) debe entonces ser desplazado por el número determinado de píxeles en cada dirección para asegurar la alineación precisa entre el V y m SR Ca 2+ conjuntos de datos. </li></ol>

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The authors declare that they have no competing financial interests.

Las claves del éxito Fluo-5N colorante de carga son la configuración de perfusión de recirculación de pequeño volumen, lo que permite una alta concentración Fluo-5N sin la necesidad de grandes cantidades de colorante, la longitud de tiempo de carga (1 hr), y la realización de la carga a TA. Si la carga se realiza a temperaturas fisiológicas, la actividad enzimática celular rápidamente escinde la etiqueta -AM cuando el colorante atraviesa la membrana celular, atrapando las moléculas de colorante en el citosol ...

Dual optical mapping of intracellular Ca2+ and transmembrane potential (Vm) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between Vm and intracellular Ca2+. However, optical mapping of intracellular Ca2+ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca2+ and is unable to distinguish whether these changes are due to transmembrane Ca2+ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca2+ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca2+ concentration5.
Each action potential triggers a rise in intracellular Ca2+, known as the intracellular Ca2+ transient (CaT). In the mammalian heart, approximately 70 &#8211; 90% of the total CaT is due to release of Ca2+ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)6. Within the SR, approximately half of the total Ca2+ is bound to calsequestrin (CSQ) and other intra-SR buffers7, which play an important role in SR Ca2+ homeostasis8,9. The amount of free SR Ca2+ dictates the driving force for SR Ca2+ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca2+ release or reuptake can, in turn, impact Vm via the electrogenic Na+-Ca2+ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca2+ can provide important insights into contractile and electrophysiological dysfunction.
Over the past several years, investigators have made significant advances in the monitoring of SR Ca2+ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca2+ content is then inferred or calculated from the immediate rise in intracellular Ca2+10. Another intriguing approach uses low-affinity Ca2+ indicators, such as Fluo-5N11 or Mag-Fluo412, which bind to free SR Ca2+. These indicators have dissociation constants (Kd) in the range of 10 &#8211; 400 &#956;M and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ concentration ([Ca2+]SR) is in the millimolar range. Using low-affinity Ca2+ indicators, several aspects of SR Ca2+ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca2+ release and the mechanisms of Ca2+ alternans13,14. However, in order to fully understand the heterogeneous nature of SR Ca2+ cycling in the intact heart and the role of SR Ca2+ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca2+ across the epicardial surface of the intact heart are required15.
This article describes methodology for dual optical mapping of free SR Ca2+ and Vm in the intact Langendorff-perfused rabbit heart with the low-affinity Ca2+ indicator Fluo-5N. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S271-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">CaCl2 (2H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">C79-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">KCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S217-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MgCl2 (6H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">M33-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaH2PO4 (H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S369-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaHCO3</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S233-3</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">D-Glucose</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">D16-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">95% O2 5% CO2</td>
			<td style="width:167px;">AirGas</td>
			<td style="width:151px;">carbogen</td>
			<td style="width:423px;">For oxygenation and pH of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Blebbistatin</td>
			<td style="width:167px;">Tocris Bioscience</td>
			<td style="width:151px;">1760</td>
			<td style="width:423px;">Excitation-contraction uncoupler</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">RH237</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">61018</td>
			<td style="width:423px;">Voltage-sensitive dye</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Fluo-5N AM</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:151px;">F-26915</td>
			<td style="width:423px;">Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pluronic F127</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">59004</td>
			<td style="width:423px;">For Ca2+ indicator loading; Warm until the solutiion is clear before use</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td style="width:423px;">For dissolving blebbistatin and dyes</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Filter</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:151px;">NY1104700</td>
			<td style="width:423px;">11um in-line filter</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pressure Transducer</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">BLPR2</td>
			<td style="width:423px;">For measuring perfusion pressure</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Transbridge Transducer Amplifier</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">SYS-TBM4M</td>
			<td style="width:423px;">For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">PowerLab 26T</td>
			<td style="width:167px;">ADInstruments</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">For continuous recording of pressure and ECG signals</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">THT Macroscope</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1x3.1 cm, depth of focus = ~1.5mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MiCam Ultima-L CMOS</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Optical mapping cameras</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Precision LED Spot Light</td>
			<td style="width:167px;">Mightex</td>
			<td style="width:151px;">PLS-0470-030-15-S</td>
			<td style="width:423px;">LED light source</td>
		</tr>
	</tbody>
</table>

optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

La figura 1A muestra un diagrama esquemático de la configuración óptica para la doble V m y SR Ca 2+ mapeo. Con esta configuración, no hay separación espectral completa de la V my SR Ca 2 + señales (Figura 1B). Un diagrama del sistema de perfusión de doble bucle utilizado para Fluo-5N colorante de carga se ilustra en la Figura 1C. Figura 1D muestra la orientación horizontal del corazón en el plato de ...

Watch this Scientific Journal Video about Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart at JoVE.com

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

En este artículo se describe el protocolo detallado y equipos necesarios para el mapeo óptico dual de potencial transmembrana (V m) y libre retículo intra-sarcoplásmico (SR) Ca 2+ en el corazón de conejo perfundidos Langendorff. Este método permite la observación directa y la cuantificación de V m y SR Ca 2+ dinámica en el corazón intacto.

Mapeo óptico de Intra-retículo sarcoplásmico Ca<sup&gt; 2 +</sup&gt; Y potencial transmembrana en el corazón de conejo perfundidos Langendorff

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

optical-mapping-intra-sarcoplasmic-reticulum-ca2-transmembrane

Research

JoVE Journal

Biology

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

9.2K vistas.  University of California Davis.  En este artículo se describe el protocolo detallado y equipos necesarios para el mapeo óptico dual de potencial transmembrana (V m) y libre retículo intra-sarcoplásmico (SR) Ca 2+ en el corazón de conejo perfundidos Langendorff. Este método permite la observación directa y la cuantificación de V m y SR Ca 2+ dinámica en el corazón intacto. 

Video: Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Limpieza y cuidado apropiado del microscopio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Investigación

Biología

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Mapeo óptico de corazones de ratas Langendorff perfundidos

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automatizado de análisis óptico latido de los corazones pequeños

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

La cuantificación de γH2AX focos en muestras de tejido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Evaluación de la distribución espacial de la radiación ionizante γH2AX siguientes

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.