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1. Mica y Preparación Diapositivas <ol><li> Lugar N º 1 ½ (0,17 mm) en cubreobjetos gradilla de tinción. </li><li> Someter a ultrasonidos durante 30 min en 2% de detergente a 60 ° C. </li><li> Lavar 20 veces con agua desionizada. </li><li> Retirar los portaobjetos usando pinzas y secar con aire comprimido o nitrógeno. </li><li> Cortar la hoja de mica en 10 x 10 mm piezas cuadradas con unas tijeras o cuchilla de afeitar. </li><li> Corte cada pedazo de mica en 2-3 foliolos delgados utilizando una hoja de afeitar. NOTA: Este paso requiere el uso de la hoja afilada. </li></ol> 2. Asamblea Mica y la Cámara de montaje <ol><li> Limpio portaobjetos de vidrio microscópico con etanol. </li><li> Pegamento folleto de mica cortado en el paso de 1,6 a portaobjetos de vidrio utilizando adhesivo óptico. Se aconseja adhesivo de baja viscosidad, para difundir mejor la caída y la cola de la mica. </li><li> Curar bajo la lámpara UV, 10 min. NOTA: El adhesivo se curó con luz UV con un máximo de absorción en el intervalo de 350 a 380 nm y la energía recomendada required para el curado total es de 4,5 J / cm 2. Sin embargo, diferentes fuentes de luz se pueden utilizar para este paso (ver materiales proporcionados por proveedor del adhesivo). </li><li> Exponer una superficie de mica limpio mediante la eliminación de las primeras capas con cinta Scotch. </li><li> Coloque una pequeña gota (~ 20 m) de adhesivo óptico sobre la superficie de mica. NOTA: En este paso, se aconseja alto pegamento viscosidad con bajo nivel de autofluorescencia. Nuestra experiencia ha demostrado que el uso de combinación de baja (paso 2.2) y los adhesivos de alta viscosidad aumenta significativamente la eficacia de la escisión mica (paso 2.7). </li><li> Coloque suavemente el cubreobjetos recién limpiada en la gota de adhesivo, evitando las burbujas de aire, dejar reposar durante 1 minuto. </li><li> Curar bajo la lámpara UV, 10 min. NOTA: después de este paso, el sándwich de lámina de vidrio, mica y cubreobjetos se puede almacenar durante un período relativamente largo de tiempo (hasta varias semanas). Continúe con el siguiente paso justo antes de la preparación efectiva SLB, para asegurarse de que la superficie de la mica es fresco. NOTA: Adhesivo se cura por la luz UV con la máxima absorción en el intervalo de 350 a 380 nm y la energía recomendada necesaria para el curado completo es 4.5J/cm 2. Sin embargo, diferentes fuentes de luz se pueden utilizar para este paso (ver materiales proporcionados por proveedor del adhesivo). </li><li> Usando un cuchillo exacto, desmontar suavemente el cubreobjetos de portaobjetos de vidrio lateral como se muestra en el video. En la mayoría de los casos, una capa delgada y plana de la mica permanecerá unido a la cubreobjetos. Mica adjunta a portaobjetos de vidrio pueden ser reutilizados (repita desde el paso 2.4). </li><li> Compruebe la calidad de la superficie a simple vista o bajo un microscopio de disección, para asegurarse de que la capa de mica está todavía pegada a la cubreobjetos y no se ha eliminado completamente durante la división en el paso 2.8. Haciendo un pequeño rasguño con una pinza o aguja de disección ayudará a distinguir entre el adhesivo que tiene una consistencia de forma detectable diferente de mica. </li><li> Retire el sello de goma de un ml frasco tapa 1,5 y pegar la tapa boca abajo a la superficie utilizando ópticaadhesivo o esmalte de uñas, y curar con lámpara UV, o dejar secar al aire 10 minutos, respectivamente. NOTA: Si se prepara la muestra para su uso simultáneo óptica (TIRFM o confocal) con imágenes de AFM, un ml frasco tapa 1.5 puede ser demasiado pequeño para montar la cabeza AFM en la platina del microscopio. En ese caso, la tapa podría ser sustituida por cualquier junta tórica de plástico con un diámetro más grande, adecuada para el montaje de la cabeza de AFM. En caso de que el experimento requiere quitar la tapa sin dañar la superficie de la mica, grasa de silicona se puede utilizar en lugar de pegamento o esmalte de uñas. </li></ol> 3. Apoyado bicapa lipídica (SLB) Formación <ol><li> Coloque solución de liposomas recién preparada en la cámara con la superficie. El volumen mínimo requerido para la formación de SLB es ~ 30 l. NOTA: Para más detalles sobre el liposoma y la formación de SLB, se refieren a los protocolos publicados, como por ejemplo, bolsa y otros, 2014 15.. </li><li> Proceda con la formación de SLB con el protocolo deseado. Durante la incubación yde formación de imágenes, la cámara se puede colocar en un bloque de calor o platina del microscopio calentado para mantener la temperatura necesaria para mantener los lípidos que se utilizan por encima de su temperatura de fusión. </li></ol>

<ol>
	<li>Giocondi, M. -. C., 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=Surface+topography+of+membrane+domains.">Surface topography of membrane domains.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 703-718 (2010).</li><li>Garcia-Saez, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+scanning+probe+and+total+internal+reflection+fluorescence+microscopy.">Combined scanning probe and total internal reflection fluorescence microscopy.</a> Methods. 46, 2-10 (2008).</li><li>Shaw, J. E., Slade, A., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+total+internal+reflectance+fluorescence/atomic+force+microscopy+studies+of+DPPC/dPOPC+microdomains+in+supported+planar+lipid+bilayers.">Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.</a> J Am Chem Soc. 125, 11838-11839 (2003).</li><li>Skaug, M. J., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+anomalous+diffusion+with+lipid+bilayer+membrane+structure+using+single+molecule+tracking+and+atomic+force+microscopy.">Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy.</a> J Chem Phys. 134, (2011).</li><li>Bag, N., Yap, D. H., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+diffusion+in+model+and+live+cell+membranes+characterized+by+imaging+fluorescence+correlation+spectroscopy.">Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy.</a> Biochimica et Biophysica Acta. , (2013).</li><li>Sbalzarini, I. F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> J Struct Biol. 151, 182-195 (2005).</li><li>Linkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li><li>Schutz, G. J., Schindler, H., Schmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+microscopy+on+model+membranes+reveals+anomalous+diffusion.">Single-molecule microscopy on model membranes reveals anomalous diffusion.</a> Biophys J. 73, 1073-1080 (1997).</li><li>Matysik, A., Kraut, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackArt:+the+user+friendly+interface+for+single+molecule+tracking+data+analysis+and+simulation+applied+to+complex+diffusion+in+mica+supported+lipid+bilayers.">TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.</a> BMC Research Notes. 7, (2014).</li></ol>

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Este protocolo describe un método para la preparación de superficies lisas y finas de mica para la deposición de bicapa de lípidos y formación de imágenes de alta resolución. La técnica requiere habilidades manuales mínimas, limitadas principalmente a la desmontaje cuidadoso de la sándwich de vidrio-mica y vidrio (paso 2.8), que es crítico para la obtención de una superficie de mica de alta calidad. Inspección de la mica recién escindido siempre se requiere en este punto, ya que es posible para la mica se ...

El objetivo general del presente protocolo es mostrar un método para la preparación de superficies de mica de alta resolución de imagen de mica apoyados bicapas lipídicas (SLBS) mediante microscopía óptica total de fluorescencia de reflexión interna (TIRFM) o microscopía confocal, que también se podría combinar con la fuerza atómica microscopía (AFM). SLBS son un modelo ampliamente utilizado para numerosos estudios de la agrupación de lípidos, la separación de fases, la dinámica de componentes de la bicapa o sus interacciones con péptidos, proteínas u otros compuestos 1-5. Diferentes sustratos pueden ser utilizados para la formación de SLB (es decir, vidrio, mica, dióxido de silicio, polímeros) dependiendo de la naturaleza del estudio 4,6-8. Estudios de membrana típicos se basan en técnicas de formación de imágenes basada en microscopía, tales como TIRFM y AFM. Por lo tanto, para obtener imágenes TIRFM, una superficie de vidrio es una opción típica porque el vidrio es transparente. Preparación de vidrio es relativamente fácil, y la calidad de los resultados es principalmentedeterminado por la superficie de limpieza a fondo antes de la deposición de las vesículas de lípidos. AFM, debido a su elevada resolución axial requiere superficies de mica. La mica es un mineral de silicato, con cerca de clivaje basal perfecto. Así, el recién exfoliados mica es atómicamente plana, lo que permite la observación de las diferencias de altura de la membrana, incluso a escala sub-nanométrica 9. Los estudios de difusión utilizando métodos tales como la espectroscopia de correlación de fluorescencia (FCS), sola molécula de seguimiento (SMT), y la recuperación de la fluorescencia después de photobleaching (FRAP) mostraron sin embargo, que la dinámica de la membrana de lípidos dependen en gran medida del tipo de superficie sobre la que se depositan, en el que el vidrio y la mica puede dar resultados muy variables 10,11. Estas diferencias incluyen no sólo los coeficientes de difusión de las sondas de membrana, sino también la detección de poblaciones separadas de las partículas que se difunden con diferentes tasas, y posiblemente la conmutación entre diferentes estados. Por lo tanto,la comparación directa de los resultados obtenidos utilizando técnicas TIRFM y AFM es a menudo problemática, a menos que la misma superficie (en este caso la mica) se utiliza. Aunque hay algunos estudios en los que se llevó a cabo TIRFM y AFM de imágenes bicapa en la misma superficie de mica 12,13, mica se utiliza muy poco para la microscopía óptica, sobre todo debido a problemas de manejo. Preparación Mica requiere de corte a mano en folletos delgadas, que luego se pegan a la cubreobjetos usando adhesivo óptico 12. Este método, sin embargo requiere un poco de práctica para lograr resultados satisfactorios. Además, las superficies obtenidas son a menudo ondulado y grueso, lo que dificulta su uso con distancia de trabajo bajo, lentes de alta apertura numérica. Superficies de mica preparados como se describe en este protocolo son muy delgadas (~ 220 m, incluyendo el espesor cubreobjetos de 170 m) y extremadamente plana, evitando &quot;ondulación&quot;, que es fundamental para la imagen de alta resolución con éxito. Se pueden utilizarpara TIRFM o confocal configuraciones. Por otra parte, las mismas muestras se pueden transferir a AFM, e incluso reflejados simultáneamente con TIRFM / confocal y AFM. La combinación de estas dos técnicas permite la correlación directa de comportamiento de la difusión con la estructura 14 de la membrana bicapa. Debido a que las superficies de mica están recién troceados, que estén limpias y no requieren mucho tiempo, poco reproducibles, y los procedimientos de limpieza potencialmente peligrosos (protocolos de limpieza de vidrio por lo general incluyen productos químicos como solución Piranha, ácido sulfúrico, hidróxido de sodio / potasio). Montaje de una pequeña cámara, también se describe en el protocolo, reduce el volumen de vesículas necesarios para la formación de bicapa eficaz a menos de 50 l. Finalmente, todo el proceso de montaje de superficie no es mucho tiempo (preparación tarda menos de 30 min), y no requiere un alto grado de habilidad manual, al igual que la escisión de mica convencional y encolado.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bath Sonicator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">FB15051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Coverslips 24 x 50mm - No H1.5</td>
			<td style="width:135px;">Marienfeld</td>
			<td style="width:119px;">102222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DOPC</td>
			<td style="width:135px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hellmanex III (detergent)</td>
			<td style="width:135px;">Hellma Analytics</td>
			<td style="width:119px;">320.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mica V-1 Grade</td>
			<td style="width:135px;">SPI Suppliers</td>
			<td style="width:119px;">1872-CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (high viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA63</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (low viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphingomyelin-ATTO647N</td>
			<td style="width:135px;">AttoTec</td>
			<td>AD 647N-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">UV lamp</td>
			<td style="width:135px;">Synoptics Ltd.</td>
			<td style="width:119px;">GelVue GVM20</td>
			<td>The lamp was set to 100% power</td>
		</tr>
	</tbody>
</table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

Bicapas lipídicas soportadas (SLBS) son ampliamente utilizados como modelo para el estudio de propiedades de la membrana (separación de fases, la agrupación, la dinámica) y su interacción con otros compuestos, como los fármacos o péptidos. Sin embargo las características SLB difieren dependiendo del soporte utilizado. Técnicas utilizadas comúnmente para SLB óptica y mediciones son microscopía de fluorescencia de una sola molécula, de FCS y microscopía de fuerza atómica (AFM). Debido a que la mayoría de los estudios de formación de imágenes ópticas se llevan a cabo en un soporte de vidrio, mientras que AFM requiere una superficie extremadamente plana (generalmente de mica), los resultados de estas técnicas no se pueden comparar directamente, ya que las propiedades de carga y la suavidad de estos materiales influyen fuertemente en la difusión. Desafortunadamente, el alto nivel de destreza manual necesaria para el corte y pegado de láminas finas de mica para el portaobjetos de vidrio presenta un obstáculo para el uso rutinario de la mica para la preparación SLB. Aunque esto sería el método de elección, tales mica preparadosuperficies con frecuencia terminan siendo desigual (ondulado) y difícil de la imagen, especialmente con la pequeña distancia de trabajo, lentes de alta apertura numérica. Aquí presentamos un método simple y reproducible para la preparación de superficies planas delgadas de mica para la deposición de vesículas de lípidos y preparación SLB. Además, nuestra cámara por encargo requiere sólo pequeñas cantidades de vesículas para la formación de SLB. Los resultados del procedimiento general en la producción eficiente, simple y barato de superficies bicapa de lípidos de alta calidad que son directamente comparables a los utilizados en los estudios de AFM.

El comportamiento de la difusión de sondas fluorescentes de lípidos en SLBS es diferente dependiendo del sustrato. TIRFM combinado con la técnica SMT es un método valioso para la visualización de movimientos de partículas y la extracción de sus coeficientes de difusión. Señales de moléculas individuales de una sonda de esfingomielina-ATTO647N de difusión en un DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) bicapa apoyado sobre el vidrio y mica se muestran en la figura animada adjunto. La superficie de ...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Presentamos un método de preparación de mica apoyado bicapas lipídicas de microscopía de alta resolución. La mica es transparente y plana en una escala atómica, pero rara vez se utiliza en la formación de imágenes debido a dificultades de manejo; nuestra preparación resulta en la deposición uniforme de la hoja de mica, y reduce el material utilizado en la preparación bicapa.

Preparación de Mica Soportado bicapa lipídica de Alta Resolución Microscopía Óptica de Imagen

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

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JoVE Journal

Bioengineering

13.3K vistas.  Nanyang Technological University.  Presentamos un método de preparación de mica apoyado bicapas lipídicas de microscopía de alta resolución. La mica es transparente y plana en una escala atómica, pero rara vez se utiliza en la formación de imágenes debido a dificultades de manejo; nuestra preparación resulta en la deposición uniforme de la hoja de mica, y reduce el material utilizado en la preparación bicapa. 

Video: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy 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 resolución de fibra óptica para Microendoscopy In situ Celular imágenes

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

Investigación

Bioingeniería

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 óptico resolución microscopía fotoacústica

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

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Banda ancha Detector óptico de ultrasonido para aplicaciones de imágenes médicas

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolución espacio-temporal Análisis de la Dinámica del receptor por Casa molécula de microscopía de fluorescencia

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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.

Fluorescencia de extinción de un fluoróforo encapsulada en liposomas en infrarrojo cercano como herramienta para<em&gt; En Vivo</em&gt; Imagen 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 ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Fuerza Atómica Microscopía Imaging y Fuerza Espectroscopia de admitidos Bilayers lípidos

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bilayers Individual Molécula microscopía de fluorescencia en planar compatibles

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Arrays Ligando Nano-racimo en un Apoyado bicapa lipídica

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Microscopía fotoacústica de resolución óptica y acústica conmutable para<em&gt; In Vivo</em&gt; Imágenes de vasos sanguíneos de pequeños animales

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.