Yazarlar herhangi bildirimleri var.

1.. Mica ve Slaytlar Hazırlık <ol><li> Sıra No: 1 ½ (0.17 mm) boyama rafa lamelleri. </li><li> 60 ° C&#39;de% 2 deterjan içinde 30 dakika boyunca sonikasyon </li><li> Iyonu giderilmiş su ile 20 kez yıkanır. </li><li> Forseps kullanarak slaytları çıkarın ve sıkıştırılmış hava veya nitrojen kullanarak kuru darbe. </li><li> Mika levha makas veya jilet kullanarak 10 x 10 mm kare parçalar halinde kesilir. </li><li> Jilet kullanarak 2-3 ince broşürler halinde her mika parça kesin. NOT: Bu adım keskin bıçak kullanımını gerektirir. </li></ol> 2.. Mika Montaj ve Montaj Odası <ol><li> Etanol ile temizleyin mikroskobik cam slayt. </li><li> Optik yapıştırıcı kullanılarak cam slayt adım 1.6 kesilmiş mika Tutkal broşür. Düşük viskoziteli yapıştırıcı iyi damla yaymak ve mika tutkal tavsiye edilir. </li><li> , UV lamba altında 10 dk Cure. Not: Yapışkan 380nm 350 aralığında maksimum emme ve requ önerilen enerji ile UV ışığı ile sertleştirilirTam tedavi için ired 4,5 J / cm 2 &#39;dir. Bununla birlikte, farklı ışık kaynakları (yapıştırıcı malzeme, satıcı tarafından sunulan bakınız) Bu adım için kullanılabilir. </li><li> Scotch bant ile ilk birkaç katmanları kaldırarak temiz bir mika yüzey Açığa. </li><li> Mika yüzeye optik yapıştırıcı küçük bir damla (~ 20 mikron) yerleştirin. NOT: Bu aşamada, düşük otofloresan düzeyi ile yüksek viskoziteli yapıştırıcı tavsiye edilir. Deneyimlerimiz düşük (adım 2.2) ve yüksek viskoziteli yapıştırıcı kombinasyonunu kullanarak mika bölme anlamlı etkinliği (adım 2.7) arttırdığını göstermiştir. </li><li> Yavaşça hava kabarcıkları kaçınarak, yapıştırıcı damla üzerine taze temizlenmiş lamel yerleştirin, 1 dakika razı olsun. </li><li> , UV lamba altında 10 dk Cure. NOT: Bu aşamadan sonra cam slayt, mika ve lamel sandviç (birkaç hafta kadar) bir zaman nispeten uzun bir süre boyunca saklanabilir. Mika yüzey taze olduğundan emin olmak için, sadece gerçek SLB öncesi hazırlık sonraki adıma geçin. NOTYapışkan 380nm 350 aralığında maksimum emme ve tam tedavisi için gerekli olan enerji ile önerilen UV ışığı ile sertleştirilir 4.5J/cm 2&#39;dir. Bununla birlikte, farklı ışık kaynakları (yapıştırıcı malzeme, satıcı tarafından sunulan bakınız) Bu adım için kullanılabilir. </li><li> Videoda gösterildiği gibi bir exacto bıçak kullanarak, hafifçe yan cam slayt lamel kaldırılıyor. Çoğu durumda, mika, ince ve düz bir tabaka lamel takılı kalır. (Adım 2.4 tekrarlayın) tekrar edilebilir Mika cam slayt bağlı. </li><li> Mika tabakası hala lamel yapıştırılmış ve adım 2.8 &#39;yarma sırasında tamamen kaldırıldı değildi emin olmak için, çıplak gözle ya da mikroskop altında yüzey kalitesini kontrol edin. Bir forseps ile küçük bir çizik yapma veya iğne diseksiyon mika algılanabilir, farklı bir tutarlılık vardır yapıştırıcı ayırt yardımcı olacaktır. </li><li> 1.5 ml şişe kapağından lastik conta çıkarın ve optik kullanarak yüzeye ters kapağı tutkalyapışkan veya tırnak cilası, ve UV lamba ile tedavi veya hava sırasıyla, 10 dakika kurumasını bekleyin. NOT: örnek AFM görüntüleme ile eş zamanlı optik (TIRFM veya konfokal) için hazırlanmış ise, bir 1.5 ml şişe kapağı mikroskop sahnede AFM baş bağlamaya çok küçük olabilir. Bu durumda, kapak AFM başlığı monte etmek için uygun olan daha büyük bir çapa sahip herhangi bir plastik O-halka ile ikame edilmiş olabilir. Deney mika yüzeye zarar vermeden kapağı çıkarmadan gerektiren bir durumda, silikon yağ yerine, yapıştırıcı veya oje kullanılabilir. </li></ol> 3.. Desteklenen çift katlı lipid (SLB) Oluşumu <ol><li> Yüzeyli odasına taze hazırlanmış lipozom solüsyonu yerleştirin. SLB oluşumu için gerekli olan minimum hacmi ~ 30 ul. NOT: lipozom ve SLB oluşumu hakkında daha fazla bilgi için, örneğin Çanta ark olarak yayınlanan protokolleri, bakınız, 2014 15... </li><li> İstenilen protokolü kullanılarak SLB oluşumu ile devam edin. İnkübasyon sırasında veGörüntüleme, odacık bir ısı-blok veya erime sıcaklığının üstünde kullanılan lipitleri tutmak için gerekli sıcaklığı sağlamak için ısıtıldı mikroskop aşamasında yerleştirilebilir. </li></ol>

<ol>
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F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> J Struct Biol. 151, 182-195 (2005).</li><li>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li><li>Schutz, G. 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Yazarlar ifşa hiçbir şey yok.

Bu protokol, lipid iki katmanlı birikimi ve yüksek çözünürlüklü görüntüleme için düz ve ince mika yüzeylerin hazırlanması için bir yöntemi tarif etmektedir. Bu teknik, çok yüksek kalitede bir yüzey elde etmek için mika kritik olan cam mika cam sandviç (adım 2.8), dikkatli sökme sınırlı az manuel beceri gerektirir. Mika yaran olmadan optik yapıştırıcı ayırmak için mümkün olduğu Taze kırılmış mika Denetim zaman optik yapıştırıcı maruz kalan bölgeleri bırakarak, bu noktada g...

Mevcut protokolün genel amacı da atomik kuvvet ile kombine edilebilir optik toplam iç yansıma floresan mikroskobu (TIRFM) veya konfokal mikroskopi kullanılarak mika desteklenen lipit tabakalarının (SLBS) yüksek çözünürlüklü görüntüleme için mika yüzeyleri hazırlamak için bir yöntem göstermektir mikroskobu (AFM). SLBS lipid kümeleme, faz ayrılması, peptidler, proteinler veya diğer bileşikler 1-5 ile iki tabakalı bileşenler veya bunların etkileşimleri ve dinamikleri çok sayıda çalışmalar için yaygın olarak kullanılan bir model vardır. Farklı alt tabakalar SLB oluşumu çalışmanın 4,6-8 doğasına bağlı olarak, (örneğin, cam, mika, silikon dioksit, polimerler) için kullanılabilir. Tipik membran çalışmalar, TIRFM ve AFM gibi mikroskopi tabanlı görüntüleme teknikleri, güveniyor. Cam şeffaf olduğu için Böylece TIRFM görüntüleme için, bir cam yüzeyi, tipik bir seçimdir. Cam imalatı nispeten kolay ve sonuçların kalitesi öncelikleönce lipid veziküllerin çökeltilmesinden temizleme ayrıntılı yüzey tarafından belirlenir. Yüksek çözünürlük nedeniyle eksenel AFM mika yüzeyleri gerektirir. Mika mükemmel bazal bölünme yakın olan, bir silikat mineraldir. Böylece, taze yarılmış mika hatta alt-nanometre ölçeğinde 9 zar yükseklik farklarının gözlem sağlayan atomik düz. Örneğin flüoresan korelasyon spektroskopisi (FCS), izleme tek bir molekül (SMT) ve (sıkı bağlamak) ışıkla ağartma sonra floresan kurtarma gibi yöntemler kullanılarak difüzyon çalışmalar, lipid zarı dinamikleri tevdi hangi üzerine yüzey tipine, burada cam büyük ölçüde bağlıdır, ancak gösterdi ve mika yaygın olarak farklı sonuçlar 10,11 verebilir. Bu farklılıklar membran sondaların difüzyon katsayıları, aynı zamanda farklı oranları ile difüzyon parçacıkların ayrı popülasyonlarının algılama ve muhtemelen farklı durumları arasında geçiş sadece içerir. Bu durumda,Aynı yüzey (bu durumda mika) kullanılmadıkça TIRFM ve AFM teknikleri kullanılarak elde edilen sonuçların doğrudan bir karşılaştırma, genellikle sorunludur. TIRFM ve AFM bilayeri görüntüleme aynı mika yüzeye 12,13 yürütülmüştür bazı çalışmalar olmasına karşın, mika nadiren çoğunlukla nedeniyle taşıma problemleri, optik mikroskopi için kullanılır. Mika hazırlık sonra optik yapıştırıcı 12 kullanarak lamel yapıştırılmış ince broşürler, içine elle kesim gerektirir. Bu yöntem, bununla birlikte tatmin edici sonuçlar elde etmek için bazı pratik gerektirir. Dahası, elde yüzeyleri onları zor düşük çalışma mesafesi, yüksek sayısal diyafram lensler ile kullanmak için yapım, genellikle dalgalı ve kalın. Bu protokolde tarif edildiği gibi hazırlanmıştır mika yüzeyler çok ince (170 um lamel kalınlığı dahil ~ 220 um) ve son derece düz, başarılı bir yüksek çözünürlüklü görüntüleme için kritik olan &quot;saç, dalgalı&quot; önlenir. Bunlar kullanılabilirTIRFM veya konfokal kurulumları için. Ayrıca, aynı örnekler AFM transfer edilebilir ve hatta TIRFM / konfokal ve AFM ile eş zamanlı olarak görüntülenebilir. Bu iki teknik birleştiren iki katmanlı zar yapısı 14 ile difüzyon davranışı doğrudan bir ilişki sağlar. Mika yüzeyleri taze yarılır, çünkü onlar temiz ve (cam temizleme protokollerinin genellikle Piranha çözüm, sülfürik asit, sodyum / potasyum hidroksit gibi kimyasallar dahil) zaman alıcı, kötü tekrarlanabilir, ve potansiyel olarak tehlikeli temizlik prosedürlerini gerekmez. De tarif edilen bir protokol küçük odası, montajı, en az 50 ul için etkili iki katmanlı oluşumu için gerekli veziküllerin hacmini azaltır. Son olarak, yüzey montaj tüm süreç zaman tüketen geleneksel mika bölünme ve tutkallama işlemi yaptığı gibi, (hazırlık az 30 dakika sürer), ve manuel beceri yüksek derecede gerektirmez değildir.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bath Sonicator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">FB15051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Coverslips 24 x 50mm - No H1.5</td>
			<td style="width:135px;">Marienfeld</td>
			<td style="width:119px;">102222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DOPC</td>
			<td style="width:135px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hellmanex III (detergent)</td>
			<td style="width:135px;">Hellma Analytics</td>
			<td style="width:119px;">320.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mica V-1 Grade</td>
			<td style="width:135px;">SPI Suppliers</td>
			<td style="width:119px;">1872-CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (high viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA63</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (low viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphingomyelin-ATTO647N</td>
			<td style="width:135px;">AttoTec</td>
			<td>AD 647N-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">UV lamp</td>
			<td style="width:135px;">Synoptics Ltd.</td>
			<td style="width:119px;">GelVue GVM20</td>
			<td>The lamp was set to 100% power</td>
		</tr>
	</tbody>
</table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

Desteklenen ikili lipid tabakalarıdır (SLBS) yaygın membran özellikleri (faz ayrımı, kümeleme dinamikleri) ve bu tür ilaçların veya peptidler gibi başka bileşikler ile etkileşimi incelemek için bir model olarak kullanılır. Ancak SLB özellikleri, kullanılan desteğine bağlı olarak farklılık gösterir. SLB görüntüleme ve ölçümler için yaygın olarak kullanılan teknikler tek bir molekülün floresan mikroskopi, FCS ve atomik kuvvet mikroskobu (AFM) vardır. AFM son derece düz bir yüzeye (genellikle mika) gerektirir ise en çok optik görüntüleme çalışmaları, bir cam destek üzerinde gerçekleştirildiği için bu maddelerin şarj ve yumuşaklık özellikleri güçlü difüzyon etkileyebilir, çünkü bu teknikleri sonuçları, doğrudan karşılaştırılamaz. Ne yazık ki, cam slayt kesme ve mika ince dilim yapıştırma için gerekli olan el becerisi yüksek SLB hazırlanması için mika rutin kullanımı için bir engel oluşturur. Bu tercih edilen bir yöntem, bu tür hazır mika olmasına karşınyüzeyleri sık sık özellikle küçük çalışma mesafesi, yüksek sayısal diyafram lensler ile, düzensiz (dalgalı) ve görüntü zor olarak bitirmek. Burada vezikül lipid birikimi ve SLB hazırlanması için, ince, düz bir mika yüzeylerin hazırlanması için basit ve tekrarlanabilir bir yöntem sunarlar. Ayrıca, bizim ölçüye oda SLB veziküller formasyonu için sadece çok küçük miktarlar gerektirir. AFM çalışmalarda kullanılan doğrudan karşılaştırılabilir olan yüksek kaliteli lipid iki katmanlı yüzeylerin, etkili, basit ve ucuz bir üretim genel prosedürü ile sonuçlanır.

SLBS floresan lipid sondaların difüzyon davranışı alt maddeye bağlı olarak farklıdır. SMT tekniği ile birleştirilmiş TIRFM parçacık hareketleri görselleştirme ve difüzyon katsayıları ayıklamak için değerli bir yöntemdir. Bir DOPC cam ve mika üzerinde desteklenen (1,2-dioleoil-sn-glisero-3-fosfokolin) iki katmanlı olarak bir difüzyon Sfingomiyelin-ATTO647N prob tek molekül sinyalleri bağlı hareketli şekilde gösterilir. Burada yer yüzeyi mika edilen protokole göre hazırlandı. Op...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Biz yüksek çözünürlüklü mikroskopi için mika desteklenen lipit katmanlarını hazırlamanın bir yöntem mevcut. Mica bir atomik ölçekte şeffaf ve düz, ancak nadiren nedeniyle zorluklar ele görüntülenmesinde kullanılan; bizim hazırlık mika tabakanın da birikimi ile sonuçlanır, ve iki katmanlı hazırlanmasında kullanılan malzemeyi azaltır.

Mika hazırlanması Yüksek Çözünürlüklü Optik Mikroskopi Görüntüleme için lipit tabakalarının Desteklenen

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Research

JoVE Journal

Bioengineering

13.3K Görüntüleme.  Nanyang Technological University.  Biz yüksek çözünürlüklü mikroskopi için mika desteklenen lipit katmanlarını hazırlamanın bir yöntem mevcut. Mica bir atomik ölçekte şeffaf ve düz, ancak nadiren nedeniyle zorluklar ele görüntülenmesinde kullanılan; bizim hazırlık mika tabakanın da birikimi ile sonuçlanır, ve iki katmanlı hazırlanmasında kullanılan malzemeyi azaltır. 

Video: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Biyomühendislik

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 ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Geniş bant Tıbbi Görüntüleme Uygulamaları Ultrason Optik Dedektörü

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

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., ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Aracı Olarak bir lipozomal-kapsüllü Yakın-Kızılötesi floroforun Floresan-söndürme<em&gt; İn Vivo</em&gt; Optik Görüntüleme

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomik Kuvvet Mikroskobu Görüntüleme ve Desteklenen Lipid Bilayers Kuvvet Spektroskopisi

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Bir Desteklenen lipit iki tabakalı içinde Ligand Nano küme Diziler

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Için değiştirilebilir Akustik ve Optik Çözünürlük Fotoakustik Mikroskopi<emIn vivo</em&gt; Küçük Hayvan Kan Vaskülatür Görüntüleme

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.