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.

倫理の声明：ヒト被験者、インフォームドコンセントと倫理の承認から細胞を採取する前に取得する必要があります。 1.ステインを準備注：実験日の汚れを準備します。 <ol><li> 15mlの遠心チューブに2.5ミリリットルのリン酸緩衝生理食塩水（PBS）に5μlのエチジウム（または4μM）および5μlのカルセインAM（または4μM）を追加します。ミックス。使用するまで室温で光とストアから遮蔽するためにアルミホイルで包みます。 </li></ol> 2.細胞を回収<ol><li>そのパッケージから細胞培養インサートを取り外し、各まぶたをサンプリングするためにラベルを付けます。永久的なマーカーを用いた膜プラスチックホルダー側の細胞収集のマークの向き。 ICを受ける地域の適切な健康状態を確認するために、適度な倍率（約20倍）で、スリットランプ検査を実施します。 </li><li> EAの下結膜嚢に局所麻酔薬（0.5％プロパラカイン塩酸塩）の1滴を分配chの目と目を保つために、参加者に指示するには、1分間閉じました。 </li><li>エバート上部眼瞼とまつ毛により所定の位置に保持します。蓋ワイパー領域との接触を避けます。 オプション：第二研究者の援助は、この手順を実行する必要があります。 </li><li>最小限の圧力で蓋ワイパー領域の中央部に垂直に膜を適用します。 </li><li> 5秒 - 3のための場所で膜を保持します。接触領域に半透明になった膜を観察します。この時点で、優しく膜を除去し、元の位置に戻し、「フリップ」に参加者のふたを可能にします。 </li><li>速やかに乾燥するのを防止するために、サンプルをPBSの一滴を適用します。膜は半透明に変わり、これが乾燥を示すように、いつでも不透明回すべきではありません。二次調査員は、サンプルの処理（セクション3）ですぐに進んでいます。 </li><li>結膜の整合性を確保するために、最終的な眼の検査を実施します。眼潤滑剤を分配し、参加者に指示麻酔効果が切れるまで、彼らの目をこすり避けるために（約15分）。 </li></ol> 3.サンプル処理<ol><li>マイクロはさみを使用して、垂直な細胞採取の中央から、半分（下流の分析のそれぞれに1つの半分）で膜をカット。プラスチック製のホルダーからそれを分離するための一つの膜半分の外縁に沿って切断。膜は、それ自体の上に折り畳まないことを保証を含む膜上に収集された細胞との相互作用を避けます。 </li><li>完全ホルダーから取り外す前に、ピンセットを用いて膜を固定します。 95％（v / v）エタノールを500μlを含む標識2 ml遠心チューブに膜を置き、（セクション4.2）を処理する前に、2時間の最大20分の最低固定するために残します。 </li><li>すぐに（セクション3.2で説明したように）膜の第二の半分を分離し、速やかにセクション4.1に進んでください。 </li></ol> 4.サンプル染色<ol><li> ImmunocytochemiCAL染色<ol><li>慎重に収集側を下に向けて35ミリメートルのガラスボトム培養皿に膜を置きます。膜が平坦であることを確認します。 </li><li>膜へのセクション1で組み立てられた免疫細胞化学的染色組成物のピペットを20μl。サンプルカールアップは、慎重にその縁部を押し下げることにより膜を平坦化する使い捨てのピペットチップを使用している場合。細胞収集領域との接触を避けます。 </li><li>ゆっくりとガラスカバースリップでメンブレンをカバーしています。サンプルの無駄な動きを避けてください。蓋付き皿を覆い、エッジの周りラボ・フィルムで密封します。ディッシュ（上部と下部）と試料の可視性の透明性を妨げないでください。ラボのマーカーでラベルを付けます。 </li><li>すぐにイメージング（セクション5.1）に進み、その後、膜の他の半分の細胞学的染色を継続するためにセクション4.2に戻ります。 </li></ol></li><li> 細胞学的染色 <ol><li>徐々にゆっくりとチューブのuに蒸留水500μLを加えることによって膜を水和セクション3.4に固定するためのsed。中身を取り出した後、混合するピペットチップに吸引する内容を数回。 </li><li>蒸留水500μLを加えます。削除した後、数秒間放置します。アルシアンブルー染色の500μLを加え、3分間のまま。汚れを削除し、3回連続500μlの蒸留水リンスや液体が透明にリンスするまで行います。 </li><li>ヘマトキシリン​​＃1染色の500μLを加え、3分間のまま。汚れを削除して、液体が透明リンスまで水リンスを蒸留または3回連続500μLを行います。透明な液体を吸引することにより、（セクション4.2.1の逆）を脱水。 </li><li>パパニコロウOG-6染色の500μLを加え、3分間のまま。汚れを削除し、1500μlの95％（v / v）エタノールリンスを行います。 </li><li>パパニコロウEA-65染色の500μLを加え、3分間のまま。汚れを削除し、（v / v）エタノールリンスの連続した​​3500μlの95％を実行します。 </li><li>完全に3回連続500μlの100％エタノールリンスを使用して脱水します。 TWEを使用しましたezers、溶液から試料を取り出します。感動細胞収集領域を避けます。 </li><li>スライドガラス上に膜を置きます。そのセルのコレクションラインを確保することは、並列またはイメージング中に容易に位置合わせのためのマージンをスライドする垂直のいずれかです。 </li><li> 100％エタノールのドロップを適用し、ガラススリップでカバーしています。すぐにイメージング（5.2節）に進みます。 </li></ol></li></ol> 5.サンプル画像<ol><li> イメージング蛍光免疫組織化学染色 <ol><li>画像取得ソフトウェアを実行しているコンピュータに接続されたデジタルカラーカメラを装備した11または蛍光顕微鏡12、共焦点レーザー走査顕微鏡（CLSM）を使用します。培養皿内に含まれるサンプルに、倒立顕微鏡を必要とすることができます。 </li><li>エチジウムホモダイマー-1（DNAの存在下で、617分の528 nmのEX /エム最大値）の吸収スペクトルおよび発光スペクトルとカルセインAM（517分の494 nmのEに係る顕微鏡11,12を設定しますX /エム最大値）。 </li><li> （ 例えば、2.5倍対物レンズ）顕微鏡で最も低い倍率を選択し、デジタルイメージングシステムを活性化させます。コンピュータのモニタ上に表示された試料を観察します。 </li><li>目的の細胞機能のサンプルを検査します。 </li><li>希望顕微鏡の倍率を選択し、イメージングソフトウェアを使用して画像を取得します。 </li><li>ネイティブ顕微鏡ファイル形式または非圧縮ファイル形式（ 例えば 、*の.raw、* .TIFF）のいずれかでファイルを保存します。 </li></ol></li><li> イメージング細胞学的染色 <ol><li>画像取得ソフトウェアを実行しているコンピュータに接続されたデジタルカラーカメラを装備していないフィルタを備えた標準的な実験明視野顕微鏡を、使用してください。 </li><li>顕微鏡ステージ上に配置し（ステップ4.2.8から）ロックスライドガラス。必要に応じて、全体のイメージング手順を通して、100％エタノールで試料を再水和します。 </li><li> （ 例えば 、2.5倍対物レンズ）顕微鏡で最も低い倍率を選択して、目を活性化させますEデジタルイメージングシステム。コンピュータのモニタ上に表示された試料を観察します。 </li><li>確保細胞収集は、顕​​微鏡ステージ制御のXまたはY軸のいずれかと整列されます。カバースリップを除去し、ピペットチップやピンセットを用いて試料を回転させることにより、必要に応じて調整してください。 </li><li>イメージングソフトウェアを使用して、最低の顕微鏡倍率で試料全体の画像（複数可）を取り込みます。 </li><li>適度な顕微鏡倍率（ 例えば、10 - 20X対物レンズ）に切り替えて、顕微鏡の光源とソフトウェア撮影パラメータ（露出、コントラスト、ホワイトバランスなど ）を調整し、その自動化されたソフトウェアメータリングを無効にします。将来の使用のためにこれらの設定を保存します。 </li><li>スキャンの開始点と終了点（細胞採取の例えば 、左上と右下の角）を決定します。 </li><li>イメージングソフトウェアを使用して、始点と終点内全体細胞収集領域の画像（複数可）を取り込みます。 betw 20％の側サイドと上から下への重複を確認してくださいすべての隣接する画像EEN。 </li><li>ネイティブ顕微鏡ファイル形式または非圧縮ファイル形式（ 例えば 、*の.raw、* .TIFF）を使用してファイルを保存します。 </li><li> （ 例えば、アドビPhotoshop Elementsの）選択肢の自動ステッチソフトウェアを使用して、最終的なパノラマ画像を生成します。最終的な画像に自動化されたステッチの精度を確認するために、ステップ5.2.5で撮影された参照画像）を使用します。 </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

印象の細胞学は、典型的には、混合セルロースエステル膜を用いて、眼球結膜上で実行されます。サンプルは、バルク材料のシートのサイズに切断し、滅菌、適用及び鉗子を使用して、結膜の表面から除去されます。同一の膜材料を用い、ピストン制御ICデバイスは、最近眼球結膜の表面形状に適合するように設計されており、アプリケーション13との間の一貫した圧...

眼球と対向する2ミリメートル狭い結膜地域-人間の上まぶたはわずか1で、毎日1約1万点滅を実行します。まばたきの間のワイピング動作による、眼瞼縁のこの部分は、「蓋ワイパー「領域2と呼ばれています。摩擦が原因で地球上ワイピング蓋に、常習的な点滅の間にこの領域で増加することが想定されます。これは、眼の快適で重要な役割を果たしている可能性があります。特定の経験眼の不快感のコンタクトレンズ装用者、これは3を着用レンズを停止するための主要な理由の一つです。コンタクトレンズが眼に配置されると、レンズ材料と眼の表面との間の摩擦係数を4に変更することが示されています。乾燥状態の症状は、この変更された摩擦2,5,6-に関連することができることを示唆する証拠があります。 この関連付けはまた、臨床的に観察現象に反映される可能性があり、特に蓋のwi上皮症（LWE）あたり、また上蓋マージン染色（ULMS）6と呼ばれます。 LWEは、眼の刺激の初期兆候とドライアイ疾患のための潜在的な予測因子です。これは、観察された上下の眼瞼縁の領域の生体染色によって測定されます。このグレーディングシステム2とその臨床的妥当性（自覚症状を持つ、すなわち、相関が）議論の下にまだあるが、眼の不快感は、最も重要なのは、患者にとって不便似臨床医と科学者のための難問のままと。 現在まで、少しは蓋ワイパー領域7の（サブ）携帯解剖学と生理学において臨床的に関連のバリエーションについては知られており、1つのレポートだけでは細胞学的方法8を利用します。印象細胞診（IC）は、膜9,10の適用によって眼球や瞼板結膜の上皮表面から細胞を収集するために40年以上にわたって使用されています。除去の際、接着細胞は、cを受けますytological染色および顕微鏡イメージング。蓋ワイパー領域における解剖学的および細胞性の違いのために、この技術は、最適化を必要とします。

<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.

プラスチックホルダーの上に張られた細胞培養膜は、蓋ワイパー結膜からの上皮細胞のハンドヘルド収集のための便利なツールです。この方法は、典型的には、IC膜を作製し、処理するために使用される追加の滅菌器具の必要性を排除する。 図1は、細胞培養インサートを使用して、蓋ワイパー領域のICを示します。我々は、新規な方法で蓋ワイパー領域?...

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.

蓋ワイパーエリアの印象細胞診

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 ビュー.  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. 

ビデオ: 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.

ホルマリン固定癌組織標本の腫瘍免疫研究の自動多重蛍光パネル

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

がん研究

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.

マウスの脳の側脳室における上衣繊毛のライブイメージング

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

神経科学

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.

脈絡叢上皮細胞の繊毛運動の観測<em&gt;エクスビボ</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.

ニワトリ頭蓋神経堤細胞培養物の作製と形態解析

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

発生生物学

前眼部HR-OCTを用いた眼表面扁平上皮腫瘍の評価

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.

著者スポットライト:眼表面扁平上皮腫瘍を特徴付けるための非侵襲的ツールとしての前方HR-OCT

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.

眼表面扁平上皮腫瘍の診断と治療モニタリングにおける前方高解像度光干渉断層撮影法

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

工学

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.

結膜から細胞を単離して表現する非侵襲的方法

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

免疫・感染学

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.

デジタル高速毛様体ビデオ顕微鏡を使用した鼻ブラッシングサンプリングと処理–COVID-19パンデミックへの適応

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.

イメージングと免疫組織化学のための網膜ホールマウントの準備のために最適化されたプロトコル

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

蓋ワイパーエリアの印象細胞診

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ホルマリン固定癌組織標本の腫瘍免疫研究の自動多重蛍光パネル

マウスの脳の側脳室における上衣繊毛のライブイメージング

脈絡叢上皮細胞の繊毛運動の観測

ニワトリ頭蓋神経堤細胞培養物の作製と形態解析

眼表面扁平上皮腫瘍の診断と治療モニタリングにおける前方高解像度光干渉断層撮影法

眼表面扁平上皮腫瘍の診断と治療モニタリングにおける前方高解像度光干渉断層撮影法

眼表面扁平上皮腫瘍の診断と治療モニタリングにおける前方高解像度光干渉断層撮影法

結膜から細胞を単離して表現する非侵襲的方法

デジタル高速毛様体ビデオ顕微鏡を使用した鼻ブラッシングサンプリングと処理–COVID-19パンデミックへの適応

イメージングと免疫組織化学のための網膜ホールマウントの準備のために最適化されたプロトコル