Questo lavoro è supportato dal National Cancer Institute presso il National Institute of Health di sovvenzione R01 CA140257-01.

<html> NOTA: Il consenso informato è stato ottenuto dai pazienti. Inoltre, questa ricerca è stata effettuata in conformità a tutte le linee guida istituzionali, nazionali e internazionali per il benessere umano. 1. Preparazione del computer <ol><li> Accendere il computer portatile e collegare USB dalla scheda di acquisizione DVI2USB. </li></ol> 2. Preparare Monitor <ol><li> Collegare il cavo DVI per monitorare e PinP per consentire l&#39;endoscopista di visualizzare video da questi schermi, piuttosto che al computer. </li><li> Assicurarsi che il monitor è in piedi su ed è impostato su DVI. </li><li> Collegare PinP sul retro del sistema Olympus quindi premere il pulsante di ingresso sul sistema Olympus per visualizzare l&#39;immagine sul grande schermo montato sulla parete. </li></ol> 3. Laser Diode Driver Setting <ol><li> Verificare che il driver diodo laser è spento. Dovrebbe essere acceso soltanto un paio di minuti prima di eseguire l&#39;imaging. </li></ol>e_title &quot;&gt; 4. Power Strip <ol><li> Collegare il computer portatile, processore, autista diodo laser, splitter DVI, e Epiphan scheda di acquisizione nella ciabatta. </li></ol> 5. Eseguire MDE Widefield sul Desktop Laptop <ol><li> Hit &#39;Cartella corrente&#39;. </li><li> Digitare il numero del paziente e le iniziali e premere &#39;Crea cartella del paziente&#39;. </li></ol> 6. Preparare Cap e Filter <ol><li> Durante la manipolazione filtro, usare sempre guanti e Kimwipes ridurre al minimo il trasferimento di petrolio e detriti al filtro. Garza o alcool tamponi possono lasciare fibre che possono interferire con l&#39;imaging. </li><li> Mettere un paio di Kimwipes su un tavolo per creare una piattaforma per fissare il filtro e tappo. Questa zona disinfettata dovrebbe essere facilmente accessibile al gastroenterologo durante la procedura. </li><li> Adagiare il filtro e tappo e mantenere Kimwipes chiuderà per l&#39;uso durante la procedura. </li></ol> 7. Preparazione del paziente <ol><li> Consenso del paziente sull&#39;usodi VFI e proflavina colorante prima di arrivare alla suite endo. </li><li> Posizione del paziente per la procedura endoscopia superiore. </li><li> Procedere con l&#39;imaging a luce bianca standard utilizzando un microscopio digitale multispettrale (MDM) 7. </li></ol> 8. Inserire e Spray proflavina <ol><li> Dopo aver terminato con l&#39;imaging a luce bianca, inserire e spray colorante proflavina su tessuto di interesse. 1-5 mm dovrebbe essere sufficiente a seconda della zona del tessuto di Barrett. Spruzzando il colorante proflavina in questo passaggio, il tempo sufficiente (almeno 1 min) è data per l&#39;assorbimento del tessuto sufficiente prima VFI. Proflavina, che è coperto da una nuova applicazione d&#39;investigazione della droga da parte della FDA (IND 102.217), è un agente di contrasto fluorescente che concentra in nuclei delle cellule poco dopo l&#39;applicazione. Sebbene VFI non può risolvere morfologia cellulare individuale, quando le padelle diodo laser nel tessuto, la luce riflessa consente la endoscopist apprezzare morpholo ghiandolare complessivagy. </li></ol> 9. Accendere diodo laser <ol><li> Accendere il diodo laser. Fate questo almeno 2 minuti prima dell&#39;inizio della rappresentazione. </li></ol> 10. Preparare endoscopio per VFI <ol><li> Ritirarsi completamente l&#39;endoscopio. </li><li> Prima di maneggiare la endoscopio, accertarsi che l&#39;assistente ha due paia di guanti. </li><li> Hanno l&#39;endoscopista tenere l&#39;endoscopio davanti alla assistente che si trova accanto alla piattaforma Kimwipe sopra indicato. </li><li> Con Kimwipes, hanno l&#39;assistente pulisce la punta dell&#39;endoscopio. Assicurarsi di pulire la superficie frontale con delicatezza e anche pulire a pochi centimetri lungo il lato dell&#39;endoscopio per facilitarne la movimentazione. </li><li> Dopo la pulizia, hanno l&#39;assistente smaltire la coppia esterna di guanti. </li><li> Hanno l&#39;assistente messo sul filtro. Inserire il breve protuberanza cilindrica sul filtro nel foro complementare in dell&#39;endoscopio. Mantenere l&#39;endoscopio verticale per aiutare il processo. </li><li> Tenendo il filter in posto, far scorrere il tappo sul filtro e spingerlo verso il basso sopra la punta dell&#39;endoscopio. Assicurarsi che il tappo è sicuro e spinto completamente con i bordi a filo con l&#39;endoscopio. Assicurarsi che il bordo della calotta si estende leggermente sopra la punta dell&#39;endoscopio. Infine, assicurarsi che il filtro sia ancora al suo posto e lavare con la punta dell&#39;endoscopio. </li></ol> 11. Inserire l&#39;endoscopio indietro nell&#39;esofago e immagine 12. Rimuovere endoscopio da Esofago <ol><li> Utilizzando Kimwipes tirare il tappo con attenzione e filtrare fuori l&#39;endoscopio. Buttare via il tappo e pulire il filtro per il prossimo caso. </li></ol> 13. Pulire il filtro <ol><li> Pulire delicatamente il filtro con Kimwipes. </li><li> Riempire una piccola tazza con CyDex e immergere il filtro per 12 min. Ogni pochi minuti girare il filtro sopra. </li><li> Pulire delicatamente il filtro con Kimwipes. </li><li> Immergere il filtro, questa volta in acqua sterile. Dopo 5 minuti, utilizzare uno schizzo bottle e inumidire acqua sterile sul filtro. </li><li> Posizionare il filtro su alcune Kimwipes e lasciare asciugare. </li><li> Una volta asciutto, posto in contenitore tra 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>

Non c&#39;è nulla da rivelare.

Con la sorveglianza endoscopica standard neoplasia in BE è spesso mancato 8 perché metaplasia benigna può essere indistinguibile da displasia di alto grado e adenocarcinoma. Come strumento che meglio consentire gastroenterologi per rimediare a questo errore al momento inevitabile, vitale-dye avanzata di imaging di fluorescenza evidenzia la morfologia ghiandolare di un tessuto fornendo così un tratto distintivo per differenziare i tipi di tessuto. Inoltre, prevedendo un campo visivo fino a 2,5 cm, VFI perm...

Negli ultimi 40 anni, l&#39;incidenza di adenocarcinoma esofageo (EAC) è notevolmente aumentato 1,2; ma a causa di una diagnosi tardiva, il tasso di sopravvivenza a cinque anni è inferiore al 20% 3. L&#39;attuale standard di sorveglianza endoscopica in BE, il precursore di EAC, è bianco endoscopia chiaro con biopsie del segmento casuali pinze a quattro quandrant. Purtroppo, questa tecnica spesso non trova neoplasia, che può essere piana, sottile e difficile distinguere il livello di imaging luce bianca 4. Mentre si è riusciti a usando microscopia laser confocale per evidenziare le caratteristiche cellulari in vivo, lesioni possono ancora essere persa a causa della diminuita campo di vista 5. Avere una tecnologia di &#39;ponte&#39; che possa evidenziare le aree di ulteriore microendoscopica l&#39;imaging confocale sarebbe notevolmente prezioso. Di conseguenza, una migliore modalità di imaging bandiera rossa che migliora la capacità di target e la biopsia neoplasia presto BE sarebbe fondamentale per individuare EAC in una fase iniziale e curabile e potrebbe portare a un trattamento più efficace e successivamente migliorato i tassi di sopravvivenza. VFI è una nuova tecnica che combina immagini ad alta risoluzione epiteliale con esogeno attualità fluorescente di contrasto, proflavina, per evidenziare la morfologia ghiandolare e delineare neoplasia (displasia di alto grado e cancro) nell&#39;esofago distale nella speranza di migliorare la diagnosi in vivo 6. Su eccitazione del proflavina, che concentra in nuclei cellulari poco dopo l&#39;applicazione, le immagini fluorescenti offrono una risoluzione spaziale di 50 a 100 um e un campo visivo fino a 2,5 cm, consentendo di visualizzare endoscopists morfologia ghiandolare. Di conseguenza, questo approccio permette di distinguere gastroenterologi metaplasia classico di Barrett, che ha continue, ghiandole uniformemente distanziate e una morfologia omogenea generale, da BE con neoplasia, che ha obliterazione dell&#39;architettura ghiandolare. Qui si descrive il protocollo di questa nuova tecnica con un endoscopio multispettrale, e fornire risultati rappresentativi per dimostrare l&#39;utilità di questo dispositivo in raffigurante la trasformazione morfologica da metaplasia benigna a displasia di alto grado e cancro.

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

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

La possibilità di differenziare metaplasia benigna in esofago di Barrett (BE) da neoplasia in vivo resta difficile in quanto entrambi i tipi di tessuto possono essere piatta e indistinguibile con il bianco della luce di imaging da solo. Come risultato, una modalità che evidenzia architettura ghiandolare sarebbe utile per discriminare neoplasia da epitelio benigno nell&#39;esofago distale. VFI è una tecnica innovativa che utilizza un agente di contrasto fluorescente topico esogeno per delineare displasia di alto grado e cancro da epitelio benigno. In particolare, le immagini fluorescenti offrono una risoluzione spaziale di 50 a 100 um e un campo visivo fino a 2,5 cm, consentendo di visualizzare endoscopists morfologia ghiandolare. Al momento di eccitazione, metaplasia classico di Barrett appare come continuo, ghiandole uniformemente distanziate e una morfologia omogenea globale; in contrasto, tessuto neoplastico appare affollata con completa obliterazione del quadro ghiandolare. Qui forniamo una panoramica dellela strumentazione ed enumerare il protocollo di questa nuova tecnica. Mentre VFI offre un gastroenterologo con l&#39;architettura ghiandolare del tessuto sospetto, displasia cellulare non può essere risolto con questa modalità. In quanto tale, non si può distinguere morfologicamente metaplasia di Barrett da BE con displasia a basso grado attraverso questa modalità di imaging. Decide di acquistare una diminuzione della risoluzione con un maggior campo di vista, questo sistema di imaging può essere utilizzato almeno un dispositivo di imaging rosso-bandiera a bersaglio e biopsia lesioni sospette; tuttavia, se le misure di precisione sono promettenti, VFI può diventare la tecnica di imaging standard per la diagnosi di neoplasia (definita sia come displasia di alto grado o cancro) nell&#39;esofago distale.

Figura 1B illustra classico Esofago di Barrett con displasia senza circondato sui confini da una normale epitelio squamoso. Cominciando con il tessuto squamoso piatta, che si trova perifericamente e indicato dalle frecce blu, una superficie omogenea di fluorescenza opaco è presente senza architettura ghiandolare. Le frecce verdi indicano una linea verde circolare che circonda il tessuto squamoso. Questo schema è artefatto risultante dal tappo dell&#39;endoscopio. Spostamento di tessuto di Barrett posi...

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-dye avanzata imaging di fluorescenza (VFI) è un romanzo in tecnica vivo che unisce imaging ad alta risoluzione epiteliale con contrasto fluorescente attualità esogeno per evidenziare la morfologia ghiandolare e delineare neoplasia (displasia di alto grado e cancro) nell&#39;esofago distale.

La diagnosi di neoplasia in esofago di Barrett con Vital-dye avanzata di imaging di fluorescenza

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

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

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Unit 1

Disorders of the Esophagus

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12.0K Views.  Icahn School of Medicine at Mount Sinai.  Vital-dye avanzata imaging di fluorescenza (VFI) è un romanzo in tecnica vivo che unisce imaging ad alta risoluzione epiteliale con contrasto fluorescente attualità esogeno per evidenziare la morfologia ghiandolare e delineare neoplasia (displasia di alto grado e cancro) nell'esofago distale. 

Video: 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.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

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

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Bioingegneria

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.

Lifetime Imaging di fluorescenza di rotori molecolari nelle cellule viventi

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

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.

Ad alta risoluzione Spatiotemporal Analisi del recettore Dynamics per singola molecola di microscopia a fluorescenza

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 Orbital Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

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.

Da Veloce imaging di fluorescenza di Molecular legge Diffusione su membrane cellulare dal vivo in un microscopio commerciale

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.

Electrotaxis Studi di polmone cellule tumorali usando un Multicanale Dual-campo elettrico Microfluidic 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.

Lo svolgimento di diverse modalità di Imaging con un microscopio a fluorescenza

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.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

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.

Utilizzando Nanoplasmon-diffusione migliorata e imaging del microscopio a basso ingrandimento per quantificare gli esosomi derivati dal tumore

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

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.