Les auteurs n&#39;ont aucun remerciements.

1. Mica et diapositives Préparation <ol><li> Lieu n ° 1 ½ (0,17 mm) des lamelles couvre en support de coloration. </li><li> Ultrasons pendant 30 min à 2% de détergent à 60 ° C. </li><li> Laver 20 fois avec de l&#39;eau désionisée. </li><li> Retirer les lames en utilisant des pinces et sécher à l&#39;air comprimé ou de l&#39;azote. </li><li> Couper la feuille de mica en 10 x 10 mm morceaux carrés à l&#39;aide de ciseaux ou d&#39;une lame de rasoir. </li><li> Couper chaque morceau de mica en 2-3 folioles minces en utilisant une lame de rasoir. NOTE: Cette étape nécessite l&#39;utilisation de lame tranchante. </li></ol> 2. Assemblée Mica et de la Chambre de montage <ol><li> Clean lame de verre microscopiques avec de l&#39;éthanol. </li><li> Colle notice de mica couper à l&#39;étape 1.6 de lame de verre avec de la colle optique. Adhésif à faible viscosité est conseillé de mieux étaler la goutte et coller le mica. </li><li> Durcir sous la lampe UV, 10 min. NOTE: L&#39;adhésif est durci par la lumière UV avec une absorption maximale dans la plage de 350 à 380 nm et l&#39;énergie recommandé requIRED pour la guérison complète est de 4,5 J / cm 2. Cependant, les différentes sources de lumière peuvent être utilisés pour cette étape (voir documents fournis par le fournisseur de l&#39;adhésif). </li><li> Exposer une surface de mica nettoyer en enlevant les quelques premières couches avec du scotch. </li><li> Placer petite goutte (~ 20 um) d&#39;adhésif optique sur la surface du mica. NOTE: A ce stade, colle à haute viscosité avec le niveau d&#39;autofluorescence faible est conseillé. Notre expérience a montré que l&#39;utilisation de la combinaison de bas (étape 2.2) et les adhésifs haute viscosité augmente de manière significative l&#39;efficacité de mica fractionnement (étape 2.7). </li><li> Placer doucement la lamelle fraîchement nettoyée sur la goutte de colle, en évitant les bulles d&#39;air, laisser reposer pendant 1 min. </li><li> Durcir sous la lampe UV, 10 min. REMARQUE: après cette étape, le sandwich de lame de verre, le mica et la lamelle peut être stocké pendant une relativement longue période de temps (jusqu&#39;à quelques semaines). Passez à l&#39;étape suivante, juste avant la préparation SLB réelle, pour s&#39;assurer que la surface du mica est frais. REMARQUE: Adhésif est durci par la lumière UV avec une absorption maximale de l&#39;ordre de 350 à 380 nm et l&#39;énergie recommandé nécessaire pour la guérison complète est 4.5J/cm 2. Cependant, les différentes sources de lumière peuvent être utilisés pour cette étape (voir documents fournis par le fournisseur de l&#39;adhésif). </li><li> L&#39;aide d&#39;un couteau exacto, démonter doucement la lamelle de lame de verre de côté comme le montre la vidéo. Dans la plupart des cas, une couche mince et plat de mica restera attaché à la lamelle. Mica attaché à lame de verre peut être réutilisé (répéter l&#39;étape 2.4). </li><li> Vérifier la qualité de surface à l&#39;oeil nu ou sous un microscope à dissection, afin de s&#39;assurer que la couche de mica est encore collée sur la lamelle couvre-objet et n&#39;a pas été complètement enlevé au cours de fractionnement à l&#39;étape 2.8. Faire une petite égratignure avec une pince à dissection ou aiguille aidera à distinguer entre l&#39;adhésif qui a une consistance détectable différente de mica. </li><li> Retirer le joint en caoutchouc du flacon d&#39;un plafond de 1,5 ml et coller le bouchon à l&#39;envers à la surface à l&#39;aide optiqueadhésif ou vernis à ongles, et traitement avec la lampe UV, ou laissez sécher à l&#39;air 10 min, respectivement. NOTE: Si l&#39;échantillon est préparé pour une utilisation simultanée optique (TIRFM ou confocale) avec l&#39;imagerie AFM, un ml flacon plafond de 1,5 pourrait être trop petit pour monter la tête AFM sur la platine du microscope. Dans ce cas, le bouchon peut être substitué par un joint torique en plastique avec un diamètre plus grand, adapté pour le montage tête AFM. Dans le cas où l&#39;expérience, il faut enlever le bouchon sans endommager la surface de mica, de la graisse de silicone peut être utilisé à la place de la colle ou le vernis à ongles. </li></ol> 3. Pris en charge bicouche lipidique (SLB) Formation <ol><li> Placez solution de liposomes fraîchement préparé dans la chambre avec la surface. Le volume minimum requis pour la formation SLB est ~ 30 pl. NOTE: Pour plus de détails sur le liposome et formation SLB, se référer aux protocoles publiés, tels que tels que sac et al, 2014 15.. </li><li> Procéder à la formation SLB utilisant le protocole souhaité. Pendant l&#39;incubation etl&#39;imagerie, la chambre peut être placé dans un bloc thermique ou platine chauffante de microscope pour maintenir une température requise pour maintenir les lipides utilisés au-dessus de leur température de fusion. </li></ol>

<ol>
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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. 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Les auteurs n&#39;ont rien à révéler.

Ce protocole décrit un procédé pour la préparation de surfaces de mica lisses et minces pour le dépôt de la bicouche lipidique et l&#39;imagerie haute résolution. La technique nécessite des compétences manuelles minimales, limitées essentiellement à la démontage attention du sandwich verre-verre-mica (étape 2.8), ce qui est essentiel pour obtenir une surface de mica de haute qualité. L&#39;inspection du mica fraîchement clivé est toujours nécessaire, à ce stade, car il est possible que le mica se déta...

L&#39;objectif global du présent Protocole est de montrer une méthode pour la préparation des surfaces de mica pour l&#39;imagerie haute résolution de mica soutenu bicouches lipidiques (BLP) à l&#39;aide de la microscopie optique de fluorescence totale de réflexion interne (TIRFM) ou la microscopie confocale, qui pourrait également être combiné avec force atomique microscopie (AFM). BLP sont un modèle largement utilisé pour de nombreuses études de groupement lipidique, séparation des phases, la dynamique des composants de bicouche ou de leurs interactions avec des peptides, des protéines ou d&#39;autres composés 5.1. Différents substrats peuvent être utilisés pour la formation SLB (c.-à-verre, le mica, le dioxyde de silicium, des polymères) en fonction de la nature de l&#39;étude 4,6-8. Des études de membranes typiques reposent sur des techniques d&#39;imagerie à base de microscopie, tels que TIRFM et AFM. Ainsi, pour l&#39;imagerie par TIRFM, une surface de verre est un choix typique parce que le verre est transparent. Préparation de verre est relativement facile, et la qualité des résultats est essentiellementdéterminé par la surface de nettoyage en profondeur avant le dépôt de vésicules lipidiques. AFM en raison de sa haute résolution axiale nécessite des surfaces de mica. Mica est un minéral de silicate, avec près de parfait clivage basal. Ainsi, le mica fraîchement clivé est atomiquement plat, permettant l&#39;observation des différences de hauteur de la membrane même à l&#39;échelle sub-nanométrique 9. études sur la diffusion en utilisant des méthodes telles que la spectroscopie de corrélation de fluorescence (FCS), seul le suivi molécule (SMT), et la récupération de fluorescence après photoblanchiment (FRAP) ont montré cependant que la dynamique de la membrane lipidique dépendent fortement du type de surface sur laquelle ils sont déposés, lequel verre et le mica peut donner des résultats très différents 10,11. Ces différences comprennent non seulement les coefficients de diffusion des sondes membranaires, mais aussi la détection des populations distinctes de particules diffusantes avec des vitesses différentes, et éventuellement entre les différents états de commutation. Ainsi,la comparaison directe des résultats obtenus en utilisant des techniques de TIRFM et AFM est souvent problématique, à moins que la même surface (dans ce cas, mica) est utilisé. Bien qu&#39;il existe quelques études où TIRFM et AFM bicouche imagerie a été effectuée sur la même surface de mica 12,13, mica est rarement utilisé pour la microscopie optique, la plupart du temps en raison de problèmes de manutention. préparation de Mica nécessite la coupe à la main en folioles minces, qui sont ensuite collées sur la lamelle en utilisant un adhésif optique 12. Cette méthode nécessite cependant une certaine pratique pour obtenir des résultats satisfaisants. En outre, les surfaces obtenues sont souvent ondulés et épais, ce qui les rend difficiles à utiliser à faible distance de travail, des lentilles de grande ouverture numérique. Surfaces de mica préparés comme décrit dans ce protocole sont très minces (~ 220 um, y compris l&#39;épaisseur de la lamelle de 170 um) et extrêmement plat, en évitant &quot;ondulation&quot;, ce qui est essentiel pour la réussite de l&#39;imagerie de haute résolution. Ils peuvent être utiliséspour TIRFM ou confocale configurations. De plus, les mêmes échantillons peuvent être transférés à l&#39;AFM, et même imagées simultanément avec TIRFM / confocale et AFM. La combinaison de ces deux techniques permet une corrélation directe du comportement de diffusion avec la structure de membrane à double couche 14. Parce que les surfaces de mica sont fraîchement coupés, ils sont propres et ne nécessitent pas beaucoup de temps, peu reproductible, et les procédures de nettoyage potentiellement dangereuses (protocoles de nettoyage de verre sont habituellement des produits chimiques tels que solution Piranha, l&#39;acide sulfurique, hydroxyde de sodium / potassium). Montage d&#39;une petite chambre, également décrit dans le protocole, de réduire le volume des vésicules nécessaires à la formation de la bicouche efficace à moins de 50 pi. Enfin, l&#39;ensemble du processus de montage de surface n&#39;est pas de temps (préparation prend moins de 30 minutes), et ne nécessite pas un degré élevé d&#39;habileté manuelle, de même que le clivage du mica et de collage classique.

<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

Bicouches lipidiques supportées (BLP) sont largement utilisés comme un modèle pour l&#39;étude des propriétés de la membrane (de séparation de phase, regroupement, dynamique) et son interaction avec d&#39;autres composés, tels que des médicaments ou des peptides. Cependant caractéristiques SLB diffèrent selon le support utilisé. Techniques couramment utilisées pour l&#39;imagerie SLB seul et les mesures sont la microscopie de fluorescence de la molécule, FCS et microscopie à force atomique (AFM). Parce que la plupart des études d&#39;imagerie optique sont réalisées sur un support en verre, tandis que l&#39;AFM nécessite une surface extrêmement plate (généralement de mica), les résultats de ces techniques ne peuvent pas être comparés directement, étant donné que les propriétés de charge et la douceur de ces matériaux influencent fortement la diffusion. Malheureusement, le niveau élevé de la dextérité manuelle nécessaire pour le découpage et le collage de fines tranches de mica à la lame de verre présente un obstacle à l&#39;utilisation systématique de mica pour la préparation SLB. Bien que ce serait la méthode de choix, par exemple le mica préparésurfaces finissent souvent par être inégale (ondulé) et difficile à l&#39;image, en particulier avec la petite distance de travail, des lentilles de grande ouverture numérique. Nous présentons ici une méthode simple et reproductible pour la préparation minces, les surfaces de mica plats pour le dépôt de la vésicule lipidique et la préparation SLB. De plus, notre chambre sur mesure ne nécessite que de très petits volumes de vésicules de formation SLB. Les résultats de la procédure globale de la production efficace, simple et peu coûteux de surfaces de bicouche lipidique de haute qualité qui sont directement comparables à celles utilisées dans les études de l&#39;AFM.

Le comportement de diffusion de sondes lipidiques fluorescents dans BLP est différente en fonction du substrat. TIRFM combinée avec la technique SMT est une méthode utile pour visualiser les mouvements de particules et d&#39;extraire leurs coefficients de diffusion. Signaux de molécules simples d&#39;une sonde Sphingomyelin-ATTO647N diffusant dans un DOPC (1,2-dioléoyl-sn-glycéro-3-phosphocholine) bicouche supportée sur verre et le mica sont indiqués sur la figure ci-jointe animée. La surface de mica a...

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

Nous présentons une méthode de préparation de mica soutenu bicouches lipidiques pour microscopie à haute résolution. Mica est transparent et plat à l&#39;échelle atomique, mais rarement utilisé dans l&#39;imagerie en raison des difficultés de manipulation; notre préparation résulte en un dépôt régulier de la feuille de mica, et de réduire la matière utilisée dans la préparation de bicouche.

Préparation de Mica charge bicouches lipidiques pour haute résolution microscopie optique d&#39;imagerie

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

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13.3K vues.  Nanyang Technological University.  Nous présentons une méthode de préparation de mica soutenu bicouches lipidiques pour microscopie à haute résolution. Mica est transparent et plat à l'échelle atomique, mais rarement utilisé dans l'imagerie en raison des difficultés de manipulation; notre préparation résulte en un dépôt régulier de la feuille de mica, et de réduire la matière utilisée dans la préparation de bicouche. 

Vidéo: 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.

Haute résolution à fibre optique pour les Microendoscopy In situ Cellular Imaging

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

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Bio-ingénierie

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.

Tridimensionnelle optique résolution microscopie photoacoustique

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.

Wideband détecteur optique des ultrasons pour les applications d&#39;imagerie médicale

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.

Haute résolution spatio-temporelle d&#39;analyse de la dynamique des récepteurs par simple molécule microscopie de fluorescence

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.

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

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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.

Microscopie à force atomique imagerie et spectroscopie de force de bicouches lipidiques supportées

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 seule molécule microscopie à fluorescence sur Planar pris en charge

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.

Nano-Ligand Arrays cluster dans un pris en charge Lipid bicouches

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.

Microscopie photoacoustique à résolution acoustique et optique à commutation commutable pour<em&gt; In vivo</em&gt; Imagerie vasculaire de sang de petit animal

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.