この作品は、医科大学グラーツの衛生フォンによってサポートされていました。すべての被験者は、彼らのインフォームドコンセントを与えた。治験実施計画書の制度的承認は、医科大学グラーツから入手したものです。

1。検体収集<ol><li>口腔内のサービスの4ヶ月後に固定口蓋パンダを取り外します。 （図1および図2）、それらを削除する前にアプライアンスを分離するためにNOLAドライフィールドのシステムを使用してください。 </li><li>汚染（図3と図4）を追加することなく、パンダを除去する滅菌ペンチ、手袋、トレーを使用してください。 </li><li> -20℃でザルスタット120ミリリットル瓶で保管してオブジェクト24時間以内に氷やプロセス上の実験室に連れて行く。 </li></ol> 2。バイオフィルムの固定<ol><li>滅菌メスでバイオフィルムフレークを削り取る、または滅菌ピンセットで作品を収集​​。 </li><li>サンプルが十分に覆われるまで氷冷4％PFA溶液を加える。 </li><li> 3〜12時間のために4 ° C（凍結しない）で混合物をインキュベートする。長い固定時間以上固定温度は、オリゴヌクレオチドプローブへの透過性が低いグラム陰性細胞の細胞エンベロープをレンダリングしてもよい。 </li><li> PFA溶液を除去し、氷冷した1 × PBSで洗浄する。 rにこの手順を2〜3回繰り返す残留PFAを残したまま削除する。 </li><li> 1巻のサンプルを再懸濁します。氷冷した1 × PBSと1容量を加える。氷冷96％（v / v）エタノール。 </li><li> -20℃でサンプルを保存するこのプロトコルに従って固定サンプルは、年に数ヶ月間保存することができます。 </li></ol> 3。固定試料の脱水<ol><li>顕微鏡スライドにPFA固定試料物質5〜30μlを適用します。 </li><li> 46で乾燥℃で約15分間以上室温で。リゾチームとホルムアミドを解凍。 </li><li>したがって、細胞壁の部分的な破壊によって細胞内へのプローブの浸透を促進する、10分間室温でリゾチーム（1mg/ml）250μlを添加する。 </li><li> 50％、3分ごとに80％、96％（v / v）エタノールに浸漬スライド。エタノールの濃度系列の脱水効果は、細胞膜を崩壊されます。 </li><li> ℃で10分間、46でスライドを乾燥させる。 </li></ol> （4） のin - situハイブリダイゼーション<ol><li> 1 mlの準備新鮮なハイブリダイゼーション緩衝液（濃度は表1を参照）。本研究で使用したホルムアミド濃度：10％（EUBmix）、20％（Bac303、POGI、MUT590）または45％（LGCmix）。 </li><li>オリゴヌクレオチドプローブ溶液を融解。解凍したプローブは氷上で保存し、光から保護する必要があります。 </li><li>ハイブリダイゼーション緩衝液200μlに各プローブ2μl加え、よく混ぜ、顕微鏡スライド上に脱水試料に混合物を適用する。 </li><li>正方形のペトリ皿にティッシュを一枚の紙を置き、ティッシュペーパーの上に残っているハイブリダイゼーション緩衝液を注ぐ。 </li><li>すぐに皿に水平にスライドを置き、皿を閉じます。 46℃で1〜5時間（90分、ほとんどのケースで十分です）のためにオーブンでインキュベートする。スライドからハイブリダイゼーション溶液の蒸発を防止する水分室など、料理機能。特に、ホルムアミドの蒸発は、非標的細胞への非特異的プローブを引き起こす可能性があります。 </li><li> （conceは表2を参照の洗浄バッファー50 mlを調製ntrations）。 48に50 mlチューブおよび予熱の洗浄バッファーを準備℃の水浴中。洗浄工程は48℃で行われ</li><li>ハイブリダイゼーションオーブンからスライドして皿を取り外します。すぐに予め温めておいた洗浄液は少量でハイブリダイゼーション緩衝液を洗い流し、そして残りの洗浄バッファにスライドを移す。 </li><li> 10〜15分間48℃で水浴し、インキュベートに戻し、洗浄バッファとスライドを含むチューブを置きます。 </li><li>チューブからスライドを削除し、残留洗浄バッファーを除去するために2〜3秒間氷冷のddH 2 Oに手をつける。 </li><li> （圧縮空気の使用を推奨）可能な限り迅速にスライドを風乾する。速乾性は、プローブ解離が削減されます。 </li><li>乾燥スライドはプローブ与え蛍光シグナルをあまり失うことなく、数週間のために-20℃で暗所に保存することができます。 </li></ol> 5。顕微鏡検査<ol><li> FISHと洗浄後に、APPLスライドの左右の端（凍結切片を前にこのステップに室温に温める必要があります）にantifadent近いのy 2滴。 </li><li>上に顕微鏡のカバーガラスを置き、antifadentはスライド全体に広がるまで待ちます。 antifadent量が過剰となり、顕微鏡の画像をぼかすことができることに注意してください。 </li><li>適切なフィルターまたはレーザーを搭載した共焦点レーザー走査型顕微鏡で試料を観察。私たちは、ライカTCSユニット（; NA 1.2 HCX PL APO/63x）を使用。データは、IMARISやアミラなどのソフトウェアで解析することができます。 </li><li>プローブ与え蛍光が減少し始める前にantifadentに埋め込まれているスライドは、° C（​​凍結しない）までの7日間まで4で保存することができます。また、antifadentはのddH 2 Oを用いて削除することができます、そして乾燥させたスライドは、長時間にわたって-20℃で保存することができます。 </li></ol> 6。代表的な結果： （固定矯正装置からバイオフィルムを掻き図5）顕微鏡用コーティングガラススライド上に直接ハイブリダイズすることができる適切なフレーク（図6）が得られます。このように、orobiome細菌の異なるグループに異なる名前が付けられ、特定のプローブ（図7及び図8）とタギング細菌のリボソームRNAによって彼らの自然な三次元環境で識別することができます。図7においては、バイオフィルムはEUBmix（緑、すべての細菌）とLGCmix（黄色、Firmicutes）で染色した。彼らは、黄色とブルーで染色したとしてFirmicutesは、​​緑色で表示されます。図8では、バイオフィルムはEUBmix（赤、すべての細菌）、Bac303（青、Bacteroidetes）とPOGI（黄色、 ポルフィロ歯周 ）で染色した。すべての3つのプローブは、そのDNAと重複するバインドとしてポルフィロ歯周が 、黄白色で表示されます白信号での色の結果。細菌のグループとの間の形態的な違いは、（図9と図10）を同定することができます。球状​​の細菌の大規模なクラスタは、染色がEUBmix（緑、すべてのBAで実施された図9に示されていますcteria）。口腔内細菌の異なる形状は球状と糸状菌がEUBmix（赤）で染色することによって区別す​​ることができます図10に可視化した。また、バイオフィルムの典型的なキノコ状の構造がCLSMのデータの3Dモデリング（図11および12）を介して処理することができると、 <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">3Dモデリングの映画を鑑賞するためにはここをクリックしてください。</a> <img alt="図1" src="/files/ftp_upload/2967/2967fig1.jpg" /> 図1は、。 場で口蓋エキスパンダーを修正しました。 <img alt="図2" src="/files/ftp_upload/2967/2967fig2.jpg" /> 図2。Nolaのドライフィールドシステム。 <img alt="図3" src="/files/ftp_upload/2967/2967fig3.jpg" /> 図3は、。ペンチ、手袋とトレイを滅菌。 <img alt="図4" src="/files/ftp_upload/2967/2967fig4.jpg" /> 図4削除パンダ。 <img alt="図5" src="/files/ftp_upload/2967/2967fig5.jpg" /> 図5。滅菌メスでバイオフィルムを掻き。 <img alt="図6" src="/files/ftp_upload/2967/2967fig6.jpg" /> 図6。樹脂フレークに直接コーティングされたガラススライド上にハイブリダイズさせた。 <img alt="図7" src="/files/ftp_upload/2967/2967fig7.jpg" /> 図7 CLSM像：特定の細菌群の分化。 3D CLSMのスタックデータの2Dオーバーレイ。バイオフィルムはEUBmix（緑、すべての細菌）とLGCmix（黄色、Firmicutes）で染色。 <img alt="図8" src="/files/ftp_upload/2967/2967fig8.jpg" /> 図8 CLSM像：特定の細菌群の分化。 3D CLSMのスタックデータの2Dオーバーレイ。 EUBmix（赤、すべての細菌）、Bac303（青、Bacteroiで染色したバイオフィルムdetes）とPOGI（黄色、 ポルフィロ歯周 ）。 <img alt="図9" src="/files/ftp_upload/2967/2967fig9.jpg" /> 図9 CLSM像：特定の形態の分化。 3D CLSMのスタックデータの2Dオーバーレイ。バイオフィルムはEUBmix（緑、すべての細菌）で染色。球状​​の細菌の大規模なクラスタ（矢印）。 <img alt="図10" src="/files/ftp_upload/2967/2967fig10.jpg" /> 図10 CLSM画像：特定の形態の分化。 3D CLSMのスタックデータの2Dオーバーレイ。バイオフィルムはEUBmix（赤、すべての細菌）で染色。球状​​（下矢印）と糸状菌（上記の矢印）を区別することができます。 <img alt="図11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 図11。キノコの構造、下から3Dビュー。 EUBmixと手順で染色したバイオフィルムフレークのスタック（矯正装置の表面掻き）、IMARIS（CLSM像）とssed。 <img alt="図11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 図12。キノコの構造、3D側面図。 IMARIS（CLSM像）で処理したバイオフィルムフレークのスタック（矯正装置の表面掻き）。 <table border="1"><tbody><tr><td colspan="4"> ハイブリダイゼーション緩衝液（200μl） </td></tr><tr><td> ホルムアミド濃度 </td><td> 10パーセント </td><td> 20パーセント </td><td> 45パーセント </td></tr><tr><td> 5 M NaClを </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1 Mトリス塩酸 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2％SDS </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> FA </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt;のddH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> 任意のFISHプローブ </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> 表1。ハイブリダイゼーションバッファーの成分（μlの濃度）。 <table border="1"><tbody><tr><td colspan="4"> 洗浄バッファー（50 ml）を </td></tr><tr><td> ホルムアミド濃度 </td><td> 10パーセント </td><td> 20パーセント </td><td> 45パーセント </td></tr><tr><td> 5 M NaClを </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1 Mトリス塩酸 </td><td> 1000年</td><td> 1000年</td><td> 1000年</td></tr><tr><td> 0.5 M EDTA </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> のddH 2 O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> 表2。洗浄バッファーの成分（μlの濃度）。 映画は1。 <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">映画を鑑賞するためにはここをクリックしてください。</a>

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利害の衝突は宣言されません。

ここに記述されたプロトコルは、硬い材料から採取した染色のバイオフィルムへの高加工性アプローチです。サンプリングとハイブリダイゼーションは、最も重要なステップです。サンプリング時には、注意は十分な厚さのスライスがバイオフィルムの構造が損なわれないということを確実に収集されることを注意する必要があります。ハイブリダイゼーションの間に、それはこのように蛍光標識プローブの非特異的結合、または結合の損失を、避け、温度の過度の変動を避けるために不可欠です。このFISHプロトコルは、その組成を中断することなく、直接スライドガラス上に正常異種のバイオフィルムを染色するエレガントな方法です。スライドはすぐに再度サンプルを移動することなく、顕微鏡に使用することができます。このように、チューブ内の染色以上の改善がバイオフィルムに適用される以下のせん断力によって達成されます。 &gt;50μm厚のスライスは、この方法で調製し、調べることができます。

<table border="1">
	<tbody>
		<tr>
			<th>Oligonucleotides</th>
			<th>Target organism</th>
			<th>Fluorochrome</th>
		</tr>
		<tr>
			<td>EUB338 - 338III</td>
			<td>Eubacteria</td>
			<td>Cy3</td>
		</tr>
		<tr>
			<td>LGC354 A-C</td>
			<td>Firmicutes</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>Bac303</td>
			<td>Bacteroidetes</td>
			<td>Cy5</td>
		</tr>
		<tr>
			<td>Mut590</td>
			<td>Streptococcus mutans</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>POGI</td>
			<td>Porphyromonas gingivalis</td>
			<td>FITC</td>
		</tr>
	</tbody>
</table>
Table 3. Oligonucleotides (refer to probeBase13 for details).
<table border="1">
	<tbody>
		<tr>
			<th>Component</th>
			<th>Company</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>&nbsp;</td>
			<td>4% solution in PBS</td>
		</tr>
		<tr>
			<td>1 x PBS</td>
			<td>&nbsp;</td>
			<td>3 x solution prepared</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Millipore</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>Merck</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Roth</td>
			<td>100 mg/ml stock solution</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Roth</td>
			<td>99.5% solution</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% SDS</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FISH probes (oligonucleotides)</td>
			<td>Invitrogen, Sigma</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold antifadent</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<th>Equipment</th>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>IKA Tez</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Sarstedt</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polysine-coated slides</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 4. Materials and equipment.

oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy

自然の異種バイオフィルムの共焦点レーザー走査顕微鏡（CLSM）は本日、そのうちの一つは、in situハイブリダイゼーション（FISH） の蛍光であること、染色技術の包括的な範囲によって促進される。1,2は、我々は、口腔バイオフィルムのサンプルは、固定から収集したパイロットスタディを実施矯正装置（口蓋パンダは）自然なバイオフィルムとプラークの蓄積の三次元組織を評価するという目的、FISHによって染色した。3,4 FISHが蛍光標識16Sを使用することにより、ネイティブのバイオフィルム環境で細胞を染色するための機会を作成するrRNAを標的とするプローブを。4-7,19は、免疫蛍光ラベルのような代替技術と比較すると、これは、混合バイオフィルムのコンソーシアムに異なる細菌のグループを調査するために安価な、正確でわかりやすい標識法である。18,20一般的なプローブが使用されていたその真正細菌に結合する（ EUB338 + EUB338II + EUB338III、以下EUBmix）、80〜10 Firmicutes（LGC354 AC;以下LGCmix）、9,10およびBacteroidetes（Bac303）さらに11、 ストレプトコッカスミュータンス （MUT590）12,13と病菌歯周への結合を特異的プローブ（POGI）13,14が使用された。関与する表面物質の極端な硬さは（ステンレスとアクリル樹脂）フィルムを準備する新しい方法を見つけるために私たちを強要。これらの表面の材料が容易にcryotomeで切断することができなかったとして、様々なサンプリング方法がそのまま口腔バイオフィルムを得るために検討された。これらのアプローチの最も有効なのは、この通信で表示されます。バイオフィルムを運ぶアクリル樹脂の小さなフレークは、バイオフィルムの構造を傷つけないように注意しながら、滅菌メスでこすり取った。鉗子は、鋼表面からバイオフィルムを収集するために使用された。が収集されると、サンプルは固定されており、polysineコーティングされたガラススライド上に直接配置されました。 FISHは、プローブのmで、これらのスライド上で直接実行され上記entioned。様々な魚のプロトコルは扱いやすくなった新しいプロトコルを作成するために結合し、変更されました。5,10,14,15その後試料は共焦点レーザー走査顕微鏡によって分析した。よく知られている構成は3,4,16,17は、チャネルによって浸透し球状の細菌のマッシュルームスタイルの形成およびクラスタなど、可視化することができます。さらに、これらの典型的なバイオフィルムの構造の細菌の組成を分析し、2Dと3D画像が作成されました。

Watch this Scientific Journal Video about Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy at JoVE.com

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

我々は、と矯正装置から自然口腔バイオフィルムの構造と組成分析のためのプロトコルを提示<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）と共焦点レーザー走査顕微鏡（CLSM）。口腔バイオフィルムのサンプルは、その表面からアクリル樹脂フレークをこすると分子の処理のためにそれらを参照して、口蓋パンダから採取した。

蛍光in situハイブリダイゼーションおよび共焦点レーザー走査顕微鏡による口蓋パンダの口腔バイオフィルムの解析

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

oral-biofilm-analysis-palatal-expanders-fluorescence-situ

Research

JoVE Journal

Medicine

15.9K ビュー.  Medical University of Graz. 我々は、と矯正装置から自然口腔バイオフィルムの構造と組成分析のためのプロトコルを提示その場でハイブリダイゼーション（FISH）と共焦点レーザー走査顕微鏡（CLSM）。口腔バイオフィルムのサンプルは、その表面からアクリル樹脂フレークをこすると分子の処理のためにそれらを参照して、口蓋パンダから採取した。

ビデオ: Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

尿路のプローブベースの共焦点レーザーEndomicroscopy：テクニック

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

医学

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNAを用いた乳がん組織におけるmiRNA発現の臨床病理学的解析<em&gt;その場で</em&gt;ハイブリダイゼーション

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

マイクロバイにおける健常児の口腔バイオ フィルム サンプリング

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

架橋 (TXL) 近視治療の組織の評価ツールとしてウサギの強膜の第 2 高調波発生信号

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

歯科用インプラント ・異種材料の口腔バイオ フィルム形成

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

高齢者のレイ・スタッフによる口腔健康評価

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

走査型電子顕微鏡による膜ラッフル形成の可視化

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

多発性骨髄腫の骨髄塗抹標本の その場での 相間蛍光ハイブリダイゼーション

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

口腔内蛍光カメラを用いた歯垢の半自動平面定量

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

空間オミクス解析のための口腔粘膜下線維症組織サンプルの採取

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

著者スポットライト:経口潜在的な悪性腫瘍の謎を解き明かす 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

レーザーキャプチャーマイクロダイセクションによる口腔粘膜下線維症サンプルからの形態学的および空間的情報による細胞の分離

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...