Diese Arbeit wird durch das National Cancer Institute der National Institutes of Health Zuschuss R01 CA140257-01 unterstützt.

<html> HINWEIS: Informierte Zustimmung wurde von den Patienten erhalten. Auch hat diese Forschung in Übereinstimmung mit allen institutionellen, nationalen und internationalen Richtlinien für die menschliche Wohlfahrt durchgeführt worden. 1. Bereiten Computer- <ol><li> Schalten Laptop auf und verbinden USB aus dem DVI2USB Capture Card. </li></ol> 2. Bereiten Überwachung <ol><li> DVI-Kabel anschließen zu überwachen und PinP, damit der Endoskopiker, Videos von diesen Bildschirmen anzuzeigen, anstatt den Computer. </li><li> Stellen Sie sicher, die stehende Monitor auf und ist mit DVI eingestellt. </li><li> Schließen PinP an der Rückseite der Olympus System drücken Sie dann die Eingabe-Taste auf der Olympus-System, um das Bild auf dem großen Monitor an der Wand montiert zu sehen. </li></ol> 3. Laserdiodentreiber-Einstellung <ol><li> Überprüfen Sie, ob die Laserdioden-Treiber ist AUS. Es sollte nur für ein paar Minuten eingeschaltet, bevor Bildgebung durchgeführt werden. </li></ol>e_title &quot;&gt; 4. Power Strip <ol><li> Stecken Laptop, Prozessor, Laserdiodentreiber, DVI-Splitter, und epiphan-Capture-Karte in die Steckerleiste. </li></ol> 5. Führen MDE Weitfeld auf dem Laptop Desktop <ol><li> Hit &#39;Current Folder &quot;. </li><li> Geben Patientennummer und Initialen und drücken Sie &quot;Neues Patienten Folder &#39;. </li></ol> 6. Bereiten Cap und Filter <ol><li> Handhabung von Filter immer Schutzhandschuhe und Kimwipes verwenden, um die Übertragung von Öl und Schmutz auf dem Filter zu minimieren. Gaze-oder Alkoholtupfer können Fasern, die mit bildgebenden stören lassen. </li><li> Legen Sie ein paar Kimwipes auf einen Tisch, um eine Plattform, um den Filter und Kappe legen erstellen. Diese desinfizierten Bereich sollte während des Verfahrens leicht zugänglich für die Gastroenterologen sein. </li><li> Den Filter und Kappe fest und halten Kimwipes während des Verfahrens in der Nähe für den Einsatz. </li></ol> 7. Patientenvorbereitung <ol><li> Consent Patienten auf den Einsatzvon VFI und Proflavin Farbstoff vor der Ankunft in endo-Suite. </li><li> Position für Patienten oberen Endoskopie Verfahren. </li><li> Fahren Sie mit der Weißlicht-Standard mit einem multispektralen Bildgebung Digital-Mikroskop (MDM) 7. </li></ol> 8. Einfügen und Spray Proflavin <ol><li> Nach dem Abschluss mit Weißlicht-Bildgebung, legen und sprühen Proflavin Farbstoff über Gewebe von Interesse. 1-5 mm sollte ausreichend sein, abhängig von dem Bereich des Barretts-Gewebe. Durch Sprühen der Proflavin Farbstoff bei diesem Schritt wird genügend Zeit (mindestens 1 min) für eine ausreichende Gewebeabsorption vor VFI gegeben. Proflavin, die unter einem Prüfpräparat-Anwendung von der FDA (IND 102217) abgedeckt ist, eine fluoreszierende Kontrastmittel, die in Zellkerne kurz nach der Anwendung konzentriert. Obwohl VFI nicht lösen können einzelne Zellmorphologie, wenn die Laserdiode Pfannen über ein Gewebe, das reflektierte Licht ermöglicht es dem Endoskopiker, um die Gesamtdrüsen Morphologien schätzengy. </li></ol> 9. Auf Laser Diode drehen <ol><li> Einschalten der Laserdiode. Tun Sie dies mindestens 2 min vor der Belichtung beginnt. </li></ol> 10. Bereiten Endoskop für die VFI <ol><li> Ziehen Sie das Endoskop vollständig. </li><li> Vor der Handhabung des Endoskops, dass der Assistent zwei Paare von Handschuhen. </li><li> Haben die Untersucher halten das Endoskop vor der Assistentin, die neben dem Kimwipe Plattform oben beschrieben ist. </li><li> Mit Kimwipes, haben die Assistentin reinigt die Spitze des Endoskops. Achten Sie darauf, die Frontfläche sanft reinigen und reinigen auch ein paar Zentimeter an der Seite des Endoskops zur leichteren Handhabung. </li><li> Nach der Reinigung müssen Sie den Assistenten verfügen über die äußere Paar Handschuhe. </li><li> Haben der Assistent setzen auf dem Filter. Legen Sie die kurzen zylindrischen Vorsprung auf den Filter in seine Komplementär Loch in das Endoskop. Halten Sie das Endoskop vertikal, um den Prozess zu unterstützen. </li><li> Halten Sie die filter an Ort und Stelle, schieben Sie die Kappe über dem Filter und drücken Sie ihn über die Spitze des Endoskops. Achten Sie darauf, die Kappe sicher und mit den Kanten bündig mit dem Endoskop geschoben vollständig auf. Sicherstellen, dass die Lippe der Kappe leicht über die Spitze des Endoskops erstreckt. Schließlich, stellen Sie sicher, dass der Filter ist noch an Ort und bündig mit der Spitze des Endoskops. </li></ol> 11. Legen Sie die Endoskop zurück in die Speiseröhre und Image- 12. Entfernen Endoskop aus Ösophagus <ol><li> Mit Kimwipes ziehen vorsichtig den Deckel und Filter aus dem Endoskop. Werfen Sie die Schutzkappe und reinigen Sie den Filter für den nächsten Fall. </li></ol> 13. Filter reinigen <ol><li> Den Filter mit Kimwipes reinigen sanft. </li><li> Füllen Sie eine kleine Tasse mit CyDex und tauchen Sie den Filter für 12 min. Alle paar Minuten drehen Sie den Filter über. </li><li> Den Filter mit Kimwipes reinigen sanft. </li><li> Tauchen Sie Filter, diesmal in sterilem Wasser. Nach 5 min, verwenden Sie einen Spritzer bottle und sprühen sterilem Wasser über den Filter. </li><li> Legen Sie Filter auf ein paar Kimwipes und trocknen lassen. </li><li> Nach dem Trocknen in Lagerbehälter zwischen 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>

Es gibt nichts zu offenbaren.

Mit Standard-endoskopische Überwachung wird in BE Neoplasien oft übersehen, weil 8 gutartige Metaplasie kann nicht von hochgradiger Dysplasie und Adenokarzinom. Als Instrument, das besser ermöglichen würde, Gastroenterologen, um diesen Fehler zu beheben derzeit unvermeidbar, vital-Farbstoff-Fluoreszenz-Bildgebung verbessert hebt ein Drüsengewebe Morphologie, wodurch ein besonderes Merkmal, um die Gewebetypen differenzieren. Darüber hinaus, indem ein Sichtfeld von bis zu 2,5 cm, VFI ermöglicht Endoskopi...

In den letzten vierzig Jahren hat sich die Häufigkeit von Adenokarzinom des Ösophagus (EAC) signifikant 1,2 erhöht; noch durch die verspätete Diagnose, ist die Fünf-Jahres-Überlebensrate von weniger als 20% 3. Der aktuelle Standard der endoskopischen Überwachung in BE, die Vorstufe zu EAC, weißes Licht Endoskopie mit Zufalls vier Quadranten Zange &#39;Biopsien des Segments. Leider ist diese Technik oft vermisst Neoplasie, die flache, subtil und schwer zu auf Standard-Weißlicht-Bild 4 unterscheiden können. Zwar gab es mit Erfolg in der konfokalen Lasermikroskopie, um zelluläre Funktionen in vivo zu markieren, können Läsionen immer noch aufgrund der verringerten Sichtfeld 5 verpasst werden. Mit einer &quot;Brücke&quot;-Technologie, die für das weitere konfokale Bildgebung mikroendoskopischen markieren kann, wäre deutlich wertvoll sein. Folglich eine erweiterte Rote-Fahne bildgebendes Verfahren, das die Fähigkeit verbessert target und Biopsie frühen Neoplasien in BE werden, wäre zur Erfassung EAC in einem frühen, heilbaren Stadium und könnte zu wirksamere Behandlung und anschließend verbesserte Überlebensraten führen. VFI ist eine neuartige Technik, die hochauflösende Bildgebung mit epithelialen exogene aktuelle fluoreszierenden Kontrast, Proflavin kombiniert, um Drüsenmorphologie markieren und abzugrenzen Neoplasie (hochgradige Dysplasie und Krebs) im distalen Ösophagus in der Hoffnung auf eine Verbesserung der in-vivo-Diagnose 6. Bei Erregung der Proflavin, die Zellkerne innerhalb kurz nach der Anwendung konzentriert, die Fluoreszenzbilder eine räumliche Auflösung von 50 bis 100 um und ein Sichtfeld von bis zu 2,5 cm, so dass endoscopists Drüsenmorphologie sichtbar. Im Ergebnis ermöglicht dieser Ansatz Gastroenterologen zu klassischen Barrett-Metaplasie, die kontinuierlich, gleichmäßig angeordneten Drüsen und eine Gesamt homogene Morphologie hat von BE mit Neoplasien, die oblitera hat zu unterscheidention des Drüsen Architektur. Hier beschreiben wir das Protokoll dieser neuen Technik mit einem multispektralen Endoskop und repräsentative Ergebnisse liefern, um die Nutzung dieses Systems in der Darstellung der morphologischen Transformation von gutartigen Metaplasie hochgradige Dysplasie und Krebs zu demonstrieren.

<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

Die Fähigkeit, gutartige Metaplasie im Barrett-Ösophagus (BE) von Neoplasien in vivo differenzieren bleibt schwierig, da beide Gewebearten kann flach und nicht zu unterscheiden mit weißem Licht Bildgebung allein sein. Als Ergebnis würde eine Modalität, Drüsen Architektur hebt nützliche Neoplasie von gutartigen Epithel im distalen Ösophagus zu unterscheiden. VFI ist eine neuartige Technik, die eine exogene aktuelle fluoreszierende Kontrastmittel verwendet, um hochgradige Dysplasie und Krebs aus gutartigen Epithel abzugrenzen. Insbesondere werden die Fluoreszenzbilder eine räumliche Auflösung von 50 bis 100 um und ein Sichtfeld von bis zu 2,5 cm, so dass endoscopists Drüsenmorphologie sichtbar. Bei Erregung, erscheint klassischen Barrett-Metaplasie als kontinuierliche, gleichmäßig angeordneten Drüsen und eine Gesamt homogene Morphologie; Im Gegensatz dazu scheint Tumorgewebe überfüllt mit kompletten Auslöschung des Drüsen Rahmen. Hier bieten wir Ihnen einen Überblick überdie Instrumentierung und aufzuzählen das Protokoll dieser neuen Technik. Während VFI bietet einen Gastroenterologen mit dem Drüsen Architektur des verdächtigen Gewebes, können zelluläre Dysplasie nicht mit dieser Modalität gelöst werden. Als solche kann man nicht morphologisch Barrett-Metaplasie unterscheiden von BE mit Low-grade-Dysplasie über diese bildgebenden Verfahren. Durch den Handel aus einer Abnahme der Auflösung mit einem größeren Sichtfeld, kann dieser Abbildungssystem zumindest als rot-Flagge Abbildungsgerät, um verdächtige Läsionen Ziel und Biopsie verwendet werden; doch, wenn die Genauigkeit Maßnahmen sind vielversprechend, VFI kann die Standard-Imaging-Technik für die Diagnose von Neoplasien (definiert als entweder hochgradige Dysplasie oder Krebs) in der distalen Speiseröhre zu werden.

1B zeigt klassische Barrett-Ösophagus ohne Dysplasie umgeben an der Grenze von normalen Plattenepithel. Beginnend mit der flacheren Plattenepithel Gewebe, die peripher gelegen und durch die blauen Pfeile angedeutet ist, ist eine homogene Fläche von langweilig Fluoreszenz ohne Drüsen-Architektur vor. Die grünen Pfeile zeigen eine kreisförmige grüne Linie um den Plattenepithelkarzinomen Gewebe. Diese Gliederung ist Artefakt aus der Kappe des Endoskops resultieren. Der Umzug in die zentral gelegene B...

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-Farbstoff verbesserte Fluoreszenz-Bildgebung (VFI) ist eine neue Technik, die in vivo hochauflösenden bildgebenden Epithelzellen kombiniert mit exogenen aktuelle Fluoreszenz Gegensatz zu Drüsenmorphologie markieren und abzugrenzen Neoplasie (hochgradige Dysplasie und Krebs) im distalen Ösophagus.

Diagnose von Neoplasien im Barrett-Ösophagus mit Vital-Farbstoff verstärktes Fluoreszenz-Imaging

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

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Bioengineering

Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

11.9K Aufrufe.  Icahn School of Medicine at Mount Sinai.  Vital-Farbstoff verbesserte Fluoreszenz-Bildgebung (VFI) ist eine neue Technik, die in vivo hochauflösenden bildgebenden Epithelzellen kombiniert mit exogenen aktuelle Fluoreszenz Gegensatz zu Drüsenmorphologie markieren und abzugrenzen Neoplasie (hochgradige Dysplasie und Krebs) im distalen Ösophagus. 

Serie: 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.

Dreidimensionale Optische Auflösung photoakustische Mikroskopie

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

Forschung

Biotechnik

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.

Fluorescence Lifetime Imaging für molekulare Rotoren in lebenden Zellen

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.

Zeitraffer-Fluoreszenz-Imaging von Arabidopsis Wurzelwachstum mit Rapid Manipulation der Root-Umgebung mit dem 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.

Hochauflösende Räumlich-zeitliche Analyse der Rezeptordynamik von Einzelmolekül-Fluoreszenzmikroskopie

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.

3D-Tracking-Orbital in einer modifizierten Zwei-Photonen-Mikroskop: einen Antrag bei der Verfolgung von intrazellulären Vesikeln

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.

Von Fast Fluoreszenz-Imaging, um molekulare Diffusion Gesetz über Live Cell Membrane in einem kommerziellen Mikroskop

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

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electro Studium der Lungenkrebszellen unter Verwendung eines Mehrkanal-Zwei elektrischen Feldes Mikrofluidik-Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

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.

Durchführung von mehreren Imaging-Modi mit einem Fluoreszenzmikroskop

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.

Visualisierung von Adhäsion Bildung in den Zellen durch erweiterte Spinning-Disk-Total Internal Reflection-Fluoreszenz-Mikroskopie

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.

Verwendung von Nanoplasmon-Enhanced Scattering und Low-Magnification Microscope Imaging zur Quantifizierung von Tumor-Derivate-Exosomen

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