Die Autoren bedanken sich bei Mauna Kea Technologies für technische Unterstützung danken. Die Autoren danken Shelly Hsiao für technische Hilfe und Kathleen E. Mach für kritische Bewertung. Diese Arbeit wurde zum Teil durch NIH R01CA160986 um JCL unterstützt

Ein. Vorbereitung des Patienten <ol><li> Consent Patienten für diagnostische Zystoskopie und andere endourologischen Verfahren wie die transurethrale Resektion der Blase Tumor (TURBT) für CLE geplant. Fügen Sie in der Zustimmung eine Beschreibung der Verwendung von intravesikale und / oder intravenöse Fluorescein als Kontrastmittel. Erkundigen Geschichte der Überempfindlichkeitsreaktion auf Fluorescein. </li><li> Der Patient wird für die Zystoskopie (typischerweise in Steinschnittlage) positioniert und vorbereitet in einem sterilen Mode. </li><li> Fahren Sie mit dem Standard-Weißlicht-Zystoskopie (WLC) mit einem starren oder flexiblen Zystoskop durch die Harnröhre. </li><li> Umfrage alle Regionen der Blase unter weißem Licht (Abbildung 1), und notieren Sie Regionen von Interesse für die CLE Untersuchung. </li><li> Neben WLC kann die Blase mit anderen makroskopischen optischen Bildgebungsverfahren einschließlich Fluoreszenz Zystoskopie (2A) oder Schmalband Bildgebung (2B) zu id abzubildendenentify weitere Bereiche der Verdacht durch Charakterisierung mit CLE gefolgt. </li><li> Verwenden eine Elektrode oder Kauterisierung Resektionsschlinge einen kleinen Kauter Markierung an der interessierenden Bereiche, die sowohl bebildert und für pathologische Bestätigung biopsiert platzieren. Der Kauter mark ermöglicht Re-Lokalisierung für nachfolgende CLE Bildgebung und Biopsie. </li></ol> 2. Kontrastmittel <ol><li> Intravesikale Instillation des Kontrastmittels <ol><li> Planen durch Verdünnen klinischer Qualität 10% Fluorescein-Natrium in steriler physiologischer Kochsalzlösung (0,9% NaCl) auf die gewünschte Konzentration. Beispielsweise vorzubereiten 400 ml 0,1% Fluorescein durch Verdünnen von 4 ml 10% Fluorescein in 396 ml physiologischer Kochsalzlösung. </li><li> Eine sterile Harnkatheters in die Blase und instill verdünnten Kontrastmittels durch Schwerkraft mit einer 60 ml Spritze Katheterspitze. </li><li> Klemmen Sie den Blasenkatheter, um Kontrastmittel innewohnenden für 5 min zu halten. </li><li> Lassen Sie Kontrastmittel aus der Blase. Verwenden Sie die 60 ml ca.theter-Spitze Spritze zu waschen Restkontrast aus der Blase mit physiologischer Kochsalzlösung, ca. 240-300 ml. </li><li> Fahren Sie mit CLE Bildgebung. </li></ol></li><li> Intravenöse Injektion von Fluorescein <ol><li> Zeichnen Sie 1,0 ml Fluorescein in die Spritze. Spritze zu befestigen intravenösen Zugang und injizieren 0,5 - 1,0 ml des Fluoresceins intravenös als Bolus. Spülleitung mit physiologischer Kochsalzlösung. Dies wird typischerweise durch die Anästhesieteam im Operationssaal erfolgen. </li><li> Fahren Sie mit CLE Bildgebung. </li></ol></li></ol> 3. CLE Imaging <ol><li> Entfernen Sie die sterilisiert CLE-Sonde (Cellvizio) aus der Verpackung und verbinden Sie es mit dem Laser-Scanning-Einheit (Mauna Kea Technologies, Paris, Frankreich) </li><li> Warm up und Kalibrierung des bildgebenden Sonde, indem Sie die Anweisungen des Herstellers. </li><li> Setzen Sie den Zystoskop oder Resektoskop in die Blase. Verwenden Sie eine 0-Grad-Objektiv mit dem starren Zystoskop. </li><li> Schieben Sie den CLE-Sonde entlang der WOrking des Zystoskops mit der Spitze direkt hinter dem Zystoskop. </li><li> Unter weißem Licht, suchen Sie die zuvor markierten Regionen von Interesse. Verwenden zusätzlicher physiologischer Kochsalzlösung Bewässerung zur Visualisierung der Schleimhaut erforderlich und aufrechtzuerhalten moderaten Distension der Blase. </li><li> Manipulieren CLE-Sonde mit der Hand des Bedieners für direkte en face Kontakt der Regionen von Interesse. Für optische Schnitte von den interessierenden Bereichen, sanft zu erhöhen und Druck auf das bildgebende Sonde unter Beibehaltung direkten Kontakt. <ol><li> Tumore an der anterioren Blase zu erreichen, kann ein Standard Albarran Brücke erleichtern Auslenkung der Bildgebungssonde für direkten Kontakt mit dem interessierenden Bereich (2C). </li></ol></li><li> Gemeinsame Regionen von Interesse sind papillären Tumor, nicht-papillären Tumor Übergang zwischen normal erscheinenden Schleimhaut und Tumor und gerötete Flecken, die alle abgebildet werden können, die entweder intravenös werdensischen oder intravenöse Fluorescein Verwaltung. Intravenöse Fluorescein ist zum Abbilden der Resektion Bett, prostatische Urethra und penile Harnröhre erforderlich. </li><li> Da die bildgebenden Sonde tastet Region von Interesse, die Aufnahme von Bildern für eine spätere Analyse. Hinweis gesamten zellulären Morphologie, Grenze, Organisation und Präsenz von Neo-Angiogenese. </li><li> Nach Abschluss der Bildgebung, entfernen CLE-Sonde von der Arbeit Kanal. </li><li> Wenn gewünscht, erhalten kalt-cup Biopsien aus entsprechenden Regionen von Interesse, die von CLE gescannt wurden. Verwenden Kauter Markierungen als Landmarken für Co-Registrierung der abgebildeten Bereiche, die biopsiert. </li><li> Füllen TURBT pro Routine. Bei Patienten, die intravenöse Fluorescein erhalten, sollten Sie kontinuierlichen Resektoskopen zur Visualisierung während der Resektion zu verbessern, wie Fluorescein wird von der Niere in die Blase ausgeschieden ca. 5 min nach intravenöser Verabreichung. </li><li> Rufen Sie gespeicherte Bild Aufnahmen von konfokalen Prozessor und Breitenwirkungct Bildanalyse. </li></ol>

<ol>
	<li>Thiberville, L., 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=Human+in+vivo+fluorescence+microimaging+of+the+alveolar+ducts+and+sacs+during+bronchoscopy.">Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.</a> Eur. Respir. J. 33, 974-985 (2009).</li><li>Dunbar, K. B., Okolo, P., Montgomery, E., Canto, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+endomicroscopy+in+Barrett's+esophagus+and+endoscopically+inapparent+Barrett's+neoplasia:+a+prospective,+randomized,+double-blind,+controlled,+crossover+trial.">Confocal laser endomicroscopy in Barrett's esophagus and endoscopically inapparent Barrett's neoplasia: a prospective, randomized, double-blind, controlled, crossover trial.</a> Gastrointest. Endosc. 70, 645-654 (2009).</li><li>Buchner, A. 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=Comparison+of+probe-based+confocal+laser+endomicroscopy+with+virtual+chromoendoscopy+for+classification+of+colon+polyps.">Comparison of probe-based confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps.</a> Gastroenterology. 138, 834-842 (2010).</li><li>Pech, O., 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=Confocal+laser+endomicroscopy+for+in+vivo+diagnosis+of+early+squamous+cell+carcinoma+in+the+esophagus.">Confocal laser endomicroscopy for in vivo diagnosis of early squamous cell carcinoma in the esophagus.</a> Clin. Gastroenterol. Hepatol. 6, 89-94 (2008).</li><li>Goetz, 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=In+vivo+confocal+laser+endo+microscopy+of+the+human+liver:+a+novel+method+for+assessing+liver+microarchitecture+in+real+time.">In vivo confocal laser endo microscopy of the human liver: a novel method for assessing liver microarchitecture in real time.</a> Endoscopy. 40, 554-562 (2008).</li><li>Meining, A., 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=Detection+of+cholangiocarcinoma+in+vivo+using+miniprobe-based+confocal+fluorescence+microscopy.">Detection of cholangiocarcinoma in vivo using miniprobe-based confocal fluorescence microscopy.</a> Clin. Gastroenterol. Hepatol. 6, 1057-1060 (2008).</li><li>Wu, 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=Dynamic+real-time+microscopy+of+the+urinary+tract+using+confocal+laser+endomicroscopy.">Dynamic real-time microscopy of the urinary tract using confocal laser endomicroscopy.</a> Urology. 78, 225-231 (2011).</li><li>Sonn, G. A., 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=Optical+biopsy+of+human+bladder+neoplasia+with+in+vivo+confocal+laser+endomicroscopy.">Optical biopsy of human bladder neoplasia with in vivo confocal laser endomicroscopy.</a> J. Urol. 182, 1299-1305 (2009).</li><li>Sonn, G. A., 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=Fibered+confocal+microscopy+of+bladder+tumors:+an+ex+vivo+study.">Fibered confocal microscopy of bladder tumors: an ex vivo study.</a> J. Endourol. 23, 197-201 (2009).</li><li>Adams, W., 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=Comparison+of+2.6-+and+1.4-mm+imaging+probes+for+confocal+laser+endomicroscopy+of+the+urinary+tract.">Comparison of 2.6- and 1.4-mm imaging probes for confocal laser endomicroscopy of the urinary tract.</a> J. Endourol. 25, 917-921 (2011).</li><li>Wiesner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+endomicroscopy+for+the+diagnosis+of+urothelial+bladder+neoplasia:+a+technology+of+the+future?.">Confocal laser endomicroscopy for the diagnosis of urothelial bladder neoplasia: a technology of the future?.</a> BJU Int. 107, 399-403 (2011).</li><li>Wallace, M. B., 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=The+safety+of+intravenous+fluorescein+for+confocal+laser+endomicroscopy+in+the+gastrointestinal+tract.">The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract.</a> Aliment. Pharmacol. Ther. 31, 548-552 (2010).</li><li>Konda, V. J. A., 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=First+assessment+of+needle-based+confocal+laser+endomicroscopy+during+EUS-FNA+procedures+of+the+pancreas+(with+videos.">First assessment of needle-based confocal laser endomicroscopy during EUS-FNA procedures of the pancreas (with videos.</a> Gastrointest. Endosc. 74, 1049-1060 (2011).</li><li>Liu, J. -. J., Droller, M., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Optical+Imaging+Technologies+in+Bladder+Cancer:+Considerations+and+Perspectives.">New Optical Imaging Technologies in Bladder Cancer: Considerations and Perspectives.</a> J. Urol. 188, 361-368 (2012).</li><li>Hsiung, P. -. L., 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=Detection+of+colonic+dysplasia+in+vivo+using+a+targeted+heptapeptide+and+confocal+microendoscopy.">Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy.</a> Nat. Med. 14, 454-458 (2008).</li></ol>

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Erreichen und Aufrechterhalten festen en face Kontakt zwischen der Sonde zur Abbildung und der Blasenschleimhaut ist der wichtigste Schritt in Erfassen optimaler Bildqualität. Es ist etwa 3-5 Patienten Lernkurve, um die Geschicklichkeit zu entwickeln, um die bildgebende Sonde zu manipulieren und zu halten die Sonde konstant während der Bildaufnahme. Darüber, wie dieses Verfahren in vivo durchgeführt wird, kann Bewegungen des Patienten (dh der Atemwege) und vaskuläre Pulsationen auf die bi...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Cellvizio 100 Series </td><td> Mauna Kea Technologies </td><td> 100 Series </td><td> Inklusive konfokalen Prozessor und LSU: F400-v2 bei 488 nm </td></tr><tr><td> Cellvizio Konfokale Minisonden </td><td> Mauna Kea Technologies </td><td> Gastroflex UHD </td><td></td></tr><tr><td> AK-FLUOR 10% </td><td> Akorn, Inc. </td><td> NDC 17478-253-10 </td><td></td></tr></tbody></table>

probe-based confocal laser endomicroscopy of the urinary tract: the technique

Probe-basierte konfokale Laser Endomikroskopie (CLE) ist eine aufstrebende optische Imaging-Technologie, die in Echtzeit ermöglicht in vivo Mikroskopie von Schleimhäuten im Standard-Endoskopie. Mit Anwendungen, die derzeit in den Atemwegen 1 und Gastrointestinaltrakt hat 2-6 CLE auch in den Harnwegen für Blasenkrebs Diagnose untersucht. 7-10 Cellular Morphologie und Gewebe Mikroarchitektur mit Mikrometer-Skala Auflösung in Echtzeit gelöst werden, zusätzlich zu dynamische Bildgebung des normalen und pathologischen Gefäßsystem. 7 Die Sonde basierenden CLE System (Cellvizio, Mauna Kea Technologies, Frankreich) aus einem wiederverwendbaren faseroptische Sonde zur Abbildung, die mit einem 488 nm Laser-Scanning-Einheit. Das bildgebende Sonde in den Arbeitskanälen von Standard flexible und starre Endoskope eingefügt. Ein Endoskop-basierten CLE System (Optiscan, Australien), in dem der konfokale Endomikroskopie functionality auf das Endoskop integriert ist, ist auch im Magen-Darm-Trakt verwendet werden. Angesichts des größeren Umfangs Durchmesser, jedoch ist die Anwendung in den Harnwegen derzeit ex vivo Anwendung begrenzt. 11 Konfokale Bildaufnahme durch den direkten Kontakt des abbildenden Sonde mit dem Ziel-Gewebe durchgeführt und als Videosequenzen. Wie in den Gastrointestinaltrakt, erfordert Endomikroskopie der Harnwege eine exogenenous Kontrastmittel-häufigsten Fluorescein, die intravenös oder intravesikal verabreicht werden kann. Intravesikale Verabreichung ist eine gut etablierte Methode, um pharmakologische Wirkstoffe lokal einzuführen mit minimaler systemischer Toxizität, die einzigartig in der Harnwege. Fluorescein schnell Flecken, die extrazelluläre Matrix und verfügt über ein etabliertes Sicherheitsprofil. 12 Imaging-Sonden mit verschiedenen Durchmessern ermöglichen Kompatibilität mit verschiedenen Kalibers Endoskope. Bisher wurden 1,4 und 2,6 mm Sonden mit flexiblen und ri beurteiltgid Zystoskopie. 10 Aktuelle Verfügbarkeit eines &lt;1 mm Bildgebungssonde 13 eröffnet die Möglichkeit des CLE in der oberen Harnwege während Ureteroskopie. Fluoreszenz Zystoskopie (dh photodynamischen Diagnose) und Schmalband-Imaging sind zusätzliche Endoskop-basierten optischen Abbildungsmodalitäten 14, die mit CLE kombiniert werden, um multimodale Bildgebung der Harnwege erreichen kann. In der Zukunft kann CLE mit molekularem Kontrastmitteln wie fluoreszenzmarkierten Peptiden und Antikörpern für 15 Endoskopabbildungsvorrichtung von Krankheitsprozessen mit molekularem Spezifität gekoppelt sein.

CLE Bilder gespeichert werden als Video-Sequenz-Dateien Graustufen bei 12 Frames pro Sekunde. Bild Interpretation wird in Echtzeit und kann verwendet werden, um die klinische Entscheidungsfindung im Rahmen einer experimentellen Protokoll auswirken werden. Offline-Analyse, die der Überprüfung der Videosequenz, zusätzliche Bildverarbeitung wie Mosaiken, 7 und Vergleich mit Standard-Pathologie beinhaltet, die während der Lernphase Phase mit der Technologie verbunden wichtig. 1B und 1C sind Darstellungen CLE ...

Watch this Scientific Journal Video about Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique at JoVE.com

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-basierte konfokale Laser Endomikroskopie ermöglicht Echtzeit-Mikroskopie des menschlichen Harnwege während Zystoskopie, Bereitstellung dynamischer, intravital Darstellung pathologischer Zustände wie Blasenkrebs mit zellulärer Auflösung. Endomikroskopie kann ergänzen die diagnostische Genauigkeit der Standard-Weißlicht-Endoskopie und bieten intraoperative Bildführung zur chirurgischen Resektion verbessern.

Probe-basierte konfokale Laser Endomikroskopie der Harnwege: Die Technik

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

probe-based-confocal-laser-endomicroscopy-urinary-tract

Research

JoVE Journal

Medicine

20.7K Aufrufe.  Stanford University School of Medicine. Probe-basierte konfokale Laser Endomikroskopie ermöglicht Echtzeit-Mikroskopie des menschlichen Harnwege während Zystoskopie, Bereitstellung dynamischer, intravital Darstellung pathologischer Zustände wie Blasenkrebs mit zellulärer Auflösung. Endomikroskopie kann ergänzen die diagnostische Genauigkeit der Standard-Weißlicht-Endoskopie und bieten intraoperative Bildführung zur chirurgischen Resektion verbessern.

Serie: Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Hochauflösende LWL-Mikroendoskopiesysteme für In situ Cellular Imaging

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

Forschung

Biotechnik

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Video-rate konfokalen Mikroskopie und Mikroendoskopiesysteme

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

Multiplexen fokussiert Ultraschall-Stimulation mit Fluoreszenz-Mikroskopie

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Ingenieurwesen

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Optische Überprüfung der neuartige Bakterien-spezifischen Sonden auf Ex Vivo menschlichen Lungengewebe durch konfokale Laser Endomikroskopie

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Immunologie und Infektion

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Räumlich-zeitliche In-vivo-Bildgebung von okulären Wirkstoffabgabesystemen mittels faseroptischer konfokaler Lasermikroendoskopie

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

Ultrasonographie des adulten männlichen Harntraktes für Harnfunktionstests

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

Medizin

Enucleation of the Prostate for the Treatment of Benign Prostatic Hyperplasia Using a 980 nm Diode Laser

In the aging male population, the occurrence of lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is a common problem. Here, we introduce a new technique called 980 nm diode laser enucleation (DiLEP) to treat BPH1. Diode lasers can absorb both water and hemoglobin at the same time, so they are good for cutting and hemostasis2. The diode laser was approved by the FDA in 2007, and has been used in the treatment of BPH because of its effective cutting and hemostasis effect3. DiLEP presents several advantages over other techniques, such as TURP, HoLEP, and PVP. During the procedure, we define the boundary of a high-volume prostate and separate it into three lobes with a diode laser by burning two rings and one groove (like a Cupid&#39;s arrow). Compared to other procedures, mDiLEP has fewer intraoperative complications, a shorter learning curve, and achieves more tissue resection.

Here, we present a protocol for modified 980 nm diode laser enucleation to treat large volume benign prostatic hyperplasia.

Enukleation der Prostata zur Behandlung der gutartigen Prostatahyperplasie mit einem 980 nm Diodenlaser

In the aging male population, the occurrence of lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is a common problem. ...

Focal Laser Ablation of Prostate Cancer: An Office Procedure

In this article, we describe and illustrate an outpatient procedure for focal laser ablation (FLA) of prostate cancer (PCa). The procedure is conceptually similar to a fusion biopsy and is performed under local anesthesia in a clinic setting; treatment time is usually less than one hour. Laser insertion is guided by ultrasound; lesion targeting is via magnetic resonance imaging-ultrasound (MRI/US) fusion, as in targeted prostate biopsy. Real-time ablation monitoring is achieved utilizing a thermal probe adjacent to the laser fiber. The video demonstrates procedure planning, patient preparation, various steps during the procedure, and treatment monitoring. Safety, feasibility, and efficacy of this approach have been established during a previous trial. Outpatient FLA under local anesthesia is an option for management of intermediate risk prostate cancer.

This article presents and describes an outpatient treatment for prostate cancer using focal laser ablation. Laser catheter placement is guided by MRI-ultrasound fusion imaging in a fashion similar to prostate needle biopsy. Treatment is monitored in real-time with a thermal probe, placed adjacent to the laser fiber.

Fokale Laserablation von Prostatakrebs: Ein Büroverfahren

In this article, we describe and illustrate an outpatient procedure for focal laser ablation (FLA) of prostate cancer (PCa). The procedure is conceptually ...

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

Ex Vivo Analyse von mechanisch aktivierten Ca2+ Transienten in Urothelzellen

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and ...

Micro-Ultrasound Guided Transperineal Prostate Biopsy: A Clinic-Based Procedure

Prostate cancer is the most common solid malignancy in men and requires a biopsy for diagnosis. This manuscript describes a freehand micro-ultrasound guided transperineal technique performed under local anesthesia, which maintains accuracy, keeps patients comfortable, has low adverse events, and minimizes the need for disposables. Prior micro-ultrasound-guided transperineal techniques required general or spinal anesthesia. The key steps described in the protocol include (1) the placement of the local anesthesia, (2) micro-ultrasound imaging, (3) and the visualization of the anesthetic/biopsy needle while uncoupled from the insonation plane. A retrospective review of 100 patients undergoing this technique demonstrated a 68% clinically significant cancer detection rate. Pain scores were prospectively collected in a subset of patients (N = 20) and showed a median procedural pain score of 2 out of 10. The 30 day Grade III adverse event rate was 3%; one of these events was probably related to the prostate biopsy. Overall, we present a simple, accurate, and safe technique for performing a micro-ultrasound-guided transperineal prostate biopsy.

This manuscript outlines the steps for performing a micro-ultrasound-guided transperineal prostate biopsy under local anesthesia.

Prostate cancer is the most common solid malignancy in men and requires a biopsy for diagnosis. This manuscript describes a freehand micro-ultrasound ...