Y. T. Y. tient à remercier le Fonds de soutien du ministère de la Science et technologie sous numéros de subvention plus 105-2221-E-007-MY3 et de la National Tsing Hua University sous concession numéros 105N518CE1 et 106N518CE1.

1. Configuration optique Remarque : le principe de la configuration optique est illustré à la Figure 1.
Kit de pinces <ol><li>set up l’optique (voir la Table des matières) et le module de fluorescence (voir Table des matières) selon leurs manuels. Connectez une source de nm bleu lumière émettant de la lumière de la diode électroluminescente (del) 470 dans le module fluorescent.</li>
	<li>Remplacer la grande ouverture numérique (NA) (NA = 1,25, grossissement x 100) objectif à immersion d’huile par un travail de longue distance objectif de microscope (WD) (focale 3,6 mm, WD = 10,6 mm, NA = 0,5).</li>
	<li>Retirez la lentille dans la section d’extension de faisceau du kit assemblé pour atteindre lâche de focalisation du faisceau laser.</li>
	Diode <li>tournant sur l’alimentation et le courant pour le laser de longueur d’onde de 980 nm et l’utilisation de l’accusé couplée (CCD) appareil pour s’assurer que le faisceau laser est bien aligné. 
	Remarque : Si le faisceau laser est bien aligné, la caméra CCD lira une tache gaussienne.</li>
</ol> 2. Nanofabrication Fabrication de marqueur <ol><li>. 
	NOTE : Marqueurs vont aidera à placer le tableau de nanoplasmonic pendant le processus de fabrication et l’expérimentation ultérieure de piégeage. Le processus détaillé est illustré figure1 supplémentaire.
	<ol><li>Film d’oxyde d’étain (ITO) d’indium dépôt 40 nm sur une lamelle d’Epaisseur 0,17 mm bredouillement. 
		NOTE : Le film de ITO aideront à décharger des électrons durant le processus de lithographie par faisceau électronique ultérieure.</li>
		<li>Spin enduire d’une couche de 8 µm de photorésine positive avec essorage vitesse 4000 tr/min et temps 30 s avec une coucheuse spin.</li>
		<li>Doux, cuire au four de l’échantillon à 90 ° C pendant 5 min et aligner l’échantillon avec le photomasque pour marqueur et exposer l’échantillon aux UV pour 80 s dans l’aligneur de masque.</li>
		<li>Tremper l’échantillon dans le développeur de résine photosensible pour 130 s.</li>
		<li>Déposer une couche de 2 nm de chrome et une couche de 40 nm d’or sur l’échantillon à l’aide d’évaporation thermique. 18</li> <li>tremper l’échantillon dans de l’acétone et le placer dans un fonctionnement nettoyeur ultrasonique à 43 kHz et 150 W pendant 5 min pour lift off.</li>
	</ol></li> <li>Fabrication de tableau Nanoplasmonic <ol><li>manteau une couche de faisceau électronique de Spin résister à PMMA 120K avec vitesse de rotation 5000 tr/min pendant 30 s sur une coucheuse spin. Cuire au four de l’échantillon à 160 ° C pendant 3 min sur une plaque chauffante.</li>
		<li>Spin enduire une autre couche de résistent e-faisceau PMMA 960K avec vitesse de rotation 5000 tr/min pendant 30 s sur une coucheuse spin. Cuire au four de l’échantillon à 160 ° C pendant 3 min sur une plaque chauffante.</li>
		<li>Écrivain e-faisceau utilisation d’exposer le faisceau électronique résistent avec accélération tension 30 kV et dosage 400C/cm 2.</li>
		<li>Déposer une couche de 40 nm d’or dans un évaporateur thermique.</li>
		<li>Faire tremper l’échantillon dans de l’acétone et le placer dans un nettoyeur à ultrasons pendant 5 min pour lift off.</li>
	</ol></li></ol> 3. Exemple de système de refroidissement et son étalonnage de température Remarque : l’échantillon scénographie de refroidissement est illustré Figure supplémentaire 2.
<ol><li>Faire le circuit pilote pour échantillon de refroidissement <ol><li>Place les résistances, transistors à jonction bipolaire et transistors d’effet de champ électrique d’oxyde métallique sur le circuit personnalisé en suivant le schéma de câblage supplémentaire Figure 3 . Tous ces composants avec fer à souder de soudure.</li>
		<li>Connecter des câbles entre le port de contrôle du circuit imprimé et le tableau de commande électronique. Brancher les fils entre le port de sortie du circuit imprimé et thermoélectrique dissipateur (TEC). Placer l’élément TEC sur la scène de l’échantillon avec chaleur naufrage. 
		&#8203; Remarque : élément de la TEC a un trou au Centre pour permettre le faisceau laser de passer par.</li>
		Bloc d’alimentation <li>raccorder les fils du circuit à 5 V. La caméra infrarouge permet de contrôler la température pour vérifier si le refroidissement thermoélectrique correctement refroidit.</li>
	</ol></li> <li>Étalonnage de température mesurée dans la prospective caméra et résistance température détecteur (RTD) thermomètre infrarouge.
	<ol><li>Placer le thermomètre de RDT sur une lamelle de Vierge et appliquez une petite quantité de pâte thermique sur elle pour assurer le bon contact thermique entre le thermomètre RTD et lamelle.</li>
		<li>Changer le réglage de puissance de sortie du circuit de commande électronique en élément de TEC en changeant le cycle du paramètre de pulse width modulation et attendre 3 minutes pour s’assurer que la température de l’état d’équilibre est atteinte. Lire la température avec le thermomètre RTD.</li>
		<li>Tourner la caméra infrarouge regardant vers l’avant et surveiller la température. Répétez cette procédure à différents paramètres de puissance de sortie pour obtenir la courbe d’étalonnage de température. Une courbe d’étalonnage de température représentative est illustrée Figure supplémentaire 4. 
		Remarque : Il est crucial de faire l’étalonnage entre thermomètre de RDT et la caméra infrarouge vers l’avenir parce que la lecture de la température de la caméra infrarouge doit être précise afin d’assurer la bonne température est atteinte.</li>
	</ol></li></ol> 4. Piégeage de microparticules <ol><li>diluer micro particules de polystyrène de µm de diamètre 2 dans l’eau déionisée dans un microcentrifugetube avec rapport de volume suffisant. 
	Remarque : La concentration de microparticules peut être ajustée selon l’objectif de l’expérience. Alors que la plus faible concentration permet un intervalle de temps d’échantillon entre les événements de piégeage de particules uniques, une concentration plus élevée raccourcira le temps de piégeage de particules multiples. Pour la récupération d’une seule particule, une concentration typique est ~0.05% (p/v).</li>
	<li>Mettre l’échantillon avec tableau nanoplasmonic sur la scène et allumez un 470 nm LED comme source lumineuse fluorescence et définir manuellement la puissance de 5 mW pour l’imagerie champ lumineux.</li>
	<li>Utilisez le marqueur pour localiser le tableau nanoplasmonic, aligner l’échantillon et la caméra CCD pour s’assurer que le tableau est au centre de la région d’intérêt sur l’écran d’ordinateur.</li>
	<li>Distribuer 10 µL des dilués micro particules de diamètre 2 µm sur l’échantillon avec une pipette micro.</li>
	<li>Allumez l’alimentation en courant de la diode laser de longueur d’onde de 980 nm pour exciter la résonance plasmonique du tableau avec une puissance dans la gamme ~ 1 mW à 10 mW.</li>
	<li>Activer manuellement l’alimentation de l’électronique de contrôle des stupéfiants pour refroidir l’échantillon à un état d’équilibre température ~ 4 ° C.</li>
	<li>Dans le logiciel de visualisation, cliquez sur le &#34; vidéo record &#34; séquence pour ouvrir la boîte de dialogue enregistrement. Cliquez sur le &#34; dossier &#34; bouton pour lancer la vidéo enregistrement 1,5 de la motion des micro particules à une cadence de 10 images/s sur l’échantillon sous l’influence du faisceau laser à l’aide de la caméra CCD. Cliquez sur le &#34; stop &#34; le bouton pour arrêter l’enregistrement. Voir la vidéo 1.</li>
</ol>

<ol>
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Les auteurs n’ont rien à divulguer.

La procédure décrite ici permet au lecteur de façon fiable reproduire piégeage sur une base quotidienne. Règle empirique générale pour concevoir un réseau optique utilisable est d’utiliser une taille comparable pour plasmonique nanoarray, distance interdisc et pris au piège de la taille des particules. Par rapport à une nanostructure plasmonique seule, isolée, la conception de réseau optique en conjonction avec la puissance optique élevée offerte par refroidissement de l’échantillon à ~ 4 ° C utilis?...

Le piégeage optique de particules de petite échelle a été initialement développé par Arthur Askin dans les années 1970. Depuis son invention, la technique a été développée comme un outil polyvalent pour les micro - et nanomanipulations1,2. Conventionnel optique piégeage basé sur le champ-mise au point de principe est intrinsèquement limitée par la diffraction dans son confinement spatial, dans lequel la force de piégeage diminue considérablement (suivant un ~undroit de3 pour une particule de rayon un) 3. pour surmonter ces limites de diffraction, les chercheurs ont développé des techniques de piégeage optique en champ proche basés sur champ évanescent optique utilisant des nanostructures métalliques plasmoniques et, en outre, le piégeage des nano objets vers le bas pour molécules protéiques unique a été démontrée4,5,6,7,8,9,10,11. En outre, le réseau optique plasmonique est créé à partir tableaux de nanostructures plasmoniques périodique de conférer le transport à distance des micro - et nanoparticules et plusieurs particules empilement11,12. Un des principaux obstacles pour perturber le piégeage dans un réseau optique sont photothermique convection et s’est efforcé d’élucider ses effets par plusieurs groupes14,15,16,17. En utilisant la fonction de Green, baffoué et coll. ont calculé un profil de température en modélisant chaque nanostructure plasmonique comme un radiateur de point et puis expérimentalement validé leur modèle14. Groupe de toussant a également mesuré la convection induite par plasmon avec particule vélocimétrie par15. L’auteur a également caractérisé transport contigu et convection et démontré une stratégie d’ingénierie pour supprimer photothermique convection16,17.
Nous présentons ici la conception d’un montage optique et une procédure détaillée spécifiquement pour des expériences de piégeage avec treillis optique plasmoniques. Le potentiel optique a été créé en éclairant un tableau d’or nanodiscs avec un faisceau gaussien faiblement concentré. Un régime pour réprimer la convection photothermique en refroidissant l’échantillon à une température basse (~ 4 ° C) pour la récupération optimale est aussi décrire ici17. Aux termes de l’approximation de Boussinesq, une estimation de l’ordre de grandeur de vitesse de convection naturelle u est donnée par u ~L2 gβΔT / v, où L est l’échelle de la longueur de la source de chaleur et Δ T est l’augmentation de la température par rapport à la référence à cause du réchauffement.  g et β sont l’accélération gravitationnelle et coefficient de dilatation thermique, respectivement. À des températures proches de 4 ° C, la densité de l’eau milieu montre la dépendance en température anormale et cela se traduit par un coefficient de dilatation thermique proche de zéro et, partant, une convection photothermique ridiculement faible.

<table style="border-collapse:collapse;width:629pt" width="839">
	<colgroup>
		<col style="width:207pt" width="276" />
		<col style="width:131pt" width="174" />
		<col style="width:122pt" width="162" />
		<col style="width:170pt" width="227" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Thermoelectric cooling element</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">TEC 1.4-6</td>
			<td class="xl70" style="width:170pt" width="227">TEC element for sample cooling</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">RTD thermometer</td>
			<td class="xl69" style="width:131pt" width="174">Omega Engineering</td>
			<td class="xl69" style="width:122pt" width="162">RTD Thermometer 969C</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Forward looking infrared camera</td>
			<td class="xl69" style="width:131pt" width="174">FLIR&#160;</td>
			<td class="xl69" style="width:122pt" width="162">FLIR One</td>
			<td class="xl70">IR camera for temperature monitoring</td>
		</tr>
		<tr height="46" style="height:34.5pt">
			<td class="xl69" height="46" style="width:207pt;height:34.5pt" width="276">light emitting diode light source</td>
			<td class="xl69" style="width:131pt" width="174">Touchbright</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">Light source for illumination for fluorescent imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Long working distance objective</td>
			<td class="xl69" style="width:131pt" width="174">Olympus</td>
			<td class="xl69" style="width:122pt" width="162">LMPLFLN</td>
			<td class="xl70">For illuminating the sample and imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Optical trap kit</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB/M</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Cover slip</td>
			<td class="xl69" style="width:131pt" width="174"></td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">thickness 0.17 mm</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Scanning electron microscope</td>
			<td class="xl69" style="width:131pt" width="174">Hitachi</td>
			<td class="xl69" style="width:122pt" width="162">SEM-Hitachi S3400N</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="44" style="height:33.0pt">
			<td class="xl71" height="44" style="height:33.0pt">Electron beam blanker</td>
			<td class="xl69" style="width:131pt" width="174">DEBEN</td>
			<td class="xl69" style="width:122pt" width="162">PCD beam blanker</td>
			<td class="xl69" style="width:170pt" width="227">the blanker is added to the scanning electron microscope&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Thermal evaporator</td>
			<td class="xl69" style="width:131pt" width="174">SYSKEY Technology</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Mask aligner</td>
			<td class="xl69" style="width:131pt" width="174">Karl Suss</td>
			<td class="xl69" style="width:122pt" width="162">MJB 3</td>
			<td class="xl69" style="width:170pt" width="227">For marker fabrication</td>
		</tr>
		<tr height="24" style="height:18.0pt">
			<td class="xl69" height="24" style="width:207pt;height:18.0pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 120K</td>
			<td class="xl69" style="width:170pt" width="227">For e-beam lithography</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 960K</td>
			<td class="xl70">For e-beam lithography</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">Fluoresent labeled polystyrene microspheres</td>
			<td class="xl69" style="width:131pt" width="174">Polyscience</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">2 um diameter</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3904</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl69" height="28" style="width:207pt;height:21.0pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3906</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF5305</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF131ON</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">10 kOhm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 6 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">910 Ohm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Photoresist</td>
			<td class="xl69" style="width:131pt" width="174">Microchemicals</td>
			<td class="xl69" style="width:122pt" width="162">AZ4620</td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Acetone</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl69" height="41" style="width:207pt;height:30.75pt" width="276">Fluorescence Module for the OTKB/M, Metric Threads</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB-FL/M</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="49" style="height:36.75pt">
			<td class="xl69" height="49" style="width:207pt;height:36.75pt" width="276">Fluorescent filter set</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">MDF-FITC</td>
			<td class="xl70">For Fluorescein Isothiocyanate (FITC)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl70" height="21" style="height:15.75pt">Ultrasonic cleaner</td>
			<td class="xl69" style="width:131pt" width="174">Delta</td>
			<td class="xl70">DC150H</td>
			<td class="xl70">For the lift off step</td>
		</tr>
	</tbody>
</table>

trapping of micro particles in nanoplasmonic optical lattice

La pince optique plasmonique a été développé pour pallier les limites de la diffraction de la pince optique traditionnelle en champ lointain. Plasmoniques trellis optique se compose d’un tableau de nanostructures, qui présentent une variété de piégeage et des comportements de transport. Nous rapportons les procédures expérimentales pour piéger des micro particules dans un réseau optique simple nanoplasmonic carrés. Nous décrivons également la configuration optique et la nanofabrication d’un tableau de nanoplasmonic. Le potentiel optique est créé en éclairant un tableau d’or nanodiscs avec un faisceau gaussien de longueur d’onde de 980 nm et passionnant de résonance plasmonique. Le mouvement des particules est contrôlé par imagerie de fluorescence. Un schéma à supprimer photothermique convection décrit également pour augmenter la puissance optique utilisable pour la récupération optimale. Suppression de la convection est obtenue par refroidissement de l’échantillon à une température basse et en utilisant le coefficient de dilatation thermique proche de zéro d’un milieu d’eau. Les transport de particules uniques et multiples piégeage de particules sont rapportés ici.

Trajectoires de particules uniques ont été enregistrées par une caméra CCD dans notre expérience, et les images sont ensuite traitées avec un programme personnalisé pour extraire trajectoire16 de chaque particule. Résultats représentatifs sont affichés dans la Figure 3 et 1 vidéo pour micro-sphères d’un diamètre de 2 µm. multiples atours de particules à l’intérieur du réseau optique ont été obser...

Watch this Scientific Journal Video about Trapping of Micro Particles in Nanoplasmonic Optical Lattice at JoVE.com

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Nous décrivons une procédure pour intercepter optiquement micro-particules dans nanoplasmonic réseau optique.

Piégeage de Micro particules dans trellis optique Nanoplasmonic

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

trapping-of-micro-particles-in-nanoplasmonic-optical-lattice

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.5K vues.  National Tsing Hua University. Nous décrivons une procédure pour intercepter optiquement micro-particules dans nanoplasmonic réseau optique.

Vidéo: Trapping of Micro Particles in Nanoplasmonic Optical Lattice

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 ...

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Bio-ingénierie

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

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

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

Multiparamétrique cartographie optique du Cœur lapin perfusé de Langendorff

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

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

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Cartographie optique de potentiels d&#39;action et les transitoires de calcium dans le cœur de la souris

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

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

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

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

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

Une approche recombinaison Cre-Lox P pour la détection de la fusion cellulaire In Vivo</em

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

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

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

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

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

En synthèse in vitro de la maternelle, de l&#39;espacement de longue fibreux et segmentaire collagène Espacement long

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

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particules vélocimétrie par image de profil de vitesse pour mesures de débits Micro sang

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Injection intra-lymphatique de particules de polymère biodégradables

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatisé de robotique Liquid Handling assemblage de dispositifs modulaires ADN

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted Plasma Membrane Livraison d&#39;un Hydrophobe Cargo encapsulé dans un cristal liquide Nanoparticules Transporteur

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Piégeage de Micro particules dans trellis optique Nanoplasmonic

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Irrigables réseau vasculaire avec un modèle de tissu dans un dispositif microfluidique