Este trabajo es apoyado por el Instituto Nacional del Cáncer en el Instituto Nacional de Salud de subvención R01 CA140257-01.

<html> NOTA: El consentimiento informado se obtuvo de los pacientes. Además, esta investigación se ha realizado en cumplimiento de todas las directrices institucionales, nacionales e internacionales para el bienestar humano. 1. Preparar Computadora <ol><li> Apague la computadora portátil y conectarse USB de la tarjeta de captura de DVI2USB. </li></ol> 2. Preparar monitor <ol><li> Conecte el cable DVI para monitorizar y PinP para permitir que el endoscopista para ver vídeos desde estas pantallas en lugar de la computadora. </li><li> Asegúrese de que el monitor está en pie y está listo para DVI. </li><li> Conecte PinP a la parte posterior del sistema Olympus luego presione el botón de entrada del sistema de Olympus para ver la imagen en el monitor de gran tamaño montado en la pared. </li></ol> 3. Laser Diode Configuración del controlador <ol><li> Asegúrese de que el controlador de diodo láser es OFF. Sólo se debe activar un par de minutos antes de realizar la imagen. </li></ol>e_title &quot;&gt; 4. Power Strip <ol><li> Conecte el ordenador portátil, procesador, controlador de diodo láser, splitter DVI y tarjeta de captura de Epiphan a la regleta. </li></ol> 5. Ejecute MDE Widefield en el escritorio del ordenador portátil <ol><li> Hit &#39;Current Folder&#39;. </li><li> Escriba el número y las iniciales del paciente y pulse &#39;Crear carpeta del Paciente. </li></ol> 6. Preparar Cap y Filtros <ol><li> Al manipular el filtro, utilice siempre guantes y Kimwipes para reducir al mínimo la transferencia de petróleo y desechos sólidos para el filtro. Gasa o hisopos con alcohol pueden dejar fibras que pueden interferir con las imágenes. </li><li> Coloque unas pocas Kimwipes sobre una mesa para crear una plataforma para establecer el filtro y la tapa. Esta área desinfectada debe ser fácilmente accesible para el gastroenterólogo durante el procedimiento. </li><li> Establecer el filtro y la tapa y mantenga Kimwipes cerca para su uso durante el procedimiento. </li></ol> 7. Preparación del paciente <ol><li> Consentimiento del paciente sobre el usode VFI y el tinte proflavina antes de llegar a la suite endo. </li><li> Posición del paciente para el procedimiento de endoscopia superior. </li><li> Proceda con el estándar de imágenes de luz blanca utilizando un microscopio digital multiespectral (MDM) 7. </li></ol> 8. Inserte y Spray Proflavina <ol><li> Después de terminar con la imagen de luz blanca, inserte y rociar colorante proflavina sobre tejido de interés. 1-5 mm debe ser suficiente dependiendo del área de tejido de Barrett. Por pulverizar el colorante proflavina en este paso, se da tiempo suficiente (por lo menos 1 min) para la absorción de suficiente tejido antes de VFI. Proflavina, que está cubierto en virtud de una solicitud de nuevo fármaco en investigación de la FDA (IND 102 217), es un agente de contraste fluorescente que se concentra en los núcleos celulares, poco después de la aplicación. Aunque VFI no puede resolver la morfología celular individual, cuando las cacerolas de diodos láser sobre el tejido, la luz reflejada permite al endoscopista apreciar morfologías glandular generalgía. </li></ol> 9. Encienda el láser de diodo <ol><li> Encienda el láser de diodo. Haga esto por lo menos 2 minutos antes de que empiece la imagen. </li></ol> 10. Preparar endoscopio para VFI <ol><li> Retire el endoscopio por completo. </li><li> Antes de manipular el endoscopio, asegúrese de que el asistente tiene dos pares de guantes. </li><li> Haga que el endoscopista sostener el endoscopio en la parte delantera de la asistente que está al lado de la plataforma Kimwipe detallado anteriormente. </li><li> Con Kimwipes, tiene su auxiliar y limpia la punta del endoscopio. Asegúrese de limpiar la superficie frontal con suavidad y también limpiar un par de centímetros a lo largo del lado del endoscopio para facilitar su manejo. </li><li> Después de la limpieza, tener el asistente disponer del par exterior de los guantes. </li><li> Haga que el asistente ponga en el filtro. Inserte el corto protuberancia cilíndrica en el filtro en su agujero complementario en el endoscopio. Mantenga el endoscopio vertical para ayudar en el proceso. </li><li> Sosteniendo el filter en su lugar, deslice la tapa sobre el filtro y empuje hacia abajo sobre la punta del endoscopio. Asegúrese de que la tapa esté segura y empujado por completo con los bordes al ras con el endoscopio. Asegúrese de que el labio de la tapa se extiende ligeramente sobre la punta del endoscopio. Por último, asegúrese de que el filtro se encuentra todavía en su lugar y al ras con la punta del endoscopio. </li></ol> 11. Inserte el endoscopio hacia el esófago y la Imagen 12. Retire endoscopio de Esófago <ol><li> Usando Kimwipes tire con cuidado la tapa y el filtro fuera del endoscopio. Tire de la tapa y limpie el filtro para el siguiente caso. </li></ol> 13. Limpie el filtro <ol><li> Limpie suavemente el filtro con Kimwipes. </li><li> Llene una taza pequeña con CyDex y sumergir el filtro durante 12 min. Cada pocos minutos se vuelven el filtro sobre. </li><li> Limpie suavemente el filtro con Kimwipes. </li><li> Sumerja el filtro, esta vez en agua estéril. Después de 5 minutos, utilice un chorro bOttle y ligeramente rocíe agua estéril sobre el filtro. </li><li> Coloque el filtro en unas pocas Kimwipes y dejar secar. </li><li> Una vez seco, colóquelo en un recipiente de almacenamiento intermedio 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>

No hay nada que revelar.

Con la vigilancia endoscópica estándar, neoplasia en BE muchas veces se pierde 8 porque metaplasia benigno puede ser indistinguible de la displasia de alto grado y adenocarcinoma. Como una herramienta que permitirá a los gastroenterólogos mejor para remediar este error actualmente inevitable y vital-dye mejorada de imágenes de fluorescencia destaca la morfología glandular del tejido proporcionando así una característica distintiva para diferenciar los tipos de tejidos. Además, al proporcionar un camp...

Durante los últimos cuarenta años, la incidencia de adenocarcinoma de esófago (EAC) ha aumentado significativamente 1,2; sin embargo, debido al diagnóstico tardío, la tasa de supervivencia a cinco años es inferior al 20% 3. El estándar actual de la vigilancia endoscópica en BE, el precursor de la EAC, es blanco endoscopia luz con biopsias del segmento aleatorias fórceps cuatro quandrant. Por desgracia, esta técnica a menudo se pierde la neoplasia, que puede ser plana, sutiles y difíciles de diferenciar en las imágenes de luz blanca estándar 4. Si bien ha habido éxito en el uso de microscopía láser confocal para resaltar características celulares in vivo, las lesiones todavía se puede perder debido a la disminución de campo de vista 5. Tener una tecnología &quot;puente&quot; que se pueden destacar las áreas para una mayor proyección de imagen confocal microendoscópica sería notablemente valiosa. En consecuencia, una modalidad de imagen de bandera roja mejorada que mejora la capacidad de Targét y biopsia de la neoplasia temprana en BE sería fundamental en la detección de EAC en una etapa temprana y curable y podría conducir a un tratamiento más eficaz y, posteriormente, la mejora de las tasas de supervivencia. VFI es una nueva técnica que combina imágenes de alta resolución epitelial con exógenos tópico fluorescente contraste, proflavina, para destacar la morfología glandular y delinear neoplasia (displasia de alto grado y el cáncer) en el esófago distal con la esperanza de mejorar el diagnóstico in vivo 6. Tras la excitación de la proflavina, que se concentra dentro de los núcleos de células poco después de la aplicación, las imágenes fluorescentes proporcionan una resolución espacial de 50 a 100 micras y un campo de visión de hasta 2,5 cm, lo que permite endoscopistas para visualizar la morfología glandular. Como resultado, este enfoque permite a los gastroenterólogos para distinguir la metaplasia de Barrett clásico, que tiene glándulas continuas, uniformemente espaciados y una morfología homogénea en general, a partir de SER con la neoplasia, que tiene obliteraciónción de la arquitectura glandular. A continuación se describe el protocolo de esta nueva técnica con un endoscopio multiespectral, y proporcionamos resultados representativos que demuestren la utilidad de este dispositivo en la representación de la transformación morfológica de metaplasia benigna a la displasia de alto grado y el cáncer.

<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 capacidad para diferenciar metaplasia benigno en el esófago de Barrett (EB) de neoplasia in vivo sigue siendo difícil, ya que ambos tipos de tejidos pueden ser planas e indistinguible con la imagen de luz blanca solo. Como resultado, una modalidad que pone de relieve la arquitectura glandular sería útil para discriminar la neoplasia benigna del epitelio en el esófago distal. VFI es una nueva técnica que utiliza un agente de contraste fluorescente tópico exógena para delinear la displasia de alto grado y el cáncer de epitelio benigno. Específicamente, las imágenes fluorescentes proporcionan una resolución espacial de 50 a 100 micras y un campo de visión de hasta 2,5 cm, lo que permite endoscopistas para visualizar la morfología glandular. Tras la excitación, metaplasia clásico de Barrett aparece glándulas como continuas, uniformemente espaciados y una morfología homogénea en general; en contraste, el tejido neoplásico aparece llena de obliteración completa del marco glandular. Aquí le ofrecemos una visión general dela instrumentación y enumerar el protocolo de esta nueva técnica. Mientras VFI permite un gastroenterólogo con la arquitectura glandular del tejido sospechoso, displasia celular no se puede resolver con esta modalidad. Como tal, no se puede distinguir morfológicamente metaplasia de Barrett de BE con displasia de bajo grado a través de esta modalidad de imagen. Por el comercio de una disminución en la resolución con un mayor campo de visión, este sistema de formación de imágenes se puede usar al menos como un dispositivo de formación de imágenes de color rojo-bandera para apuntar y biopsia de lesiones sospechosas; sin embargo, si las medidas de precisión son prometedores, VFI puede convertirse en la técnica de imagen estándar para el diagnóstico de la neoplasia (definido como la displasia de alto grado o cáncer) en el esófago distal.

La figura 1B representa clásica esófago de Barrett sin displasia rodeado en las fronteras por epitelio escamoso normal. Comenzando con el tejido escamoso más plana, que se encuentra en la periferia y se indica mediante las flechas azules, una zona homogénea de aburrida fluorescencia está presente sin la arquitectura glandular. Las flechas verdes indican una línea verde circular que rodea el tejido escamoso. Este esquema es artefacto resultante de la tapa del endoscopio. Mover al tejido del céntri...

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 mejorada de imágenes de fluorescencia (VFI) es una novela en la técnica de vivo que combina imágenes epitelial de alta resolución con contraste fluorescente tópico exógena para destacar la morfología glandular y delinear neoplasia (displasia de alto grado y el cáncer) en el esófago distal.

El diagnóstico de la neoplasia en el esófago de Barrett utilizando Vital-dye Enhanced Imaging Fluorescencia

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

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

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Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

11.9K vistas.  Icahn School of Medicine at Mount Sinai.  Vital-dye mejorada de imágenes de fluorescencia (VFI) es una novela en la técnica de vivo que combina imágenes epitelial de alta resolución con contraste fluorescente tópico exógena para destacar la morfología glandular y delinear neoplasia (displasia de alto grado y el cáncer) en el esófago distal. 

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.

Tridimensional óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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.

Imágenes de fluorescencia vida de rotores moleculares en las células vivas

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

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

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

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

Time-lapse de imágenes de fluorescencia del crecimiento de raíces de Arabidopsis con la manipulación del ambiente rápido y raíz mediante la RootChip

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

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolución espacio-temporal Análisis de la Dinámica del receptor por Casa molécula de microscopía de fluorescencia

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.

Rastreo Orbital 3D en un microscopio de dos fotones Modificado: una aplicación para el Seguimiento de vesículas intracelulares

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

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

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

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

De Fluorescencia Fast Imaging a la Ley de Difusión Molecular en membranas de células vivas en un microscopio Comercial

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.

Estudios electrotaxis de pulmón células de cáncer utilizando un Multicanal Dual-campo eléctrico chip de microfluidos

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.

Realización de múltiples modos de la proyección de imagen con un microscopio de fluorescencia

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.

Visualizar la formación de adherencias en las células por medio de avanzados Spinning microscopía de fluorescencia de reflexión interna Total de 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.

Usando dispersión mejorada por Nanoplasmon y imágenes de microscopio de baja magnificación para cuantificar exosomas derivados del tumor

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