Gli autori desiderano ringraziare Mauna Kea Tecnologie per il supporto tecnico. Gli autori hanno anche ringraziare Shelly Hsiao per l&#39;assistenza tecnica e Kathleen E. Mach per la revisione critica. Questo lavoro è stato in parte finanziato dal NIH R01CA160986 a JCL

1. Preparazione del paziente <ol><li> Il consenso del paziente in programma per la cistoscopia diagnostica e altre procedure endourologiche come resezione transuretrale del tumore della vescica (TURBT) per CLE. Includere nella consenso una descrizione dell&#39;uso di fluoresceina intravescicale e / o endovenosa come agente di contrasto. Informarsi storia della reazione di ipersensibilità ad fluoresceina. </li><li> Paziente viene posizionato per cistoscopia (tipicamente in posizione litotomica) e preparato in modo sterile. </li><li> Procedere con lo standard cistoscopia a luce bianca (WLC) con un cistoscopio rigido o flessibile attraverso l&#39;uretra. </li><li> Indagine tutte le regioni della vescica in luce bianca (Figura 1) e annotare le regioni di interesse per le indagini CLE. </li><li> Oltre al WLC, la vescica può essere ripreso con altre modalità di imaging ottico comprendente macroscopici cistoscopia a fluorescenza (Figura 2A) o imaging banda stretta (Figura 2B) per identify ulteriori aree di sospetto seguita da caratterizzazione con CLE. </li><li> Utilizzare un elettrodo di cauterizzazione o loop resezione per inserire un segno piccolo cauterio adiacenti alle regioni di interesse, che saranno entrambi impressi e biopsia per la conferma patologica. Il marchio cauterizzazione facilita ri-localizzazione per la successiva CLE imaging e biopsia. </li></ol> 2. Di contrasto <ol><li> Instillazione intravescicale di mezzo di contrasto <ol><li> Preparare diluendo clinica sodio fluoresceina grade 10% in soluzione salina normale (0,9% NaCl) alla concentrazione desiderata. Per esempio, preparare 400 ml di 0,1% fluorescina diluendo 4 ml di 10% fluoresceina in 396 ml di soluzione salina normale. </li><li> Inserire un catetere sterile in vescica urinaria e infondere agente di contrasto diluito per gravità utilizzando un catetere da 60 ml-siringa. </li><li> Fissare il catetere urinario per contenere l&#39;agente di contrasto interiore per 5 min. </li><li> Scolare agente di contrasto di vescica. Usa la 60 catheter-siringa per lavare contrasto residua dalla vescica con soluzione fisiologica, circa 240-300 ml. </li><li> Procedere alla CLE imaging. </li></ol></li><li> L&#39;iniezione endovenosa di fluoresceina <ol><li> Disegna 1,0 ml di fluoresceina nella siringa. Collegare la siringa per iniettare l&#39;accesso per via endovenosa e 0,5-1,0 ml di fluorescina per via endovenosa in bolo. Lavare linea con soluzione fisiologica. Questo viene fatto tipicamente dal team di anestesia in sala operatoria. </li><li> Procedere alla CLE imaging. </li></ol></li></ol> 3. CLE Imaging <ol><li> Rimuovere il sterilizzato CLE immagini della sonda (Cellvizio) dalla confezione e collegarlo all&#39;unità di scansione laser (Mauna Kea Technologies, Parigi, Francia) </li><li> Riscaldare e calibrare la sonda immagini seguendo le istruzioni del produttore. </li><li> Reinserire il cistoscopio o resettoscopio nella vescica. Utilizzare uno 0 lente gradi con il cistoscopio rigido. </li><li> Far avanzare il CLE immagini della sonda lungo il working canale del cistoscopio con la punta appena oltre il cistoscopio. </li><li> In luce bianca, individuare le regioni precedentemente contrassegnati di interesse. Utilizzare ulteriori irrigazione normale soluzione salina, se necessario per la visualizzazione della mucosa e mantenere moderata distensione della vescica. </li><li> Manipolare CLE immagini della sonda con la mano dell&#39;operatore per contatto diretto en face delle regioni di interesse. Per sezionamento ottico delle regioni di interesse, delicatamente aumentare e diminuire la pressione alla sonda di imaging restando a contatto diretto. <ol><li> Per raggiungere i tumori situati nella vescica anteriore, uno standard Albarran ponte può facilitare deflessione della sonda di imaging per il contatto diretto con la regione di interesse (Figura 2C). </li></ol></li><li> Regioni comuni di interesse tumore papillare, non papillare del tumore, la transizione tra mucosa normale e tumore, e chiazze eritematose, che possono essere esposte utilizzando via endovesico o la somministrazione per via endovenosa di fluoresceina. Fluoresceina endovenosa è necessaria per l&#39;imaging del letto resezione, uretra prostatica e uretra peniena. </li><li> Mentre la sonda esegue la scansione di immagini regione di interesse, di registrare le immagini per le analisi future. Nota generale morfologia cellulare, confine, l&#39;organizzazione, e la presenza di neo-angiogenesi. </li><li> Al termine dell&#39;imaging, rimuovere CLE immagini sonda dal canale di lavoro. </li><li> Se lo si desidera, ottenere freddo tazza biopsie dalle regioni corrispondenti di interesse che sono stati analizzati da CLE. Utilizzare marcature cauterio come punti di riferimento per la co-registrazione delle aree sotto forma di immagini che vengono sottoposti a biopsia. </li><li> Completa TURBT per routine. Nei pazienti che hanno ricevuto per via endovenosa di fluoresceina, considerare l&#39;utilizzo di resettoscopi flusso continuo per migliorare la visualizzazione durante la resezione, come fluoresceina viene escreto dal rene in vescica di circa 5 minuti dopo la somministrazione per via endovenosa. </li><li> Recupera le registrazioni di immagine salvati da confocale processore e conduct analisi dell&#39;immagine. </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. -. 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Nessun conflitto di interessi dichiarati.

Raggiungere e mantenere solidi contatti en face tra la sonda di imaging e la mucosa della vescica è il passo più importante per acquisire una qualità ottimale dell&#39;immagine. C&#39;è approssimativamente 3-5 apprendimento paziente di sviluppare l&#39;abilità di manipolare la sonda imaging e tenere la sonda costante durante l&#39;acquisizione dell&#39;immagine. Inoltre, poiché questo procedimento è condotto in vivo, i movimenti del paziente (cioè respiratoria) e pulsazioni vascolari p...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Cellvizio Serie 100 </td><td> Mauna Kea Technologies </td><td> Serie 100 </td><td> Include processore confocale e LSU: F400-v2 a 488 nm </td></tr><tr><td> Cellvizio confocale Miniprobe </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-based laser confocale endomicroscopy (CLE) è una tecnologia emergente di imaging ottico che permette in tempo reale in microscopia in vivo di superfici mucose durante il normale endoscopia. Con applicazioni attualmente nelle vie respiratorie e gastrointestinali 1, 2-6 CLE è stato analizzato nel tratto urinario per la diagnosi del cancro della vescica. 7-10 morfologia cellulare e microarchitettura del tessuto può essere risolto con risoluzione scala micron in tempo reale, oltre a l&#39;imaging dinamico del sistema vascolare normale e patologico. 7 La sonda basata CLE sistema (Cellvizio, Mauna Kea Technologies, Francia) costituita da una sonda a fibre ottiche di imaging riutilizzabile accoppiato ad un&#39;unità di scansione laser 488 nm. La sonda di imaging viene inserito nei canali di lavoro standard, endoscopi flessibili e rigidi. Un endoscopio basato CLE sistema (Optiscan, Australia), in cui il functionalit endomicroscopy confocaley è integrato sulla endoscopio, è utilizzato anche nel tratto gastrointestinale. Data la portata diametro più grande, tuttavia, applicazione nel tratto urinario è attualmente limitato all&#39;uso ex vivo. 11 acquisizione immagine confocale viene fatto attraverso il contatto diretto della sonda imaging con il tessuto bersaglio e registrata come sequenze video. Come nel tratto gastrointestinale, endomicroscopy del tratto urinario richiede un exogenenous contrasto agente più comunemente fluoresceina, che può essere somministrato per via endovenosa o per via endovescicale. Somministrazione endovescicale è un metodo consolidato per introdurre agenti farmacologici localmente con minima tossicità sistemica che è unico per il tratto urinario. Fluoresceina rapidamente le macchie della matrice extracellulare e ha un profilo di sicurezza. Imaging 12 sonde di diversi diametri garantire la compatibilità con endoscopi di calibro diversi. Fino ad oggi, 1,4 e 2,6 millimetri sonde sono state valutate con il flessibile e ricistoscopia gid 10. recente disponibilità di un &lt;1 mm di imaging sonda 13 si apre la possibilità di CLE nel tratto urinario superiore durante ureteroscopia. Cistoscopia a fluorescenza (cioè diagnosi fotodinamica) e l&#39;imaging a banda stretta sono ulteriori endoscopio a base di modalità di imaging ottico 14 che possono essere combinati con CLE per raggiungere l&#39;imaging multimodale del tratto urinario. In futuro, CLE può essere accoppiato con mezzi di contrasto molecolari come peptidi contrassegnati in modo fluorescente 15 e anticorpi per l&#39;imaging endoscopico di processi di malattia con specificità molecolare.

CLE immagini sono salvate come file di sequenze di video in scala di grigi a 12 fotogrammi al secondo. L&#39;interpretazione dell&#39;immagine avviene in tempo reale e possono essere utilizzati per influenzare il processo decisionale clinico in un protocollo sperimentale. Analisi offline, che include la revisione della sequenza video, un&#39;ulteriore elaborazione delle immagini come mosaicatura, 7 e confronto con standard di patologia, è importante durante la fase di apprendimento associata con la tecnologi...

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

Sonda basata endomicroscopy laser confocale consente in tempo reale la microscopia del tratto urinario umano durante cistoscopia, fornendo dinamico, imaging intravitale di stati patologici come il cancro della vescica con risoluzione cellulare. Endomicroscopy può aumentare l&#39;accuratezza diagnostica di livello luce bianca endoscopia e fornire orientamenti immagine intraoperatoria per migliorare la resezione chirurgica.

Probe-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

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 Views.  Stanford University School of Medicine. Sonda basata endomicroscopy laser confocale consente in tempo reale la microscopia del tratto urinario umano durante cistoscopia, fornendo dinamico, imaging intravitale di stati patologici come il cancro della vescica con risoluzione cellulare. Endomicroscopy può aumentare l'accuratezza diagnostica di livello luce bianca endoscopia e fornire orientamenti immagine intraoperatoria per migliorare la resezione chirurgica.

Video: 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.

Ad alta risoluzione in fibra ottica per Microendoscopy In situ Cellular Imaging

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

Ricerca

Bioingegneria

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-tasso di Microscopia confocale e scansione Microendoscopy

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.

Multiplexing focalizzato ad ultrasuoni stimolazione con microscopia di fluorescenza

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

Ingegneria

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.

Sonde ottiche Screening di nuovi batteri specifici sul tessuto polmonare umano Ex Vivo di endomicroscopia confocale Laser

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

Immunologia e infezioni

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.

Imaging in vivo spazio-temporale di sistemi di somministrazione di farmaci oculari mediante microscopia laser confocale a fibre ottiche

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).

Ultrasonografia del tratto urinario maschile adulto per test funzionali urinari

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

Medicina

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.

Enucleazione della prostata per il trattamento dell'iperplasia prostatica benigna mediante laser a diodi da 980 nm

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.

Ablazione laser focale del cancro alla prostata: una procedura d'ufficio

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 Analisi di transitori di Ca2+ attivati meccanicamente in cellule uroteliali

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 ...