Ce travail est soutenu par l&#39;Institut national du cancer à l&#39;Institut national de la santé subvention R01 CA140257-01.

<html> NOTE: Le consentement éclairé a été obtenu à partir des patients. En outre, cette recherche a été réalisée en conformité avec toutes les directives institutionnelles, nationales et internationales pour le bien-être humain. 1. Préparer ordinateur <ol><li> Tournez ordinateur portable et se connecter USB de la carte de capture DVI2USB. </li></ol> 2. Préparer Moniteur <ol><li> Branchez le câble DVI pour surveiller et PinP pour permettre à l&#39;endoscopiste pour voir des vidéos de ces écrans plutôt que de l&#39;ordinateur. </li><li> Assurez-vous que le moniteur permanent est allumé et réglé sur DVI. </li><li> Connectez PinP à l&#39;arrière du système Olympus puis appuyez sur le bouton d&#39;entrée sur le système Olympus pour afficher l&#39;image sur le grand écran monté sur le mur. </li></ol> 3. Diode laser Cadre <ol><li> Assurez-vous que le pilote de diode laser est éteint. Il ne doit être allumé quelques minutes avant l&#39;imagerie est effectuée. </li></ol>e_title &quot;&gt; 4. Power Strip <ol><li> Branchez un ordinateur portable, le processeur, le pilote de diode laser, Répartiteur DVI, et Épiphane carte de capture dans la bande de puissance. </li></ol> 5. Exécutez MDE Widefield sur le bureau d&#39;ordinateur portable <ol><li> Hit &#39;dossier en cours ». </li><li> Tapez le numéro de patient et les initiales et appuyez sur &quot;Créer un dossier patient». </li></ol> 6. Préparer Cap et de filtre <ol><li> Lorsque le filtre de manutention, toujours utiliser des gants et Kimwipes pour minimiser le transfert de l&#39;huile et des débris sur le filtre. Gaze ou tampons imbibés d&#39;alcool peuvent laisser des fibres qui peuvent interférer avec l&#39;imagerie. </li><li> Placez quelques Kimwipes sur une table pour créer une plate-forme pour fixer le filtre et le bouchon. Cette zone désinfectée doit être facilement accessible à la gastro-entérologue au cours de la procédure. </li><li> Fixer le filtre et le bouchon et garder Kimwipes à proximité pour une utilisation pendant la procédure. </li></ol> 7. Préparation du patient <ol><li> patients de consentement sur l&#39;utilisationde VFI et proflavine colorant avant d&#39;arriver à la suite de l&#39;endo. </li><li> le patient de position pour la procédure d&#39;endoscopie digestive haute. </li><li> Passez à l&#39;imagerie en lumière blanche standard à l&#39;aide d&#39;un microscope numérique multispectrale (MDM) 7. </li></ol> 8. Insérez et Spray Proflavine <ol><li> Après avoir terminé avec l&#39;imagerie en lumière blanche, insérer et pulvériser colorant proflavine sur tissu d&#39;intérêt. 1-5 mm devrait être suffisante en fonction de la zone de la muqueuse de Barrett. En pulvérisant le colorant proflavine à cette étape, tout le temps (au moins 1 min) est donnée pour l&#39;absorption de suffisamment de tissu avant VFI. Proflavine, qui est couvert par une demande de nouveau médicament expérimental de la FDA (IND 102 217), est un agent de contraste fluorescent qui se concentre dans les noyaux des cellules peu après l&#39;application. Bien VFI ne peut pas résoudre la morphologie cellulaire individuelle, lorsque les casseroles de diode laser sur le tissu, la lumière réfléchie permet à l&#39;endoscopiste pour apprécier morpho glandulaire globalegie. </li></ol> 9. Mettez diode laser <ol><li> Allumer la diode laser. Pour ce faire, au moins 2 min avant le début de l&#39;imagerie. </li></ol> 10. Préparer endoscope pour VFI <ol><li> Retirer l&#39;endoscope complètement. </li><li> Avant le traitement de l&#39;endoscope, s&#39;assurer que l&#39;assistant a deux paires de gants. </li><li> Avez-endoscopiste tenir l&#39;endoscope en face de l&#39;assistant qui est à côté de la plate-forme Kimwipe détaillé ci-dessus. </li><li> Avec Kimwipes, ont l&#39;assistant nettoie l&#39;extrémité de l&#39;endoscope. Assurez-vous de nettoyer la surface avant doucement et aussi nettoyer quelques centimètres le long du côté de l&#39;endoscope pour une manipulation plus facile. </li><li> Après le nettoyage, avoir l&#39;assistant disposer de la paire externe de gants. </li><li> Avez-adjoint mettre sur le filtre. Insérez la protubérance cylindrique courte sur le filtre dans son trou complémentaire dans l&#39;endoscope. Gardez l&#39;endoscope verticale pour faciliter le processus. </li><li> Tenir le filter en place, faites glisser le couvercle sur le filtre et le pousser vers le bas sur l&#39;extrémité de l&#39;endoscope. Assurez-vous que le bouchon est sécurisé et complètement enfoncée sur les bords de chasse avec l&#39;endoscope. Assurez-vous que la lèvre du capuchon s&#39;étend légèrement sur la pointe de l&#39;endoscope. Enfin, assurez-vous que le filtre est toujours en place et rincer à l&#39;extrémité de l&#39;endoscope. </li></ol> 11. Insérez le endoscope dans l&#39;œsophage et de l&#39;image 12. Retirer endoscope de l&#39;oesophage <ol><li> Utilisation Kimwipes tirez doucement le bouchon et le filtre de l&#39;endoscope. Jetez le capuchon et nettoyer le filtre pour le cas suivant. </li></ol> 13. Nettoyer le filtre <ol><li> Nettoyez délicatement le filtre avec Kimwipes. </li><li> Remplir une petite tasse avec CyDex et plonger le filtre pendant 12 min. Toutes les quelques minutes tournent le filtre plus. </li><li> Nettoyez délicatement le filtre avec Kimwipes. </li><li> Immerger le filtre, cette fois dans de l&#39;eau stérile. Après 5 min, utiliser un jet bottle et vaporiser légèrement de l&#39;eau stérile sur le filtre. </li><li> Placer le filtre sur quelques Kimwipes et laisser sécher. </li><li> Une fois sec, mettre dans un conteneur de stockage entre Kimwipes. </li></ol>

<ol>
	<li>Modiano, N., Gerson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrett's+Esophagus:+Incidence,+etiology,+pathophysiology,+prevention+and+treatment.">Barrett's Esophagus: Incidence, etiology, pathophysiology, prevention and treatment.</a> Therapeutics and Clinical Risk Management. 3, 1035-1145 (2007).</li><li>Brown, L. M., Devesa, S. S., Chow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of Adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> Journal of the National Cancer Institute. 100, 1184-1187 (2008).</li><li>Siegel, R., Naishadham, D., Ahmedin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2012.">Cancer Statistics, 2012.</a> A Cancer Journal for Clinicians. 62, 10-29 (2012).</li><li>Vieth, M., Ell, C., Gossner, L., May, A., Stolte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+endoscopic+resection+specimens+from+326+patients+with+Barrett's+esophagus+and+early+neoplasia.">Histological analysis of endoscopic resection specimens from 326 patients with Barrett's esophagus and early neoplasia.</a> Endoscopy. 36, 776-781 (2004).</li><li>Pohl, H., Rosch, T., Vieth, 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=Miniprobe+confocal+laser+microscopy+for+the+detection+of+invisible+neoplasia+in+patients+with+Barrett's+oesophagus.">Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus.</a> Gut. 57, 1648-1653 (2008).</li><li>Thekkek, N., 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=Modular+video+endoscopy+for+in+vivo+cross-polarized+and+vital-dye+fluorescence+imaging+of+Barrett's-associated+neoplasia.">Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia.</a> Journal of Biomedical Optics. 18, 026007 (2013).</li><li>Roblyer, D., 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=Multispectral+optical+imaging+device+for+in+vivo+detection+of+oral+neoplasia.">Multispectral optical imaging device for in vivo detection of oral neoplasia.</a> Journal of Biomedical Optics. 13, 024019 (2008).</li><li>Egger, K., 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=Biopsy+surveillance+is+still+necessary+in+patients+with+Barrett's+oesophagus+despite+new+endoscopic+imaging+techniques.">Biopsy surveillance is still necessary in patients with Barrett's oesophagus despite new endoscopic imaging techniques.</a> Gut. 52, 18-23 (2003).</li><li>Thekkek, N., 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=Vital-dye+enhanced+fluorescence+imaging+of+GI+mucosa:+metaplasia,+neoplasia,+inflammation.">Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.</a> Gastrointestinal Endoscopy. 75, 877-887 (2012).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointestinal Endoscopy. 68, 737-744 (2008).</li></ol>

Il n&#39;y a rien à divulguer.

Avec la surveillance endoscopique standard, néoplasie en BE est souvent manqué 8 parce métaplasie bénigne peut être impossible à distinguer de la dysplasie de haut grade et adénocarcinome. Comme un outil qui permettrait de mieux permettre aux gastro-entérologues de remédier à cette erreur a inévitable, indispensable colorant amélioré l&#39;imagerie de fluorescence met en évidence la morphologie glandulaire d&#39;un tissu offrant ainsi une caractéristique distincte de différencier les types de ...

Au cours des quarante dernières années, l&#39;incidence de l&#39;adénocarcinome oesophagien (EAC) a considérablement augmenté de 1,2; encore en raison de diagnostic tardif, le taux de survie à cinq ans est inférieur à 20% 3. La norme actuelle de la surveillance endoscopique en BE, le précurseur de l&#39;EAC, est la lumière blanche endoscopie avec biopsies de du segment aléatoires pince-quatre Quandrant de. Malheureusement, cette technique manque souvent néoplasie, qui peut être plat, subtile et difficile à distinguer sur l&#39;imagerie de la lumière blanche standard 4. Bien qu&#39;il y ait eu un succès en utilisant la microscopie laser confocale à mettre en évidence les caractéristiques cellulaires in vivo, les lésions peuvent encore manquer en raison de la diminution de champ de vue 5. Avoir une technologie «pont» qui peut mettre en évidence les domaines à l&#39;imagerie confocale Microendoscopic serait nettement précieux. Par conséquent, un drapeau rouge modalité d&#39;imagerie amélioré qui améliore la capacité de Target et une biopsie néoplasie début de BE serait joué dans la détection de la CAE à un stade curable tôt et pourrait conduire à un traitement plus efficace et par la suite l&#39;amélioration des taux de survie. VFI est une nouvelle technique qui combine haute résolution épithéliale imagerie avec exogène actualité fluorescent contraste, proflavine, pour mettre en évidence la morphologie glandulaire et de délimiter la néoplasie (dysplasie de haut grade et le cancer) dans l&#39;oesophage distal dans l&#39;espoir d&#39;améliorer le diagnostic in vivo 6. Lors de l&#39;excitation de la proflavine, qui se concentre à l&#39;intérieur de noyaux de cellules peu de temps après l&#39;application, les images fluorescentes fournissent une résolution spatiale de 50 à 100 um et un champ de vision jusqu&#39;à 2,5 cm, ce qui permet de visualiser la morphologie endoscopistes glandulaire. En conséquence, cette approche permet de distinguer les gastro-entérologues métaplasie de Barrett classique, qui possède, des glandes régulièrement espacées continue et une morphologie homogène dans l&#39;ensemble, à partir de BE à une néoplasie, qui a oblitérationtion de l&#39;architecture glandulaire. Nous décrivons ici le protocole de cette nouvelle technique avec un endoscope multispectrale, et de fournir des résultats représentatifs pour démontrer l&#39;utilité de ce dispositif en dépeignant la transformation morphologique de métaplasie bénigne de dysplasie de haut grade et le cancer.

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr>
			<td>Filter*</td>
			<td>Schott North America, Inc., Duryea, Pennsylvania</td>
			<td>Not Applicable</td>
			<td>495-nm long-pass filter</td>
		</tr>
		<tr>
			<td>Halo Cap &#8211; Medium*</td>
			<td>Barrx Medical</td>
			<td>CP-002A</td>
			<td></td>
		</tr>
		<tr>
			<td>Processor*</td>
			<td>Pentax</td>
			<td>EPK-i</td>
			<td></td>
		</tr>
		<tr>
			<td>Multispectral Digital Microscope**</td>
			<td>Not Applicable</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>KimTech Science</td>
			<td>S-8115</td>
		</tr>
		<tr>
			<td>Cidex</td>
			<td>Advanced Sterilization Products&#160;</td>
			<td>CIDEX OPA Solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Proflavine hemisulfate (0.01% w/v)</td>
			<td></td>
			<td></td>
			<td>FDA (IND 102,217)</td>
		</tr>
		<tr>
			<td>Laser Diode*</td>
			<td>Nichia Corporation</td>
			<td>Not Applicable</td>
			<td>455-nm</td>
		</tr>
		<tr>
			<td>Image Capture*</td>
			<td>Labview</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray Catheter</td>
			<td>Olympus</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>*Equipment specifics within Reference 6. **Equipment specifics within Reference 7</td>
		</tr>
	</tbody>
</table>

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

La capacité de se différencier métaplasie bénigne dans l&#39;oesophage de Barrett (BE) de néoplasie in vivo reste difficile car les deux types de tissus peuvent être plats et indiscernables à l&#39;imagerie de la lumière blanche seul. Par conséquent, une modalité qui met en évidence l&#39;architecture glandulaire serait utile pour discriminer une néoplasie bénigne de l&#39;épithélium de l&#39;oesophage distal. VFI est une nouvelle technique qui utilise un agent topique de contraste fluorescent exogène pour délimiter dysplasie de haut grade et le cancer de l&#39;épithélium bénin. Plus précisément, les images fluorescentes fournissent une résolution spatiale de 50 à 100 um et un champ de vision jusqu&#39;à 2,5 cm, ce qui permet de visualiser la morphologie endoscopistes glandulaire. Lors de l&#39;excitation, la métaplasie de Barrett semble classique, les glandes régulièrement espacées comme continues et une morphologie homogène globale; en revanche, le tissu néoplasique semble encombré avec oblitération complète du cadre glandulaire. Ici, nous donnons un aperçu desl&#39;instrumentation et d&#39;énumérer le protocole de cette nouvelle technique. Bien VFI offre un gastro-entérologue à l&#39;architecture glandulaire de tissu suspect, dysplasie cellulaire ne peut pas être résolu avec cette modalité. En tant que tel, on ne peut pas distinguer morphologiquement métaplasie de Barrett de BE avec des dysplasies via cette modalité d&#39;imagerie. Par troquer une diminution de la résolution avec un plus grand champ de vision, ce système d&#39;imagerie peut être utilisé à tout le moins comme un dispositif d&#39;imagerie drapeau rouge pour cibler et biopsie des lésions suspectes; encore, si les mesures de précision sont prometteurs, VFI peut devenir la technique d&#39;imagerie standard pour le diagnostic de la néoplasie (défini comme étant dysplasie de haut grade ou de cancer) dans l&#39;oesophage distal.

Figure 1B montre classique œsophage de Barrett avec dysplasie pas entouré sur les frontières d&#39;un épithélium malpighien normal. En commençant par le tissu malpighien plat, qui est situé à la périphérie et indiqué par les flèches bleues, une zone homogène de fluorescence terne est présente avec aucune architecture glandulaire. Les flèches vertes indiquent une ligne verte circulaire entourant le tissu épidermoïde. Ce schéma est artefact résultant de la capsule de l&#39;endoscope. D?...

Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Vital-colorant amélioré l&#39;imagerie de fluorescence (VFI) est un roman de la technique in vivo qui combine épithéliale imagerie à haute résolution avec exogène contraste fluorescent actualité pour mettre en évidence la morphologie glandulaire et de délimiter la néoplasie (dysplasie de haut grade et le cancer) dans l&#39;oesophage distal.

Le diagnostic de néoplasie dans l&#39;oesophage de Barrett en utilisant Vital-colorant amélioré imagerie de fluorescence

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

diagnosis-neoplasia-barretts-esophagus-using-vital-dye-enhanced

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Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

11.9K vues.  Icahn School of Medicine at Mount Sinai.  Vital-colorant amélioré l'imagerie de fluorescence (VFI) est un roman de la technique in vivo qui combine épithéliale imagerie à haute résolution avec exogène contraste fluorescent actualité pour mettre en évidence la morphologie glandulaire et de délimiter la néoplasie (dysplasie de haut grade et le cancer) dans l'oesophage distal. 

Vidéo: Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

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

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imagerie de vie de fluorescence des rotors moléculaires dans les cellules vivantes

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse d&#39;imagerie par fluorescence de la croissance racinaire d&#39;Arabidopsis avec la manipulation rapide de l&#39;environnement root en utilisant le RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

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., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Suivi 3D orbital dans un microscope à deux photons à jour: Une application au suivi des vésicules intracellulaires

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De Fluorescence Imaging rapide à moléculaire loi Diffusion sur les membranes cellulaires en direct dans un microscope commercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Réalisation de multiples Modes d’imagerie avec un Microscope à Fluorescence

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualiser la Formation d’adhérences dans les cellules par le biais de pointe tourne disque-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Utilisation de la diffusion améliorée par Nanoplasmon et imagerie microscopique à faible grossissement pour quantifier les exosomes dérivés de tumeurs

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Estimation de la pression assistée par sous-harmonique améliorée par contraste (SHAPE) à l’aide de l’imagerie par ultrasons en mettant l’accent sur l’identification de l’hypertension portale

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...