Name: impression cytology of the lid wiper area
Uploaded: 2016-08-09T15:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

Ética declaración: Antes de la recolección de células de sujetos humanos, el consentimiento informado y se debe obtener la aprobación ética. 1. Preparar a las manchas NOTA: Preparar mancha en el día del experimento. <ol><li> Añadir 5 l de etidio (o 4 M) y 5 l de calceína AM (o 4 M) a 2,5 ml solución salina tamponada con fosfato (PBS) en 15 ml tubo de centrífuga; mezcla. Envolver en papel de aluminio para protegerlo de la luz y se almacena a temperatura ambiente hasta su uso. </li></ol> 2. Las células Collect <ol><li> Retire insertos de cultivo celular de su paquete y la etiqueta de cada párpado a muestrear. Marca de orientación de recogida de células en el lado del soporte de plástico de la membrana usando un marcador permanente. Conducta cortó la inspección de la lámpara con un aumento moderado (aproximadamente 20 veces) para confirmar la salud apropiada de la región que someterse a IC. </li><li> Deje caer una gota de anestésico tópico (0,5% de hidrocloruro de proparacaına) en el saco conjuntival inferior del eaojo ch e instruir a los participantes para mantener los ojos cerrados durante un minuto. </li><li> Evert párpado superior y mantenga en su lugar por las pestañas; evitar el contacto con la zona de la tapa del limpiaparabrisas. Opcional: la ayuda de un investigador secundario puede ser requerido para realizar este paso. </li><li> Aplicar membrana perpendicularmente sobre la parte central de la región de limpiaparabrisas tapa con una presión mínima. </li><li> Mantenga la membrana en su lugar por 3 - 5 segundos. Observar la membrana translúcida convertido en el área de contacto. En este punto, retire con cuidado la membrana y permitir la tapa del participante de &quot;flip&quot; en su lugar. </li><li> Inmediatamente aplicar una gota de PBS a la muestra, para evitar que se sequen. Membrana a su vez, translúcido y opaco no debe girar en cualquier momento, ya que esto indica secado. Tiene el investigador secundaria proceder inmediatamente con el procesamiento de las muestras (sección 3). </li><li> Realizar comprobación final del ojo para asegurar la integridad de la conjuntiva. Prescindir de los lubricantes oculares e instruir a los participantespara evitar frotarse los ojos hasta que el efecto anestésico desaparece (aprox. 15 min). </li></ol> Procesamiento 3. Muestra <ol><li> El uso de micro-tijeras, corte de la membrana en un medio (un medio para cada uno de los análisis aguas abajo), perpendicular a través de la mitad de la recogida de células. Corte a lo largo del margen exterior de una media membrana para separarla de la soporte de plástico; evitar la interacción con las células recogidas en la membrana, incluyendo asegurar que la membrana no se pliega sobre sí mismo. </li><li> Asegure la membrana con unas pinzas antes de separar por completo del soporte. Lugar membrana en la etiqueta del tubo de centrífuga de 2 ml que contenía 500 l de 95% (v / v) de etanol y dejar de fijar durante un mínimo de 20 minutos hasta un máximo de 2 horas antes del procesamiento (sección 4.2). </li><li> segunda mitad inmediatamente separada de la membrana (tal como se describe en la sección 3.2) y proceder de inmediato con la sección 4.1. </li></ol> La tinción 4. Muestra <ol><li> ImmunocytochemiLa tinción cal <ol><li> Coloque con cuidado la membrana de vidrio de placa de cultivo de 35 mm con fondo colección lado hacia abajo; asegurar membrana es plana. </li><li> Pipeta de 20 l de composición de tintura inmuno montado en la sección 1 a la membrana. Si rizos de ejemplo, utilizar puntas de pipeta desechables para aplanar cuidadosamente la membrana presionando sus bordes hacia abajo. Evitar el contacto con las zonas de recogida de células. </li><li> cubrir con cuidado la membrana con cubierta de vidrio deslizante. Evitar movimientos innecesarios de la muestra. Cubrir el plato con la tapa y el sello con el laboratorio de película alrededor de los bordes. No obstruya la transparencia del plato (superior e inferior) y la visibilidad de la muestra. La etiqueta del marcador de laboratorio. </li><li> Inmediatamente proceder con la proyección de imagen (sección 5.1), y luego volver a la sección 4.2 para continuar con la tinción citológica de la otra mitad de la membrana. </li></ol></li><li> La tinción citológica <ol><li> Poco a poco hidratar la membrana por adición lenta de 500 l de agua destilada en el tubo uSed para su fijación en el apartado 3.4. Aspirar los contenidos varias veces a punta de pipeta para mezclar, a continuación, eliminar el contenido. </li><li> Añadir 500 l de agua destilada. Dejar reposar durante unos segundos, luego retire. Añadir 500 l de tinción de azul Alcian y dejar actuar durante 3 minutos. Retire las manchas y realice 3 consecutiva de 500 l de agua destilada enjuagues o hasta que el líquido se enjuaga clara. </li><li> Añadir 500 l de hematoxilina # 1 de manchas y dejar actuar durante 3 minutos. Retire las manchas y realizar tres lavados consecutivos 500 l de agua destilada o hasta que el líquido se enjuaga clara. Deshidratar (inversa de la sección 4.2.1) aspirando el líquido claro. </li><li> Añadir 500 l de Papanicolaou OG-6 mancha y dejar actuar durante 3 minutos. Retirar la mancha y realice una 500 l 95% (v / v) de enjuague de etanol. </li><li> Añadir 500 l de Papanicolaou EA-65 mancha y dejar actuar durante 3 minutos. Quitar las manchas y realizar 3 consecutiva 500 l 95% (v / v) de etanol se enjuaga. </li><li> Totalmente deshidratar usando tres 500 l 100% aclarados consecutivos de etanol. El uso de TWEezers, retire la muestra de la solución; evitar las zonas de recolección de células tocar. </li><li> Coloque la membrana sobre portaobjetos de vidrio; asegurar que la línea de recogida de células es paralela o perpendicular a deslizarse margen para la alineación más fácil durante la exploración. </li><li> Aplicar una gota de etanol al 100% y cubrir con el resbalón de vidrio. Inmediatamente proceder con la proyección de imagen (sección 5.2). </li></ol></li></ol> 5. Muestra de Imágenes <ol><li> Las manchas fluorescentes de imágenes Inmunocitoquímico <ol><li> Use un microscopio confocal de barrido láser (CLSM) 11 o microscopio de fluorescencia 12, equipado con una cámara digital de color, conectado a un equipo que ejecuta un software de adquisición de imágenes. Debido a las muestras que se están contenidos dentro de placas de cultivo, un microscopio invertido puede ser necesario. </li><li> Configurar microscopio 11,12 de acuerdo con los espectros de absorción y emisión de etidio homodímero-1 (528/617 nm maxima Ex / Em en presencia de DNA) y calceína AM (494/517 nm Emáximos x / Em). </li><li> Seleccionar aumento más bajo del microscopio (por ejemplo, el objetivo 2,5X) y activar el sistema de imagen digital; observar la muestra exhibida en el monitor de la computadora. </li><li> Inspeccionar la muestra para características celulares de interés. </li><li> Seleccionar magnificación del microscopio deseado y adquirir imágenes utilizando software de imagen. </li><li> Guardar archivos en cualquier formato de archivo nativo de microscopio o un formato de archivo sin comprimir (por ejemplo, * .raw, * .tiff). </li></ol></li><li> Las manchas de imagen citológica <ol><li> Utilizar un microscopio estándar de laboratorio de campo brillante sin filtros, equipado con una cámara digital en color, conectado a un ordenador con un software de adquisición de imágenes. </li><li> Lugar y portaobjetos de vidrio de bloqueo (de la etapa 4.2.8) en la platina del microscopio. Rehidratar la muestra con 100% de etanol, según sea necesario, a lo largo de todo el procedimiento de formación de imágenes. </li><li> Seleccionar aumento más bajo del microscopio (por ejemplo, el objetivo 2,5X) y activar THsistema de imagen digital e; observar la muestra exhibida en el monitor de la computadora. </li><li> garantizar la recogida de células se alinea con X o Y del eje de control de platina del microscopio. Ajustar si es necesario, mediante la eliminación de la hoja de la cubierta y la rotación de la muestra usando una punta de pipeta o pinzas. </li><li> El uso de la imagen (s) de captura de la totalidad de la muestra con el microscopio óptico bajo el software de imágenes,. </li><li> Cambiar a la magnificación del microscopio moderada (por ejemplo, 10 - 20X objetivo) y ajustar la fuente de luz del microscopio y los parámetros de imagen de software (exposición, contraste, balance de blancos, etc.), y desactivar la medición de software automatizado. Guardar esta configuración para su uso futuro. </li><li> Determinar puntos de inicio y final de la exploración (por ejemplo, esquinas superiores izquierda y derecha inferior de la recolección de células). </li><li> El uso de la imagen (s) de captura de toda la zona de recogida de células dentro de los puntos inicial y final del software de imágenes,. Garantizar un 20% de lado a lado y de arriba a abajo solapamiento between todas las imágenes adyacentes. </li><li> Guardar archivos usando el formato de archivo nativo de microscopio o un formato de archivo sin comprimir (por ejemplo, * .raw, * .tiff). </li><li> Generar una imagen panorámica final utilizando el software automatizado de costura de elección (por ejemplo, Adobe Photoshop Elements). Usar imágenes de referencia tomados en el paso 5.2.5) para confirmar la exactitud de la costura automatizada en la imagen final. </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

La citología de impresión se lleva a cabo normalmente en la conjuntiva bulbar, utilizando membranas de ésteres mixtos de celulosa. Las muestras se cortan a tamaño de las hojas de material a granel, esterilizar, aplicar y retirar de la superficie conjuntival usando fórceps. Utilizando el mismo material de la membrana, un dispositivo de IC pistón controlado fue diseñado recientemente para que coincida con la geometría de la superficie de la conjuntiva bulbar, y mantener una presión constante entre las aplicacione...

El párpado superior humano ejecuta alrededor de 10.000 parpadea cada día 1, con sólo un 1 - 2 mm región conjuntival estrecha opuestos del globo ocular. Debido a su movimiento de limpieza durante el parpadeo, esta porción de la margen del párpado se ha denominado la región &quot;tapa de limpiaparabrisas&quot; 2. Se supone que la fricción se incrementa en esta región durante el parpadeo habitual, debido a la tapa limpia el mundo. Esto puede jugar un papel importante en la comodidad ocular. Los portadores de lentes de contacto, en particular, la experiencia malestar ocular y esta es una de las principales razones para cesar el uso de lentes 3. Cuando una lente de contacto se coloca en el ojo, el coeficiente de fricción entre el material de la lente y la superficie ocular se ha demostrado que cambiar 4. Hay evidencias que sugieren que los síntomas de sequedad podrían estar relacionados con esta fricción alterada 2,5,6. Esta asociación también se puede reflejar en los fenómenos clínicamente observables, especialmente wi tapapor epiteliopatía (LSA), también llamado margen del párpado superior tinción (ULMS) 6. LSA es una señal temprana de la irritación ocular y un predictor potencial para la enfermedad del ojo seco. Se observa y se mide por la tinción vital de las regiones borde del párpado superior e inferior. Si bien este sistema de clasificación 2 y su validez clínica (es decir, la correlación con síntomas subjetivos) están siendo objeto de debate, molestia ocular sigue siendo un enigma para los médicos y científicos por igual y, sobre todo, un inconveniente para los pacientes. Hasta la fecha, poco se sabe acerca de las variaciones clínicamente relevantes en el (sub) la anatomía y la fisiología celular de la zona del limpiaparabrisas tapa 7 y un único informe hace uso de métodos citológicos 8. La citología de impresión (IC) ha sido empleado durante más de 40 años para recoger las células de la superficie epitelial de la conjuntiva tarsal bulbar o mediante la aplicación de una membrana de 9,10. Tras la retirada, las células adherentes se someten a ctinción ytological y de imágenes microscópicas. Debido a las diferencias anatómicas y celulares de la región de limpiaparabrisas tapa, esta técnica requiere la optimización.

<table><tbody><tr><td>Alcian Blue, 1% in 3% Acetic Acid</td><td>Sigma</td><td>B8438-250ML</td><td></td></tr><tr><td>Hematoxylin, Gill 1</td><td>Sigma</td><td>GHS116-500ML</td><td></td></tr><tr><td>Papanicolaou stain, OG-6</td><td>Sigma</td><td>HT40116-500 ml</td><td></td></tr><tr><td>Papanicolaou stain, Modified EA</td><td>Sigma</td><td>HT40232-1L</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Life Technologies</td><td>L-3224</td><td>contains ethidium homodimer-1 and Calcein AM dyes</td></tr><tr><td>Alcaine (0.5% proparacaine hydrochloride)</td><td>Alcon</td><td></td><td></td></tr><tr><td>Millicell Cell Culture Insert, 12&amp;nbsp;mm, hydrophilic PTFE, 0.4&amp;nbsp;&amp;micro;m</td><td>EMD Millipore</td><td>PICM01250&amp;nbsp;</td><td></td></tr><tr><td>35 mm Glass Bottom Dishes</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td></td></tr></tbody></table>

impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

La membrana de cultivo celular abarcado sobre el soporte de plástico es una herramienta conveniente para la recolección de mano de las células epiteliales de la conjuntiva tapa del limpiaparabrisas. Este método elimina la necesidad de instrumentos esterilizados adicionales típicamente usados ​​para preparar y manejar las membranas de IC. La Figura 1 representa el IC de la zona de limpiaparabrisas tapa usando un inserto de cultivo celular. Hemos optimizado dos pr...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Citología de Impresión del Área de limpiaparabrisas tapa

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

impression-cytology-of-the-lid-wiper-area

Research

JoVE Journal

Medicine

10.4K vistas.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

Video: Impression Cytology of the Lid Wiper Area

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Panel de inmunofluorescencia Multiplex automatizado para estudios de inmuno-Oncología en los especímenes del tejido formalina-fijo Carcinoma

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Investigación

Investigación sobre el cáncer

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Neurociencias

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Observación del Movimiento ciliar de las células epiteliales del plexo coroideo<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

Preparación y análisis morfológico de cultivos de células de cresta neural craneal de pollo

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Biología del Desarrollo

Evaluación de la neoplasia escamosa de la superficie ocular mediante TRC-UP del segmento anterior

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

Autor destacado: HR-OCT anterior como herramienta no invasiva para caracterizar la neoplasia escamosa de la superficie ocular

Anterior segment high-resolution optical coherence tomography (HR-OCT) is a promising non-invasive modality for the diagnosis and therapeutic evaluation of ocular surface squamous neoplasia (OSSN). Here, the system setup, scanning technique, and representative diagnostic results are presented.

Tomografía de coherencia óptica anterior de alta resolución en el diagnóstico y seguimiento terapéutico de la neoplasia escamosa de la superficie ocular

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. ...

Ingeniería

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Una forma no invasiva de aislar y células fenotípicas de la conjuntiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Inmunología e Infección

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Muestreo y procesamiento del cepillado nasal mediante videomicroscopía ciliar digital de alta velocidad: adaptación para la pandemia de COVID-19

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Protocolo optimizado para Retina Preparación Wholemount de la Imagen y la inmunohistoquímica

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Citología de Impresión del Área de limpiaparabrisas tapa

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Panel de inmunofluorescencia Multiplex automatizado para estudios de inmuno-Oncología en los especímenes del tejido formalina-fijo Carcinoma

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

Observación del Movimiento ciliar de las células epiteliales del plexo coroideo

Preparación y análisis morfológico de cultivos de células de cresta neural craneal de pollo

Tomografía de coherencia óptica anterior de alta resolución en el diagnóstico y seguimiento terapéutico de la neoplasia escamosa de la superficie ocular

Tomografía de coherencia óptica anterior de alta resolución en el diagnóstico y seguimiento terapéutico de la neoplasia escamosa de la superficie ocular

Tomografía de coherencia óptica anterior de alta resolución en el diagnóstico y seguimiento terapéutico de la neoplasia escamosa de la superficie ocular

Una forma no invasiva de aislar y células fenotípicas de la conjuntiva

Muestreo y procesamiento del cepillado nasal mediante videomicroscopía ciliar digital de alta velocidad: adaptación para la pandemia de COVID-19

Protocolo optimizado para Retina Preparación Wholemount de la Imagen y la inmunohistoquímica