Este trabalho foi apoiado pelo Fonds de Higiene da Universidade Médica de Graz. Todos os atletas deram o seu consentimento informado. Aprovação institucional do protocolo do estudo foi obtido a partir da Universidade Médica de Graz.

1. Coleta de amostras <ol><li> Remover fixa palatal expansores após 4 meses de serviço intra-oral. Use NOLA Sistema de campo seco para isolar os aparelhos antes de removê-los (Figuras 1 e 2). </li><li> Use um alicate esterilizado, luvas e bandejas para remover expansores sem adição de contaminação (Figuras 3 e 4). </li><li> Armazenar objetos em Sarstedt 120 ml frascos a -20 ° C. Leve para o laboratório em gelo e processo no prazo de 24 horas. </li></ol> 2. Fixação de biofilme <ol><li> Raspe biofilme flocos com um bisturi estéril, ou coletar peças com pinças esterilizadas. </li><li> Adicionar gelada 4% solução de PFA até amostra é bem cobertos. </li><li> Incubar a mistura a +4 ° C (não congelar) durante 3 a 12 horas. Vezes maior fixação ou temperaturas mais altas de fixação pode tornar a célula de envelopes gram-negativas células menos permeável a sondas de oligonucleotídeo. </li><li> Remoção da solução de PFA e lave com gelado 1X PBS. Repita este passo 2-3 vezes ao remove PFA residual. </li><li> Ressuspender a amostra em 1 vol. gelada 1X PBS e adicionar 1 vol. gelada-96% (v / v) de etanol. </li><li> Conservar a amostra a -20 ° C. Amostras fixadas de acordo com este protocolo pode ser armazenado por vários meses a anos. </li></ol> 3. Desidratação das amostras fixas <ol><li> Aplicar mL 50-30 de PFA fixa material de amostra para uma lâmina de microscópio. </li><li> Seco a 46 ° C por cerca de 15 min ou em temperatura ambiente por mais tempo. Descongele a lisozima ea formamida. </li><li> Adicionar 250 mL de lisozima (1mg/ml) em temperatura ambiente por 10 min, facilitando assim a penetração das sondas nas células por destruição parcial das paredes celulares. </li><li> Dip slides em 50%, 80% e 96% (v / v) de etanol por 3 min cada. O efeito desidratante da série concentração de etanol irão se desintegrar as membranas celulares. </li><li> Secar as lâminas a 46 ° C por 10 min. </li></ol> 4. Hibridização in situ <ol><li> Prepare 1 ml detampão de hibridação fresco (ver Tabela 1 para as concentrações). Formamida concentrações utilizadas neste estudo: 10% (EUBmix), 20% (Bac303, IGP, MUT590) ou 45% (LGCmix). </li><li> Descongele as soluções sonda de oligonucleotídeos. Sondas descongelados devem ser mantidos em gelo e protegido da luz. </li><li> Adicionar 2 mL de cada sonda a 200 mL de tampão de hibridação, misture bem e aplique a mistura para a amostra desidratada em uma lâmina de microscópio. </li><li> Coloque um pedaço de papel de tecido em uma placa de Petri quadrados e despeje o tampão de hibridação restantes para o papel de seda. </li><li> Colocar imediatamente a lâmina horizontalmente no prato e fechar o prato. Incubar em estufa a 46 ° C por 1-5 horas (90 min será suficiente na maioria dos casos). As funções prato como uma câmara de umidade evitando a evaporação da solução de hibridização do slide. Em particular, a evaporação de formamida pode causar não-específica sonda ligação a células não-alvo. </li><li> Prepare 50 ml de tampão de lavagem (ver Tabela 2 para concentrations). Prepare a solução de lavagem em um tubo de 50 ml e pré-aquecimento a 48 ° C em banho-maria. A etapa de lavagem é realizada a 48 ° C. </li><li> Retire o prato com a lâmina do forno de hibridização. Lave imediatamente o tampão de hibridação com um pequeno volume de pré-aquecido tampão de lavagem, e transferir o deslizar para dentro do tampão de lavagem restante. </li><li> Colocar o tubo contendo a solução de lavagem eo slide volta para o banho de água e incubar a 48 ° C por 10-15 min. </li><li> Remover slide do tubo e mergulhar gelada DDH 2 O por 2-3 segundos para eliminar a tampão de lavagem residual. </li><li> De ar seco a lâmina o mais rápido possível (o uso do ar comprimido é recomendado). Secagem rápida vai reduzir sonda dissociação. </li><li> Slides secas podem ser armazenadas no escuro a -20 ° C por várias semanas sem perda significativa de sonda-conferidos sinal de fluorescência. </li></ol> 5. Microscopia <ol><li> Depois de FISH e lavagem, apply duas gotas de perto antifadent para as extremidades esquerda e direita de um slide (fatias congelado deve ser aquecido à temperatura ambiente antes de esta etapa). </li><li> Coloque uma tampa microscópio escorregar em cima e espere até que o antifadent se espalhou por todo o slide. Note-se que uma quantidade excessiva de antifadent podem borrar a imagem de microscópio. </li><li> Observe as amostras sob um microscópio confocal de varredura a laser equipado com filtros adequados ou lasers. Nós usamos uma Leica TCS unidade (HCX PL APO/63x; NA 1.2). Os dados podem ser analisados ​​com software, tais como IMARIS ou AMIRA. </li><li> Slides embutidos em antifadent podem ser armazenados a +4 ° C (não congelar) por até 7 dias antes da sonda de fluorescência conferidos começa a declinar. Alternativamente, o antifadent pode ser removido com DDH 2 O, e as lâminas secas podem ser armazenadas a -20 ° C por um período prolongado de tempo. </li></ol> 6. Resultados representativos: Raspagem biofilme off aparelhos ortodônticos fixos (Figura 5) produz flocos adequado (Figura 6) que pode ser hibridizado diretamente sobre lâminas de vidro revestidas de microscopia. Desta forma, diferentes grupos de bactérias orobiome podem ser identificadas no seu ambiente tridimensional natural por rRNA marcação bacteriana com diferentes sondas marcadas específicas (Figuras 7 e 8). Na Figura 7, o biofilme foi corado com EUBmix (verde, todas as bactérias) e LGCmix (amarelo, Firmicutes). Firmicutes aparecem em verde, como eles foram coradas com amarelo e azul. Na Figura 8, o biofilme foi corado com EUBmix (vermelho, todas as bactérias), Bac303 (azul, Bacteroidetes) e IGP (amarelo, Porphyromonas gingivalis). Porphyromonas gingivalis é mostrado em branco amarelado, como todos os três sondas se ligam ao seu DNA e da sobreposição de cores resulta em um sinal branco. Diferenças morfológicas entre os grupos de bactérias também podem ser identificados (Figuras 9 e 10). Grandes aglomerados de bactérias cocóide são mostrados na Figura 9, onde a coloração foi realizada com EUBmix (verde, todos os bacteria). Formas diferentes de bactérias orais foram visualizados na Figura 10, onde as bactérias filamentosas cocóide e podem ser distinguidos pela coloração com EUBmix (vermelho). Além disso, uma estrutura em forma de cogumelo típico do biofilme pode ser processado através de modelagem em 3D dos dados CLSM (figuras 11 e 12) <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Clique aqui para assistir a um filme da modelagem 3D.</a> <img alt="Figura 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> Figura 1. Fixed expansor palatal in situ. <img alt="Figura 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> Figura 2. Nola Sistema de campo seco. <img alt="Figura 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> Figura 3. Esterilizado alicates, luvas e bandeja. <img alt="Figura 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> Figura 4. Removido expansor. <img alt="Figura 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> Figura 5. Scraping off biofilme com um bisturi estéril. <img alt="Figura 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> Flocos de resina Figura 6. Diretamente hibridizado em lâminas de vidro revestido. <img alt="Figura 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> Figura 7 imagem CLSM:. Diferenciação de um grupo específico de bactérias. Sobreposição de dados 2D em 3D CLSM pilha. Biofilme corado com EUBmix (verde, todas as bactérias) e LGCmix (amarelo, Firmicutes). <img alt="Figura 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> Figura 8 imagem CLSM:. Diferenciação de um grupo específico de bactérias. Sobreposição de dados 2D em 3D CLSM pilha. Manchada com biofilme EUBmix (vermelho, todas as bactérias), Bac303 (azul, Bacteroidetes) e IGP (amarelo, Porphyromonas gingivalis). <img alt="Figura 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> Figura 9 imagem CLSM:. Diferenciação de morfologias específicas. Sobreposição de dados 2D em 3D CLSM pilha. Biofilme corado com EUBmix (verde, todas as bactérias). Grandes aglomerados de bactérias cocóide (setas). <img alt="Figura 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> . Figura 10 imagens CLSM: diferenciação de morfologias específicas. Sobreposição de dados 2D em 3D CLSM pilha. Biofilme corado com EUBmix (vermelho, todas as bactérias). Cocóide (seta abaixo) e bactérias filamentosas (seta acima) podem ser distinguidas. <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 11. Mushroom estrutura, vistas 3D a partir de baixo. Pilhas de biofilme flocos (raspado a superfície de um aparelho ortodôntico), coradas com EUBmix e procedimentosssed com IMARIS (imagem CLSM). <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 12. Mushroom estrutura, vista lateral 3D. Pilhas de biofilme flocos (raspado a superfície de um aparelho ortodôntico) processados ​​com IMARIS (imagem CLSM). <table border="1"><tbody><tr><td colspan="4"> Tampão de hibridação (200 mL) </td></tr><tr><td> Concentração de formamida </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 Tris-HCl </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; DDH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> Qualquer sonda FISH </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> Tabela 1. Constituintes de tampão de hibridação (concentrações em mL). <table border="1"><tbody><tr><td colspan="4"> Tampão de lavagem (50 ml) </td></tr><tr><td> Concentração de formamida </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 Tris-HCl </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> 0,5 M EDTA </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> 2 O DDH </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> Tabela 2. Constituintes de tampão de lavagem (concentrações em mL). Movie 1. <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Clique aqui para assistir o filme.</a>

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Não há conflitos de interesse declarados.

O protocolo descrito aqui é uma abordagem altamente viável para biofilmes coloração coletados de materiais duros. Amostragem e hibridação são as etapas mais críticas. Durante a amostragem, os cuidados devem ser tomados para que as fatias de espessura adequada são coletados para garantir que a estrutura do biofilme permanece intacta. Durante a hibridização, é essencial para evitar flutuações excessivas de temperatura, evitando ligação não específica, ou perda de ligação, das sondas fluorescente etiquetado. Este protocolo FISH é um método elegante para a mancha biofilme heterogêneos normal diretamente em lâminas de vidro, sem interromper sua composição. Os slides podem ser imediatamente utilizado para microscopia sem precisar mover a amostra, mais uma vez. Desta forma, uma melhoria em relação a coloração em tubos é obtido pela força de cisalhamento menor sendo aplicada ao biofilme. Fatias de&gt; 50 mm de espessura podem ser preparados e investigados dessa forma.

<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

Microscopia de varredura confocal a laser (CLSM) do biofilme heterogênea natural é hoje facilitado por uma vasta gama de técnicas de coloração, sendo um deles hibridização fluorescente in situ (FISH). 1,2 Realizamos um estudo piloto em que as amostras coletadas de biofilme oral fixa aparelhos ortodônticos (palatal expansores) foram corados por FISH, com o objectivo de avaliar a organização tridimensional de biofilme naturais e acúmulo de placa. 3,4 FISH cria uma oportunidade para corar as células em seu ambiente nativo do biofilme com o uso de 16S fluorescente etiquetado rRNA de metas de sondas. 4-7,19 Em comparação com técnicas alternativas como a rotulagem de imunofluorescência, este é um barato, a técnica de rotulagem rigorosa e direto para investigar diferentes grupos de bactérias em biofilme consórcios mistos. 18,20 sondas foram usadas Geral que se ligam a Eubacteria ( EUB338 + + EUB338II EUB338III; seguir EUBmix), 8-10 Firmicutes (LGC354 AC;. LGCmix seguir), 9,10 e Bacteroidetes (Bac303) 11 Além disso, as sondas de ligação específico para Streptococcus mutans (MUT590) 12,13 e Porphyromonas gingivalis (IGP) foram usados ​​13,14 . A extrema dureza dos materiais de superfície envolvidos (aço inoxidável e resina acrílica) obrigaram-nos a encontrar novas maneiras de preparar o biofilme. Como estes materiais de superfície não poderia ser facilmente cortado com uma cryotome, vários métodos de amostragem foram exploradas para obter biofilme oral intacta. O mais viável dessas abordagens é apresentada na presente comunicação. Pequenos flocos da resina biofilme de transporte de acrílico foram raspados com um bisturi estéril, tomando cuidado para não danificar a estrutura do biofilme. Fórceps foram usados ​​para coletar biofilme das superfícies de aço. Uma vez coletadas, as amostras foram fixadas e colocado diretamente sobre lâminas de vidro revestidas polysine. FISH foi realizada diretamente sobre esses slides com as sondas mentioned acima. FISH vários protocolos foram combinados e modificados para criar um novo protocolo que foi fácil de manusear. 5,10,14,15 Posteriormente as amostras foram analisadas por microscopia confocal de varredura a laser. Bem conhecidos 3,4,16,17 configurações podem ser visualizados, inclusive cogumelo estilo de formações e aglomerados de bactérias cocóide permeado por canais. Além disso, a composição bacteriana do biofilme típico dessas estruturas foram analisados ​​e imagens 2D e 3D criado.

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

Nós apresentamos um protocolo para análise estrutural e de composição do biofilme oral natural de aparelhos ortodônticos com<em&gt; In situ</em&gt; Hybridization (FISH) e microscopia de varredura confocal a laser (CLSM). Amostras de biofilme oral foram coletados de expansores palatinos, raspagem de resina acrílica flocos de fora de sua superfície e encaminhá-los para o processamento molecular.

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

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 visualizações.  Medical University of Graz. Nós apresentamos um protocolo para análise estrutural e de composição do biofilme oral natural de aparelhos ortodônticos com In situ Hybridization (FISH) e microscopia de varredura confocal a laser (CLSM). Amostras de biofilme oral foram coletados de expansores palatinos, raspagem de resina acrílica flocos de fora de sua superfície e encaminhá-los para o processamento molecular.

Vídeo: 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.

Sonda baseada Endomicroscopy Laser Confocal do Trato Urinário: A Técnica

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

Medicina

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.

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA<em&gt; In Situ</em&gt; Hibridização

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.

Biofilme oral amostragem para análise de Microbiome em crianças saudáveis

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.

Segunda geração harmônica sinais na esclera coelho como uma ferramenta para avaliação do tecido terapêutico do cross-linking (TXL) para miopia

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.

Formação de biofilme oral em diferentes materiais para implantes dentários

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.

Avaliação em Saúde Bucal por Leigo para Idosos

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.

Visualizando a formação de babados de membrana usando microscopia eletrônica de varredura

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.

Fluorescência interfásico in situ Hibridização de Manchas de Medula Óssea de Mieloma Múltiplo

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.

Quantificação Planimétrica Semi-Automatizada de Placa Dentária Utilizando Câmera de Fluorescência Intraoral

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

Captura de amostras de tecido de fibrose submucosa oral para análise espacial-ômica

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.

Autor em destaque: desvendando os mistérios das neoplasias potenciais orais 

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.

Isolamento de Células com Informações Morfológicas e Espaciais de Amostras de Fibrose Submucosa Oral por Microdissecção por Captura a Laser

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 ...