Bu çalışma Sağlık hibe R01 CA140257-01 Ulusal Enstitüsü Ulusal Kanser Enstitüsü tarafından destekleniyor.

<html> NOT: Aydınlatılmış onam hastalardan elde edildi. Ayrıca, bu araştırmanın insan refahı için tüm kurumsal, ulusal ve uluslararası kurallar ile uyumlu olarak gerçekleştirilmiştir. 1.. Bilgisayarınızı Hazırlanır <ol><li> Laptop açın ve DVI2USB Capture Card USB bağlantı. </li></ol> 2.. Monitor hazırlayın <ol><li> Endoskopist yerine bilgisayardan daha bu ekranlardan videoları görüntülemek için izin izlemek ve PinP DVI kablosunu bağlayın. </li><li> Ayakta Monitör ve DVI ayarlanmış olduğundan emin olun. </li><li> Olympus sistemin arkasına PinP bağlayın ardından duvara monte edilen geniş ekran üzerinde resmi görebilmek için Olympus sistemine giriş düğmesine basın. </li></ol> 3.. Lazer Diyot Sürücü Ayarı <ol><li> Emin lazer diyot sürücü OFF olmak için kontrol edin. Görüntüleme yapılmadan önce, sadece bir kaç dakika açık olmalıdır. </li></ol>E_TITLE &quot;&gt; 4.. güç şerit <ol><li> Güç şerit içine dizüstü bilgisayar, işlemci, lazer diyot sürücü, DVI splitter ve Epiphan yakalama kartı takın. </li></ol> 5.. Laptop Masaüstüne MDE Widefield çalıştırın <ol><li> &#39;Geçerli Klasör&#39; Hit. </li><li> Hasta sayısı ve baş harflerini ve basın &#39;Hasta Klasör Oluştur&#39; yazın. </li></ol> 6.. Cap hazırlayın ve Filtre <ol><li> Filtre ele aldığınızda her zaman filtre yağ ve enkaz transferini en aza indirmek için eldiven ve Kimwipes kullanın. Gazlı bez ya da alkol sürüntü görüntüleme ile müdahale lifleri bırakabilir. </li><li> Filtreyi ve kapağı aşağı koymak için bir platform oluşturmak için bir masa üzerine birkaç Kimwipes yerleştirin. Bu dezenfekte alan işlem sırasında gastroenterolog kolayca erişilebilir olmalıdır. </li><li> Filtreyi ve kapağı uzandı ve prosedürü sırasında kullanılmak için kapatın Kimwipes tutun. </li></ol> 7. Hasta Hazırlama <ol><li> Kullanımı Rıza hastaEndo paketi gelmeden önce VFI ve proflavine boya. </li><li> Üst endoskopi işlemi için pozisyon hasta. </li><li> Bir bantlı dijital mikroskop (MDM) 7 kullanılarak standart beyaz ışık görüntüleme ile devam edin. </li></ol> 8. Proflavine takın ve Sprey <ol><li> Beyaz ışık görüntüleme ile bitirdikten sonra, eklemek ve ilgi doku üzerinde proflavine boya sprey. 1-5 mm Barrett doku alanına bağlı olarak yeterli olmalıdır. Bu aşamada proflavine boya püskürtme ile, (en az 1 dakika) yeterli zaman VFI önce yeterli doku emilimi için verilir. FDA (IND 102217) bir araştırma yeni bir ilaç uygulaması kapsamındaysa Proflavine, uygulamadan kısa bir süre sonra hücre çekirdeğinin içinde yoğunlaşır floresan kontrast ajandır. Lazer diyot tava doku üzerinde, yansıyan ışık genel glandüler simler takdir endoskopist sağlar zaman VFI bireysel hücresel morfoloji, çözemezse rağmengy. </li></ol> 9. Lazer Diyot açın <ol><li> Lazer diyot açın. Görüntüleme başlamadan önce, bu en az 2 dakika yapın. </li></ol> 10. VFI için Endoskop hazırlayın <ol><li> Tamamen endoskop çekiniz. </li><li> Önce endoskop işleme, asistan iki çift eldiven sahip olmasını sağlamak. </li><li> Endoskopist yukarıda ayrıntılı Kimwipe&#39;dan platformu yanında asistan önünde endoskopu tutun var. </li><li> Kimwipes, asistan endoskop ucunu temizler var. Nazikçe ön yüzeyi temizlemek ve aynı zamanda kolay kullanım için endoskop tarafı boyunca birkaç santimetre temizlemek için emin olun. </li><li> Temizlikten sonra, eldiven dış çiftinin yardımcısı atmayın var. </li><li> Asistan filtre koyduk. Endoskop onun tamamlayıcı bir delik içine filtre üzerinde kısa silindirik çıkıntı yerleştirin. Sürecine yardımcı olmak için dikey endoskop tutun. </li><li> Filt Holdinger yerde, filtre üzerinde kapağı kaydırın ve endoskop ucu üzerinde aşağı itin. Kap, güvenli ve endoskop ile aynı hizada kenarları tamamen itti olduğundan emin olun. Başlığın dudak hafifçe endoskopun ucu boyunca uzanan emin olun. Son olarak, emin filtre yerinde hala yapmak ve endoskop ucu ile yıkayın. </li></ol> 11.. Endoskop yemek borusuna geri ve Resim Ekle 12. Özofagus dan Endoskop kaldır <ol><li> Kimwipes kullanılarak dikkatlice kapağı çekin ve endoskop filtre kapalı. Kapağı atmak ve bir sonraki durum için filtreyi temizleyin. </li></ol> 13. Clean Filtre <ol><li> Yavaşça Kimwipes filtresini temizleyin. </li><li> Cydex küçük bir fincan doldurun ve 12 dakika boyunca filtreyi daldırın. Her birkaç dakika filtreyi ters çevirin. </li><li> Yavaşça Kimwipes filtresini temizleyin. </li><li> , Steril su içinde, bu kez filtre daldırın. 5 dakika sonra, bir bücür b kullanınÖttle ve hafifçe filtre üzerinde steril su püskürtün. </li><li> Birkaç Kimwipes filtreyi yerleştirin ve kurumaya bırakın. </li><li> Kimwipes arasında saklama kabının içinde kuruyunca, yer. </li></ol>

<ol>
	<li>Modiano, N., Gerson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrett's+Esophagus:+Incidence,+etiology,+pathophysiology,+prevention+and+treatment.">Barrett's Esophagus: Incidence, etiology, pathophysiology, prevention and treatment.</a> Therapeutics and Clinical Risk Management. 3, 1035-1145 (2007).</li><li>Brown, L. M., Devesa, S. S., Chow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of Adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> Journal of the National Cancer Institute. 100, 1184-1187 (2008).</li><li>Siegel, R., Naishadham, D., Ahmedin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2012.">Cancer Statistics, 2012.</a> A Cancer Journal for Clinicians. 62, 10-29 (2012).</li><li>Vieth, M., Ell, C., Gossner, L., May, A., Stolte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+endoscopic+resection+specimens+from+326+patients+with+Barrett's+esophagus+and+early+neoplasia.">Histological analysis of endoscopic resection specimens from 326 patients with Barrett's esophagus and early neoplasia.</a> Endoscopy. 36, 776-781 (2004).</li><li>Pohl, H., Rosch, T., Vieth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniprobe+confocal+laser+microscopy+for+the+detection+of+invisible+neoplasia+in+patients+with+Barrett's+oesophagus.">Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus.</a> Gut. 57, 1648-1653 (2008).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+video+endoscopy+for+in+vivo+cross-polarized+and+vital-dye+fluorescence+imaging+of+Barrett's-associated+neoplasia.">Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia.</a> Journal of Biomedical Optics. 18, 026007 (2013).</li><li>Roblyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+optical+imaging+device+for+in+vivo+detection+of+oral+neoplasia.">Multispectral optical imaging device for in vivo detection of oral neoplasia.</a> Journal of Biomedical Optics. 13, 024019 (2008).</li><li>Egger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopsy+surveillance+is+still+necessary+in+patients+with+Barrett's+oesophagus+despite+new+endoscopic+imaging+techniques.">Biopsy surveillance is still necessary in patients with Barrett's oesophagus despite new endoscopic imaging techniques.</a> Gut. 52, 18-23 (2003).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital-dye+enhanced+fluorescence+imaging+of+GI+mucosa:+metaplasia,+neoplasia,+inflammation.">Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.</a> Gastrointestinal Endoscopy. 75, 877-887 (2012).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointestinal Endoscopy. 68, 737-744 (2008).</li></ol>

Ifşa hiçbir şey yok.

Benign metaplazi yüksek dereceli displazi ve adenokarsinom ayırt olabilir çünkü standart endoskopik gözetim, BE neoplazi genellikle 8 cevapsız. Iyi bu şu anda kaçınılmaz hatayı düzeltmek için gastroenterologlar sağlayacak bir araç olarak, yaşamsal-boya geliştirilmiş floresan görüntüleme böylece doku tiplerini ayırt etmek için ayrı bir özellik sağlayan dokusunun glandüler morfolojisi vurgulamaktadır. Ayrıca, 2.5 cm kadar bir görüş alanı sağlayarak, VFI şüpheli lezyonlar da...

Son kırk yılda, özofagus adenokarsinomu (EAC) insidansı belirgin 1,2 arttı; Henüz nedeniyle geç tanı, beş yıllık sağkalım oranı% 20 den az 3&#39;tür. BE endoskopik gözetim mevcut standart, EAC öncü, segmentinin rasgele dört quandrant forceps &#39;biyopsi ile beyaz ışık endoskopi olduğunu. Ne yazık ki, bu teknik genellikle standart beyaz ışık görüntüleme 4 ayırt etmek için, düz ince ve zor olabilir, hangi Neoplasia&#39;nm özlüyor. In vivo hücresel özelliklerini vurgulamak için konfokal lazer mikroskopi kullanılarak başarı olmuştur da, lezyonlar nedeniyle hala bakış 5 azalmış alanına atlanabilir. Daha konfokal microendoscopic görüntüleme alanları vurgulamak bir &#39;köprü&#39; teknolojiye sahip belirgin değerli olacaktır. Sonuç olarak, küçük kalkan için yeteneğini geliştirir gelişmiş bir kırmızı bayrak görüntüleme yöntemidirBE t ve biyopsi erken neoplazi erken, tedavi edilebilir aşamada EAC tespit vesile olacak ve daha etkili tedavi ve daha sonra sağkalım oranlarına yol açabilir. VFI glandüler morfolojisi vurgulamak ve in vivo tanı 6 iyileştirmek umuduyla distal özofagus neoplasia&#39;nm (yüksek dereceli displazi ve kanser) belirginleştiren, eksojen topikal floresan kontrast, proflavine ile yüksek çözünürlüklü görüntüleme epitel birleştiren yeni bir tekniktir. Uygulamadan kısa bir süre sonra hücre çekirdekleri içinde yoğunlaşır proflavine, uyarma üzerine, floresan görsel endoskopistler glandüler morfolojisi görselleştirmek için izin mekansal 50-100 um çözünürlüğü ve 2.5 cm kadar bir görüş alanı sağlar. Sonuç olarak, bu yaklaşım oblitera sahip neoplazisi, BE ikinci, sürekli, eşit aralıklı bezleri ve genel olarak homojen bir yapıya sahiptir klasik Barrett metaplazi, ayırt gastroenterologlar sağlarglandüler mimarisinin tion. Burada bir çok bantlı endoskop ile bu yeni tekniğin protokolü açıklamak, ve yüksek dereceli displazi ve kansere iyi huylu metaplazi gelen morfolojik dönüşüm resmeden bu cihazın programını göstermek için temsili sonuçlar sağlar.

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr>
			<td>Filter*</td>
			<td>Schott North America, Inc., Duryea, Pennsylvania</td>
			<td>Not Applicable</td>
			<td>495-nm long-pass filter</td>
		</tr>
		<tr>
			<td>Halo Cap &#8211; Medium*</td>
			<td>Barrx Medical</td>
			<td>CP-002A</td>
			<td></td>
		</tr>
		<tr>
			<td>Processor*</td>
			<td>Pentax</td>
			<td>EPK-i</td>
			<td></td>
		</tr>
		<tr>
			<td>Multispectral Digital Microscope**</td>
			<td>Not Applicable</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>KimTech Science</td>
			<td>S-8115</td>
		</tr>
		<tr>
			<td>Cidex</td>
			<td>Advanced Sterilization Products&#160;</td>
			<td>CIDEX OPA Solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Proflavine hemisulfate (0.01% w/v)</td>
			<td></td>
			<td></td>
			<td>FDA (IND 102,217)</td>
		</tr>
		<tr>
			<td>Laser Diode*</td>
			<td>Nichia Corporation</td>
			<td>Not Applicable</td>
			<td>455-nm</td>
		</tr>
		<tr>
			<td>Image Capture*</td>
			<td>Labview</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray Catheter</td>
			<td>Olympus</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>*Equipment specifics within Reference 6. **Equipment specifics within Reference 7</td>
		</tr>
	</tbody>
</table>

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

Her iki doku tipleri düz ve yalnız beyaz ışık görüntüleme ile ayırt olabilir gibi in vivo neoplazya Barrett Özofagus (BE) benign metaplazisini ayırt etmek yeteneği zor kalır. Sonuç olarak, glandüler mimarisini vurgular bir yöntemdir distal özofagus benign epitel neoplaziyi ayırt etmek yararlı olacaktır. VFI benign epitel yüksek dereceli displazi ve kanser belirginleştiren bir dışsal topikal floresan kontrast madde kullanan yeni bir tekniktir. Özellikle, floresan görüntüler endoskopistler glandüler morfolojisi görselleştirmek için izin, mekansal 50 ila 100 mikron çözünürlük ve 2.5 cm kadar bir görüş alanı sağlar. Uyarma üzerine, klasik Barrett metaplazi gibi sürekli, eşit aralıklı bezleri ve genel bir homojen morfoloji görünür; aksine, neoplastik doku glandüler çerçevenin tam daralmaya kalabalık görünür. Burada bir bakış sağlamakenstrümantasyon ve bu yeni tekniğin protokolünü numaralandırmak. VFI şüpheli doku glandüler mimarisi ile bir gastroenterolog tanıyor olsa da, hücresel displazi bu yöntem ile çözülemez. Gibi, bir morfolojik bu görüntüleme yöntemi ile düşük dereceli displazi BE gelen Barrett metaplazisini ayırt edemez. , Daha geniş bir alanı ile çözünürlükte bir düşüş kapalı işlem yaparak, bu görüntüleme sistemi şüpheli lezyonları hedef ve biyopsi için kırmızı-bayrak görüntüleme cihazı olarak en azından kullanılabilir; doğruluk tedbirler umut vardır, henüz, VFI distal özofagusta (yüksek dereceli displazi veya kanser ya olarak tanımlanır) neoplazi tanısı için standart görüntüleme tekniği haline gelebilir.

Şekil 1B normal skuamoz epitel ile sınırları çevrili hiçbir displazi ile klasik Barrett Özofagus gösteriyor. Çevresel olarak yer alan ve mavi oklar ile gösterilen düz yassı dokusu ile başlayarak, mat floresans homojen bir alan herhangi bir glandüler mimarinin mevcutsa. Yeşil oklar skuamöz dokuyu çevreleyen dairesel yeşil çizgiyi gösterir. Bu taslak endoskop kapağından çıkan eseridir. Merkezi bir konumda bulunan Barrett dokusu taşıma, glandüler yapılar koyu lümen çevreleyen ...

Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Vital-boya geliştirilmiş floresan görüntüleme (VFI) glandüler morfolojisi vurgulamak ve distal özofagus neoplasia&#39;nm (yüksek dereceli displazi ve kanser) belirginleştiren eksojen topikal floresan kontrast ile yüksek çözünürlüklü görüntüleme epitel birleştiren vivo tekniğin bir roman.

Vital-boya Geliştirilmiş Floresan Imaging kullanılarak Barrett Özofagus içinde Neoplazi tanısı

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

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Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

11.9K Görüntüleme.  Icahn School of Medicine at Mount Sinai.  Vital-boya geliştirilmiş floresan görüntüleme (VFI) glandüler morfolojisi vurgulamak ve distal özofagus neoplasia'nm (yüksek dereceli displazi ve kanser) belirginleştiren eksojen topikal floresan kontrast ile yüksek çözünürlüklü görüntüleme epitel birleştiren vivo tekniğin bir roman. 

Video: Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Yaşayan Hücreler Moleküler Rotorlar arasında Flüoresans Ömrü Görüntüleme

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChip kullanarak Kök Çevre Rapid manipülasyonu ile Arabidopsis Kök Büyüme Time-lapse Floresans Görüntüleme

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Tek molekül Floresan Mikroskopi Reseptör Dinamiği Yüksek çözünürlüklü Spatiotemporal Analizi

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Modifiye İki foton mikroskop 3D Orbital Takip: Hücre içi veziküllerin İzlemeye Bir Uygulama

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Ticari Mikroskop Canlı Hücre Zarları Moleküler Difüzyon Kanunu Hızlı Floresans Görüntüleme Gönderen

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Bir Çok Kanallı Çift elektrik alan mikroakışkan Chip kullanarak Akciğer Kanseri Hücreleri Electrotaxis Çalışmaları

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Çoklu görüntüleme modları bir floresan mikroskop ile iletken

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Hücreleri tarafından seçilmiş adezyon oluşumunda görselleştirme gelişmiş Disk-toplam iç yansıma floresans mikroskobu iplik

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Tümör türevi Exosomes ölçmek için Nanoplasmon-gelişmiş saçılma ve düşük büyütme mikroskop görüntüleme kullanma

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...