Bu çalışma Hijyen Viyana Tıp Üniversitesi Graz tarafından desteklenmiştir. Tüm denekler bilgilendirilmiş onam verdi. Kurumsal çalışma protokolü ile ilgili onayı Üniversitesi Tıp Fakültesi Graz elde edildi.

1. Örnek toplama <ol><li> 4 ay sonra intraoral hizmeti sabit damak genişleticiler çıkarın. NOLA Kuru Alan Sistemi (Şekil 1 ve 2) çıkarmadan önce aletleri izole etmek için kullanın. </li><li> Kontaminasyon (Şekil 3 ve 4) eklemeden genişleticiler kaldırmak için steril pense, eldiven ve tepsileri kullanın. </li><li> -20 ° C&#39;de Sarstedt 120 ml flakon Mağaza nesneleri 24 saat içinde laboratuvara buz ve süreç ele alalım. </li></ol> 2. Biyofilm fiksasyon <ol><li> Biyofilm gevreği steril bir bisturi ile kazıyın, veya steril forseps ile parçaları toplamak. </li><li> Örnek kaplıdır kadar% 4 PFA çözüm buz ekleyin. </li><li> Karışım +4 ° C&#39;de 3 ila 12 saat boyunca (donma yok). Daha uzun fiksasyon kez veya daha fazla fiksasyon sıcaklıklarda oligonükleotid problar daha az geçirgen gram-negatif hücre hücre zarflar hale getirebilir. </li><li> PFA çözüm çıkarın ve buz 1X PBS ile yıkayın. R bu adımı 2-3 kez tekrarlayınartık PFA aldır. </li><li> 1 vol örnek süspanse edin. buz 1X PBS ve eklemek 1 vol. buz% 96 (v / v) etanol. </li><li> -20 ° C&#39;de örnek Mağaza Bu protokole göre sabit Örnekleri yıl birkaç ay saklanabilir. </li></ol> 3. Sabit Örnekler, Dehidrasyon <ol><li> Bir mikroskop lamı PFA sabit numune 5-30 ul uygulayın. </li><li> Kuru 46 at ° C yaklaşık 15 dakika veya daha uzun süre oda sıcaklığında. Lizozim ve formamid çözülme. </li><li> Böylece hücre duvarlarında kısmi yıkım tarafından problar hücre içine nüfuz kolaylaştırılması, 10 dakika oda sıcaklığında lizozim (1mg/ml) 250 ul ekleyin. </li><li> % 50,% 80 ve% 96 (v / v) her biri 3 dakika etanol içine Lamı. Etanol konsantrasyonu serisi dehydrating etkisi, hücre zarları parçalanır. </li><li> 46 ° C &#39;de 10 dakika süreyle slaytlar kurulayın. </li></ol> 4. In-situ hibridizasyon <ol><li> 1 ml hazırlayıntaze hibridizasyon tamponu (bkz. Tablo 1 konsantrasyonları için). Bu çalışmada kullanılan Formamid dağılımı:% 10 (EUBmix)% 20 (Bac303, POGI, MUT590) veya% 45 (LGCmix). </li><li> Oligonükleotid prob çözümleri çözülme. Çözülmüş probları buz üzerinde tutulmalıdır ve ışıktan korunmalıdır. </li><li> Her prob hibridizasyon tampon 200 ul 2 ul ekleyin, iyice karıştırın ve kurutulmuş örnek bir mikroskop lamı üzerine karışımı uygulayabilirsiniz. </li><li> Kare bir Petri kabı içine, bir parça kağıt mendil koyun ve kalan hibridizasyon tamponu kağıt mendil üzerine dökün. </li><li> Hemen yemeğin içine slayt yatay yerleştirmek ve tabak kapatın. 46 ° C&#39;de 1-5 saat (90 dakika çoğu durumda yeterli olacaktır) bir fırında inkübe edin. Bir nem odası olarak çanak işlevleri slayt hibridizasyon çözüm buharlaşmasını önler. Özellikle, formamid, buharlaşma, non-spesifik prob bağlayıcı olmayan hedef hücreleri neden olabilir. </li><li> 50 ml yıkama tamponu (bkz. Tablo 2 conce için hazırlayınntrations). 48 50 ml tüp ve ön ısıtma yıkama tamponu hazırlayın ° C su banyosunda. Yıkama 48 ° C yapılır </li><li> Hibridizasyon fırın slayt çanak çıkarın. Hibridizasyon tamponu önceden ısıtılmış çamaşır tampon küçük bir hacmi ile hemen yıkayın ve yıkama tampon kalan slayt aktarmak. </li><li> 48 ° C&#39;de 10-15 dakika süreyle su banyosunda inkübe geri yıkama tampon ve slayt içeren tüp yerleştirin. </li><li> Tüpten slayt çıkarın ve kalan yıkama tampon ortadan kaldırmak için 2-3 saniye boyunca buz gibi GKD 2 O içine batırın. </li><li> Mümkün olduğunca çabuk slayt (basınçlı hava kullanılması tavsiye edilir) Hava kurulayın. Hızlı kuruyan prob disosiasyon azaltacaktır. </li><li> Kurutulmuş slaytlar prob haiz floresan sinyal kaybı olmadan birkaç hafta, -20 ° C&#39;de karanlıkta saklanabilir. </li></ol> 5. Mikroskopi <ol><li> Sonra FISH ve yıkama, Applantifadent yakın bir slaytın sol ve sağ uçları (dondurulmuş dilim bu adımı önce oda sıcaklığına kadar ısıtılmalıdır) y iki damla. </li><li> Mikroskop kapağının üstünde kayma ve antifadent tüm slayt yayılmış kadar bekleyin yerleştirin. Antifadent aşırı miktarda mikroskopta görüntü bulanıklığını unutmayın. </li><li> Uygun filtreler veya lazer ile donatılmış bir konfokal lazer tarama mikroskop altında örnekleri dikkate alınmalıdır. Biz Leica TCS birimi (HCX PL APO/63x, NA 1.2) kullanılır. Veriler, IMARIS veya AMIRA yazılım ile analiz edilebilir. </li><li> Prob haiz floresan düşmeye başlamadan önce antifadent içinde gömülü Slaytlar +4 ° C (donma yok) 7 güne kadar saklanabilir. Alternatif olarak, antifadent GKD 2 O ile kaldırılır ve kurutulmuş slaytlar, uzun bir süre için -20 ° C&#39;de saklanabilir . </li></ol> 6. Temsilcisi Sonuçlar: (Sabit ortodontik aletler kapalı biyofilm KazımaŞekil 5), doğrudan mikroskopi için kaplı cam Slaytlarınıza melezleşmiştir olabilir uygun gevreği (Şekil 6) elde edilir. Bu şekilde, farklı etiketli spesifik problar (Şekil 7 ve 8) ile etiketleme bakteriyel rRNA orobiome bakterilerin farklı gruplar tarafından kendi doğal üç boyutlu ortamda tespit edilebilir. Şekil 7, biyofilm EUBmix (yeşil, tüm bakteriler) ve LGCmix (sarı, Firmicutes) ile boyandı. , Sarı ve mavi ile boyandı Firmicutes, yeşil görünür. Şekil 8, biyofilm EUBmix (kırmızı, bakteri), Bac303 (mavi, Bacteroidetes) ve POGI (sarı, Porphyromonas gingivalis) ile boyandı. DNA ve üst üste üç probları bağlamak Porphyromonas gingivalis, sarımsı beyaz gösterilir renkleri beyaz bir sinyal sonuçları. Bakteri grupları arasında morfolojik farklılıklar da tespit edilebilir (Şekil 9 ve 10). Coccoid bakteriler büyük kümeler (yeşil, tüm ba EUBmix ile boyama yapıldı Şekil 9 gösterilmiştircteria). Oral bakterilerin farklı şekiller coccoid ve ipliksi bakteri EUBmix (kırmızı) ile boyama ile ayırt edilebilir Şekil 10, görüntülendi. Ayrıca, biyofilm tipik bir mantar benzeri bir yapı, 3D modelleme, CLSM veri (Şekil 11 ve 12) ile <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">işlenebilir 3D modelleme bir film izlemek için buraya tıklayın</a> . <img alt="Şekil 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> Şekil 1. in situ palatal genişletici düzeltildi. <img alt="Şekil 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> Şekil 2 Nola Kuru Alan Sistemi. <img alt="Şekil 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> Şekil 3 kerpeten, eldiven ve tepsi, sterilize edilmiştir . <img alt="Şekil 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> Şekil 4. genişletici kaldırıldı. <img alt="Şekil 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> Şekil 5 steril bir bisturi ile biyofilm kapalı Kazıma. <img alt="Şekil 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> Şekil 6 Reçine gevreği doğrudan kaplı cam Slaytlarınıza melezleşmiştir. <img alt="Şekil 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> Şekil 7 CLSM görüntü: farklılaşma belirli bir bakteri grubu. 3D CLSM veri yığınının 2B kaplama. Biyofilm EUBmix (yeşil, tüm bakteriler) ve LGCmix (sarı, Firmicutes) ile boyandı. <img alt="Şekil 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> Şekil 8 CLSM görüntü: farklılaşma belirli bir bakteri grubu. 3D CLSM veri yığınının 2B kaplama. Biyofilm EUBmix (kırmızı, bakteri), Bac303 (mavi, Bacteroi ile boyanmışdetes) ve (sarı, Porphyromonas gingivalis) POGI. <img alt="Şekil 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> Şekil 9 CLSM resim: özel morfolojileri farklılaşma. 3D CLSM veri yığınının 2B kaplama. Biyofilm EUBmix (yeşil, tüm bakteriler) ile boyandı. Coccoid bakteriler büyük kümeler (oklar). <img alt="Şekil 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> Şekil 10 CLSM görüntüler: özel morfolojileri farklılaşma. 3D CLSM veri yığınının 2B kaplama. Biyofilm EUBmix (kırmızı, bakteri) ile boyandı. Coccoid (aşağıda ok) ve ipliksi bakteri (yukarı ok) ayırt edilebilir. <img alt="Şekil 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Şekil 11 Mantar yapısı, aşağıdan 3D görünümleri. EUBmix ve prosedür ile boyanan biyofilm gevreği (ortodontik cihaz yüzey kazınmalı), yığınlarIMARIS (CLSM resim) ile ssed. <img alt="Şekil 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Şekil 12 Mantar yapısı, 3D yan görünümü. IMARIS (CLSM resim) ile işlenmiş biyofilm gevreği (ortodontik cihaz yüzey kazınmalı) yığınlar. <table border="1"><tbody><tr><td colspan="4"> Hibridizasyon tamponu (200 ul) </td></tr><tr><td> Formamid konsantrasyon </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; GKD 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> Herhangi bir FISH probu </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> Tablo 1 hibridizasyon tamponu bileşenleri (ul konsantrasyonları). <table border="1"><tbody><tr><td colspan="4"> Yıkama tamponu (50 ml) </td></tr><tr><td> Formamid konsantrasyon </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> GKD 2 O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> Tablo 2, yıkama tampon bileşenleri (ul konsantrasyonları) . Film 1 <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">film izlemek için tıklayınız.</a>

<ol>
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Çıkar çatışması ilan etti.

Açıklanan protokol burada, sert malzemeler için toplanan boyama biyofilm yüksek uygulanabilir bir yaklaşımdır. Örnekleme ve hibridizasyon en kritik adımlar. Örnekleme sırasında, bakım, yeterli kalınlıkta dilimler biyofilm yapısı bozulmadan kalmasını sağlamak için toplanan alınmalıdır. Hibridizasyon sırasında, böylece kaçınarak, non-spesifik floresan etiketli probları bağlama, ya da bağlayıcı kaybı, aşırı sıcaklık dalgalanmaları önlemek için çok önemlidir. Bu BALIK protokol Bileşiminde bozmadan cam slaytlar doğrudan normal heterojen biyofilm leke zarif bir yöntemdir. Slaytlar hemen mikroskopi için bir kez daha örnek taşımak için bir gerek kalmadan kullanılabilir. Bu yolla, Tüplerde boyama üzerinde bir iyileşme biyofilm uygulanmakta olan daha az kesme kuvveti elde edilir. &gt; 50 mikron kalınlığında Dilimleri Bu şekilde hazırlanan ve araştırılmalıdır.

<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

Doğal heterojen biyofilm Konfokal lazer tarama mikroskobu (CLSM) onları situ hibridizasyon (FISH) floresan olma boyama teknikleri, geniş bir ürün yelpazesiyle bugün kolaylaştırmıştır. 1,2 oral biyofilm örnekleri sabit toplanan hangi bir pilot çalışma gerçekleştirilmiştir FISH, üç boyutlu bir organizasyon doğal biyofilm ve plak birikimi değerlendirmek için objektif ortodontik aletler (damak genişleticiler) ile boyandı 3,4 BALIK floresan etiketli 16S kullanımı ile kendi ana biyofilm ortamında hücreleri leke için bir fırsat yaratır rRNA hedef sondalar. 4-7,19 immunofluorescent etiketleme gibi alternatif teknikler karşılaştırıldığında, bu karma biyofilm konsorsiyumların içinde farklı bakteriyel gruplar araştırmak için ucuz, hassas ve basit etiketleme tekniği. 18,20 Genel problar kullanıldı Öbakteriler bağlamak ( EUB338 + EUB338II + EUB338III bundan EUBmix), 8-10 Firmicutes (LGC354 AC; bundan LGCmix), 9,10 ve Bacteroidetes (Bac303) 11. Buna ek olarak, Streptococcus mutans bağlanarak spesifik problar (MUT590) 12,13 ve Porphyromonas gingivalis (POGI) 13,14 . Katılan yüzey malzemeleri aşırı sertlik (paslanmaz çelik ve akrilik reçine), biyofilm hazırlanması için yeni yollar bulmak için bizi zorladı. Kriyotom ile bu yüzey malzemeleri kolaylıkla kesilebilir olamazdı olarak, sağlam sözlü biyofilm elde etmek için çeşitli örnekleme yöntemleri araştırıldı. Bu yaklaşımların en uygulanabilir, bu iletişimi sunulmaktadır. Biyofilm taşıyan akrilik reçine Küçük gevreği biyofilm yapısına zarar vermemeye dikkat ederek, steril bir bisturi ile kazınmış. Forseps çelik yüzeylerde biyofilm toplamak için kullanıldı. Bir kez toplanan numunelerin sabit ve polysine kaplı cam slaytlar doğrudan yerleştirildi. FISH probları m doğrudan bu slaytları yapıldıYukarıda entioned. Çeşitli BALIK protokolleri kombine ve kullanımı kolay yeni bir protokol oluşturmak için modifiye edilmiştir. 5,10,14,15 Daha sonra numuneler konfokal lazer tarama mikroskobu ile incelendi. Tanınmış yapılandırmaları 3,4,16,17 mantar tarzı oluşumlar ve kanallar tarafından kaplanır coccoid bakteri kümeleri dahil olmak üzere, görüntülenmiştir olabilir. Buna ek olarak, bu tipik biyofilm yapıların bakteriyel bir kompozisyon analiz ve 2D ve 3D görüntüler oluşturdu.

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

Ortodontik aletler doğal oral biyofilm yapısal ve kompozisyon analizi için bir protokol mevcut<em&gt; Yerinde</em&gt; Hibridizasyon (FISH) ve konfokal lazer tarama mikroskobu (CLSM). Oral biyofilm örnekleri kapalı akrilik reçine gevreği, yüzey kazıma ve moleküler işleme için onları yönlendiren, damak genişleticiler toplanmıştır.

Floresan In-Situ Hibridizasyon ve Konfokal Lazer Tarama Mikroskopi Palatal Genişleticilerden Oral Biyofilm Analizi

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

16.0K Görüntüleme.  Medical University of Graz. Ortodontik aletler doğal oral biyofilm yapısal ve kompozisyon analizi için bir protokol mevcut Yerinde Hibridizasyon (FISH) ve konfokal lazer tarama mikroskobu (CLSM). Oral biyofilm örnekleri kapalı akrilik reçine gevreği, yüzey kazıma ve moleküler işleme için onları yönlendiren, damak genişleticiler toplanmıştır.

Video: 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.

İdrar Yolu ve Probe tabanlı Konfokal Lazer Endomikroskopi: Tekniği

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

Tıp

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Flüoresans Konfokal videomikroskopi kullanılarak tümör Angiogenesis İn vivo

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

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 kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi<em&gt; In Situ</em&gt; Melezleme

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

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Vivo Tayf alanlı optik Koherens tomografi ve lazer Ophthalmoscopy tarama Cx3cr1gfp/gfp muhabir farelerin görüntüleme

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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.

Sağlıklı çocuklarda Microbiome analiz için sözlü biyofilm örnekleme

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.

Tavşan sklera bir araç olarak ikinci harmonik üretimi sinyalleri tedavi doku (TXL) için miyopi Cross-linking değerlendirilmesi için

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.

Diş implantları için farklı malzemeler üzerinde sözlü biyofilm oluşumu

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.

Yaşlı Yetişkinler için Lay Personel tarafından Ağız Sağlığı Değerlendirmesi

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.

Taramalı Elektron Mikroskobu ile Membran Fırfırı Oluşumunu Görselleştirme

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.

İnfaz Floresan in situ Multipl Miyelomun Kemik İliği Yaymalarının Hibridizasyonu

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

Floresan In-Situ Hibridizasyon ve Konfokal Lazer Tarama Mikroskopi Palatal Genişleticilerden Oral Biyofilm Analizi

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