이 작품은 의과 대학 그라츠의 위생 Fonds에 의해 지원되었다. 모든 과목들은 정보에 동의했다. 연구 프로토콜의 기관 승인 의과 대학 그라츠로부터 얻은 것입니다.

1. 표본 수집 <ol><li> intraoral 서비스 4 개월 후 고정 맛이 좋은 확장기를 제거합니다. 그들 (그림 1 및 2) 제거하기 전에 어플 라이언 스를 분리 놀라 드라이 필드 시스템을 사용합니다. </li><li> 오염을 (그림 3, 4) 추가하지 않고 확장기를 제거하는 소독 펜치, 장갑과 쟁반을 사용합니다. </li><li> -20 ° C.에 Sarstedt 120 ML 튜브 안에 저장 개체 24 시간 이내에 얼음과 프로세스에 대한 실험실 소요됩니다. </li></ol> 2. Biofilm의 고정 <ol><li> 무균 메스 biofilm 부스러기를 긁어, 또는 멸균 집게와 조각을 수집합니다. </li><li> 샘플이 잘 적용되기 전까지 얼음처럼 차가운 4% PFA 솔루션을 추가합니다. </li><li> 4에서 혼합물을 품어 ° C 3-12시간위한 (동결하지 않음). 긴 고정 시간 이상 고정 온도는 oligonucleotide 프로브 적게 투과 그람 음성 세포의 세포 봉투를 렌더링 수 있습니다. </li><li> PFA 솔루션을 제거하고 얼음처럼 차가운 1X PBS로 세척. R이 단계에게 2-3 번 반복잔류 PFA를 emove. </li><li> 1 권에서 샘플을 Resuspend. 얼음처럼 차가운 1X PBS과 추가 1 권. 얼음처럼 차가운 96% (V / V) 에탄올. </li><li> -20 ° C.에있는 샘플을 저장 이 프로토콜에 따라 고정 샘플 년 몇 달 동안 저장할 수 있습니다. </li></ol> 3. 고정 샘플의 탈수 <ol><li> 현미경 슬라이드에 PFA - 고정 샘플 자료 5-30 μl을 적용합니다. </li><li> 46시 드라이 ° C 15 분 이상 실온에서. 라이 소 자임과 포름 아미드를 해동. </li><li> 따라서 세포 벽의 부분 파괴하여 세포에 프로브의 침투를 촉진 10 분 상온에서 라이 소 자임 (1mg/ml) 250 μl를 추가합니다. </li><li> 50 %, 80 % 96% (V / V) 3 분 각각 에탄올로 물놀이 슬라이드. 에탄올 농도 시리즈의 dehydrating 효과는 세포 세포막을 분해합니다. </li><li> ° C 10 분에 대해 46에서 슬라이드를 건조시킵니다. </li></ol> 4. 원위치 하이브리드화 <ol><li> 1 ML 준비신선한 하이브 리다이 제이션 버퍼 (농도에 대한 표 1 참조). 이 연구에 사용된 포름 아미드 농도 : 10 % (EUBmix), 20 % (Bac303, POGI, MUT590) 또는 45 % (LGCmix). </li><li> oligonucleotide 프로브 솔루션을 녹여. 해동 프로브는 얼음에 보관하고 빛으로부터 보호되어야합니다. </li><li> 하이브 리다이 제이션 버퍼 200 μl 각 프로브 2 μl를 추가, 잘 섞어 현미경 슬라이드에 건조 표본으로 혼합물을 적용합니다. </li><li> 사각형 페트리 접시에 조직 종이를 삽입하고 티슈에 남아있는 하이브 리다이 제이션 버퍼를 부어. </li><li> 바로 그릇에 가로 슬라이드를 장소와 요리를 닫습니다. 46 ° C에서 1-5시간 (90 분 대부분의 경우 충분합니다) 오븐에 품어. 수분 챔버로 요리 기능은 슬라이드에서 하이브 리다이 제이션 솔루션의 증발을 방지. 특히, 포름 아미드의 증발이 아닌 목표 세포가 아닌 특정 탐사선이 바인딩을 일으킬 수 있습니다. </li><li> (conce에 대한 표 2를 참조하십시오 세척 버퍼의 50 ML 준비ntrations). 48 50 ML 튜브와 앞서 열을 가하다에서 세탁 버퍼를 준비 ° C 물 목욕 인치 세척 단계는 48 ° C.에 수행됩니다 </li><li> 하이브 리다이 제이션 오븐에서 슬라이드로 접시를 제거합니다. 즉시 미리 예열 세척 버퍼의 작은 볼륨으로 하이브리드화 버퍼 씻어, 나머지 세척 버퍼에 슬라이드를 전송합니다. </li><li> 10-15 분 48 ° C에서 물이 욕조 및 부화로 다시 세척 버퍼와 슬라이드가 들어있는 튜브를 놓습니다. </li><li> 튜브에서 슬라이드를 제거하고 잔류 세척 버퍼를 제거하는 2~3초 위해 얼음처럼 차가운 ddH 2 O로 물놀이. </li><li> 가능한 한 빨리 슬라이드 (압축 공기의 사용이 권장됩니다) 공기 건조. 빠른 건조가 프로브 분리를 줄일 수 있습니다. </li><li> 말린 슬라이드는 프로브 - 수여 형광 신호의 상당한 손실없이 몇 주 동안 -20 ° C에서 어둠에 저장할 수 있습니다. </li></ol> 5. 현미경 사용법 <ol><li> 후 생선 세척, appl슬라이드의 왼쪽과 오른쪽 엔드 (냉동 슬라이스가 이전 단계로 실내 온도로 예열한다)에 antifadent 가까이 Y 두 방울. </li><li> 현미경 커버가 상단에 실수 antifadent가 전체 슬라이드에 걸쳐 때까지 기다려 놓습니다. antifadent의 과도한 양의 현미경 이미지를 흐려 수 있습니다. </li><li> 적절한 필터 또는 레이저를 갖춘 공촛점 레이저 스캐닝 현미경 샘플을 관찰합니다. 우리는 Leica TCS 단위 (; NA 1.2 HCX PL APO/63x)를 사용합니다. 데이터는 같은 IMARIS 또는 AMIRA와 같은 소프트웨어와 함께 분석할 수 있습니다. </li><li> 프로브 - 수여 형광이 감소하기 시작하기 전에 antifadent에 포함된 슬라이드는 ° C (동결하지 않음) 최대 7 일 동안 4에 저장할 수 있습니다. 또는 antifadent는 ddH 2 O와 제거 수 있으며, 말린 슬라이드는 오랜 기간 동안 -20 ° C에 저장할 수 있습니다. </li></ol> 6. 대표 결과 : (고정 orthodontic 기기에서 biofilm을 근근이 살아가고그림 5) 현미경에 대한 코팅 유리 슬라이드에 직접 hybridized 수 있습니다 적합 조각 (그림 6)를 산출. 이러한 방법으로 orobiome 박테리아의 다른 그룹이 다르게 표시된 특정 프로브 (그림 7 및 8)로 태그 세균성 rRNA하여 자연 입체 환경에서 확인할 수 있습니다. 그림 7에서 biofilm은 EUBmix (녹색, 모든 세균)과 LGCmix (노랑, Firmicutes)와 스테인드했습니다. 그들은 노랑, 파랑 물들일 것처럼 Firmicutes은 녹색으로 표시됩니다. 그림 8에서 biofilm은 EUBmix (빨강, 모든 박테리아), Bac303 (파란색, Bacteroidetes)과 POGI (노란색, Porphyromonas의 gingivalis)와 스테인드되었습니다. 세 프로브는 DNA와 중복에 바인딩으로 Porphyromonas의 gingivalis이, 노란 흰색에 표시됩니다 하얀 신호에 색상 결과. 박테리아의 그룹 간의 형태학의 차이는 또한 (그림 9 및 10) 확인할 수 있습니다. coccoid 박테리아의 큰 클러스터는 (녹색, 모든 바 얼룩이 EUBmix로 수행된 그림 9에 표시됩니다cteria). 구강 박테리아의 다른 모양은 coccoid 및 filamentous 박테리아가 EUBmix (적색)와 얼룩으로 구분할 수있다 그림 10에 시각되었습니다. 또한 biofilm의 전형적인 버섯 모양의 구조가 CLSM 데이터의 3D 모델링 (그림 11 및 12)를 통해 처리할 수하면 <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">3D 모델링의 동영상을 감상하려면 여기를 클릭하십시오.</a> <img alt="그림 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> 그림 1. 원위치로 맛이 좋은 확장을 수정. <img alt="그림 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> 그림 2. 노라 드라이 필드 시스템. <img alt="그림 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> 그림 3. 펜치, 장갑 및 트레이를 소독. <img alt="그림 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> 그림 4. 확장기를 제거했습니다. <img alt="그림 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> 그림 5. 무균 메스와 biofilm을 근근이 살아가고. <img alt="그림 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> 그림 6. 수지 조각은 직접 코팅 유리 슬라이드에 hybridized. <img alt="그림 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> 그림 7 CLSM 이미지 :. 특정 박테리아 그룹의 차별. 3 차원 CLSM 스택 데이터를 2D 오버레이. Biofilm은 EUBmix (녹색, 모든 세균)과 LGCmix (노랑, Firmicutes)와 얼룩. <img alt="그림 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> 그림 8 CLSM 이미지 :. 특정 박테리아 그룹의 차별. 3 차원 CLSM 스택 데이터를 2D 오버레이. EUBmix (빨강, 모든 박테리아), Bac303 (파란색, Bacteroi과 Biofilm 자국detes)와 POGI (노란색, Porphyromonas gingivalis의). <img alt="그림 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> 그림 9 CLSM 이미지 :. 특정 morphologies의 차별화. 3 차원 CLSM 스택 데이터를 2D 오버레이. Biofilm은 EUBmix (녹색, 모든 박테리아)과 얼룩. coccoid 박테리아의 대형 클러스터 (화살표). <img alt="그림 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> . 그림 10 CLSM 이미지 : 특정 morphologies의 차별화. 3 차원 CLSM 스택 데이터를 2D 오버레이. Biofilm은 EUBmix (빨강, 모든 박테리아)과 얼룩. Coccoid (아래 화살표)와 filamentous 세균 (위 화살표)를 구별하실 수 있습니다. <img alt="그림 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 그림 11. 버섯 구조 아래에서 3 차원 전망이 있습니다. EUBmix 및 proce 물들일 biofilm 조각 (orthodontic 어플 라이언 스의 표면 긁어 냈어요)의 스택IMARIS (CLSM 이미지)와 함께 ssed. <img alt="그림 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 그림 12. 버섯 구조, 3D 측면 볼 수 있습니다. IMARIS (CLSM 이미지)와 함께 처리 biofilm 조각 (orthodontic 어플 라이언 스의 표면 긁어 냈어요)의 스택. <table border="1"><tbody><tr><td colspan="4"> 하이브 리다이 제이션 버퍼 (200 μl) </td></tr><tr><td> 포름 아미드 농도 </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1 M 트리스 - HCL </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2퍼센트 SDS </td><td> 일 </td><td> 일 </td><td> 일 </td></tr><tr><td> FA </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt; ddH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> 모든 생선 프로브 </td><td> 이 </td><td> 이 </td><td> 이 </td></tr></tbody></table> 하이브 리다이 제이션 버퍼의 표 1. 성분 (μl의 농도). <table border="1"><tbody><tr><td colspan="4"> 세척 버퍼 (50 ML) </td></tr><tr><td> 포름 아미드 농도 </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1 M 트리스 - HCL </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> ddH 2 O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> 세척 버퍼의 표 2. 성분 (μl의 농도). 영화는 1. <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">영화를보고하려면 여기를 클릭하십시오.</a>

<ol>
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J., Evans, R., Noar, J., Pratten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+biofilm+formation+and+the+effects+of+antimicrobial+agents+on+orthodontic+bonding+materials+in+a+parallel+plate+flow+chamber.">Early biofilm formation and the effects of antimicrobial agents on orthodontic bonding materials in a parallel plate flow chamber.</a> Eur. J. Orthod. 28, 1-7 (2006).</li><li>Loy, A., Maixner, F., Wagner, M., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase--an+online+resource+for+rRNA-targeted+oligonucleotide+probes:+new+features+2007.">probeBase--an online resource for rRNA-targeted oligonucleotide probes: new features 2007.</a> Nucleic. Acids. Res. 35, (2007).</li><li>Diaz, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+subject-specific+oral+microflora+during+initial+colonization+of+enamel.">Molecular characterization of subject-specific oral microflora during initial colonization of enamel.</a> Appl. Environ. Microbiol. 72, 2837-2848 (2006).</li><li>Dige, I., Nilsson, H., Kilian, M., Nyvad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+identification+of+streptococci+and+other+bacteria+in+initial+dental+biofilm+by+confocal+laser+scanning+microscopy+and+fluorescence+in+situ+hybridization.">In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization.</a> Eur. J. Oral Sci. 115, 459-467 (2007).</li><li>Kolenbrander, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+interactions+and+successions+during+plaque+development.">Bacterial interactions and successions during plaque development.</a> Periodontology. 42, 47-79 (2000).</li><li>Kolenbrander, P. E., Palmer, R. J., Periasamy, S., Jakubovics, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+multispecies+biofilm+development+and+the+key+role+of+cell-cell+distance.">Oral multispecies biofilm development and the key role of cell-cell distance.</a> Nat. Rev. Microbiol. 8, 471-480 (2010).</li><li>Chalmers, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+quantum+dot+luminescent+probes+to+achieve+single-cell+resolution+of+human+oral+bacteria+in+biofilms.">Use of quantum dot luminescent probes to achieve single-cell resolution of human oral bacteria in biofilms.</a> Appl. Environ. Microbiol. 73, 630-636 (2007).</li><li>Wecke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+monitoring+the+development+of+bacterial+biofilms+in+human+periodontal+pockets.">A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets.</a> FEMS Microbiol. Lett. 191, 95-101 (2000).</li><li>Moter, A., Gobel, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH)+for+direct+visualization+of+microorganisms.">Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms.</a> J. Microbiol. Methods. 41, 85-112 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

설명한 프로토콜 계약은 하드 자료의 수집 얼룩 biofilms에 매우 가능한 접근이다. 샘플링과 하이브 리다이 제이션은 가장 중요한 단계입니다. 샘플링 기간 동안주의가 충분한 두께의 조각이 biofilm 구조가 그대로 유지하기 위해 수집되는 이동해야합니다. 하이브 리다이 제이션 동안, 그것은 따라서 찬란 분류 프로브가 아닌 특정 바인딩 또는 바인딩의 손실을 피하고, 온도 과도한 변동을 피하기 위해 필수적입니다. 이 물고기 프로토콜의 구성을 방해하지 않고 직접 유리 슬라이드에 정상적인 이기종 biofilm을 얼룩이 우아한 방법입니다. 슬라이드 바로 다시 샘플을 이동하지 않고 현미경을 위해 사용될 수 있습니다. 이러한 방식으로, 튜브에 얼룩 이상의 개선이 biofilm에 적용되는 더 적은 전단 강제로 이루어집니다. &gt; 50 μm의 두께의 조각이 방식으로 준비하고 조사 수 있습니다.

<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

자연 이기종 biofilm의 공촛점 레이저 스캐닝 현미경 (CLSM)은 얼룩 기법, 그들이 원위치 하이브리드화 (물고기)에서 형광되는 하나의 포괄적인 범위로 오늘날 용이합니다. 1,2, 우리는 구강 biofilm 예제는 고정에서 수집하는 파일럿 연구를 수행 orthodontic 가전 (맛이 좋은 확장기)는 생선, 자연 biofilm 및 상패 축적의 3 차원의 조직을 평가하는 목적에 의해 스테인드되었습니다. 3,4 물고기가 찬란 레이블 16를 사용하여 네이티브 biofilm 환경에서 세포를 얼룩 수있는 기회를 만듭니다 rRNA 타겟팅 프로브를. 4-7,19이 immunofluorescent 라벨과 같은 다른 기술에 비해,이 혼합 biofilm의 컨소시엄에서 다른 박테리아 그룹을 조사 저렴, 정확하고 간단 라벨 기법입니다. 18,20 일반 프로브가 사용하는 것을 Eubacteria에 바인딩 ( EUB338 + EUB338II + EUB338III; 이하 EUBmix), 80-10 Firmicutes는 (LGC354 AC;. 이하 LGCmix), 9,10과 Bacteroidetes (Bac303) 11 또한, 연쇄상 구균의 mutans에 바인딩을 특정 프로브 (MUT590) 12,13과 Porphyromonas gingivalis의 (POGI) 13,14 사용되었습니다 . 관련 표면 재료의 극단적인 경도 (스테인레스 스틸과 아크릴 수지)는 biofilm을 준비하는 새로운 방법을 찾는 우리에게 강요. 이러한 표면 물질이 쉽게 cryotome로 잘라 수 없습니다로서, 다양한 샘플링 방법은 그대로 구두 biofilm을 얻기 위해 탐험했다. 이러한 방식을 가장 가능한이 의사 소통에 표시됩니다. biofilm 운반 아크릴 수지의 작은 조각은 biofilm 구조를 손상하지 않도록 돌봐, 무균 메스로 긁어 냈했다. 집게는 철강 표면의 biofilm를 수집하는 데 사용되었습니다. 일단 수집, 샘플은 고정 및 polysine 코팅 유리 슬라이드에 직접 배치했다. 물고기가 프로브 m 이러한 슬라이드에서 직접 수행되었습니다위 entioned. 다양한 생선 프로토콜 결합하여 취급하기가 쉽고 새로운 프로토콜을 만들 수정되었습니다. 5,10,14,15은 이후 샘플을 공촛점 레이저 스캐닝 현미경에 의해 분석되었다. 잘 알려진 구성 3,4,16,17는 버섯 스타일의 형성 및 채널에 의해 줄다리기 coccoid 박테리아의 클러스터를 포함하여, 시각 수 있습니다. 또한, 이러한 전형적인 biofilm 구조의 세균성 조성 분석과 2D 및 3D 이미지는 만들어졌습니다.

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

우리는 함께 orthodontic 가전 제품에서 자연 구두 biofilm의 구조와 작곡 분석을 위해 프로토콜을 제시<em&gt; 현장에서</em&gt; 하이브 리다이 제이션 (물고기) 및 공촛점 레이저 스캐닝 현미경 (CLSM). 구강 biofilm 샘플들은 표면에 묻은 아크릴 - 수지 조각 근근이 살아가고와 분자 처리를 위해 그들을 추천, 맛이 좋은 확장기에서 수집되었다.

형광에 원위치 하이브리드화 및 공촛점 레이저 스캐닝 현미경에 의해 맛이 좋은 확장기의 사정 Biofilm 분석

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

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15.9K 조회수.  Medical University of Graz. 우리는 함께 orthodontic 가전 제품에서 자연 구두 biofilm의 구조와 작곡 분석을 위해 프로토콜을 제시 현장에서 하이브 리다이 제이션 (물고기) 및 공촛점 레이저 스캐닝 현미경 (CLSM). 구강 biofilm 샘플들은 표면에 묻은 아크릴 - 수지 조각 근근이 살아가고와 분자 처리를 위해 그들을 추천, 맛이 좋은 확장기에서 수집되었다.

비디오: 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.

요로의 프로브 기반 공 촛점 레이저 Endomicroscopy : 기술

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

연구

의학

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.

miRNA의를 사용하여 유방암 조직에서의 miRNA 발현의 임상 병리학 적 분석<em&gt; 현장에서</em&gt; 하이브리드

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.

건강 한 아이에서 미생물 분석을 위한 구두 Biofilm 샘플링

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.

치료 조직 (TXL) 근 시에 대 한 교차 연결의 평가 위한 도구로 토끼 Sclera에 제 2 고조파 발생 신호

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.

치과 임 플 란 트에 대 한 다른 자료에 구두 Biofilm 형성

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.

노인을위한 평신도 직원에 의한 구강 건강 평가

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.

주사 전자 현미경을 이용한 멤브레인 러플 형성 시각화

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.

다발성 골수종의 골수 얼룩의 계내 형광 혼성화

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.

구강 내 형광 카메라를 사용한 치태의 반자동 평면 정량

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

Spatial-Omics 분석을 위한 구강 점막하 섬유증 조직 샘플 캡처

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.

저자 스포트라이트: 구강 잠재적 악성 종양의 미스터리 풀기 

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.

Laser Capture Microdissection에 의한 구강 점막하 섬유증 샘플에서 형태학적 및 공간적 정보가 있는 세포 분리

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 ...