The financial support by DFG (German research foundation, project CZ 55/30-1) for parts of this work is gratefully acknowledged.

1. Deneysel Kurulum Montaj <ol><li> Vücuda yakın tarafına hazırlanması <ol><li> Yerleştirin ve bir collimated ışık demeti sağlayan lazer düzeltmek - veya fiberin çıkış faset kolimasyon optik fiber birleştiğinde lazer kullanır. </li><li> referans ve nesne ışınına lazer ışını bölmek polarize ışın ayırıcı (PBS) koyun. Referans kiriş ve (uzak tarafında) nesne ışınının gücü kabaca aynı olana kadar montaj onun rotasyon hwp çevirerek yarım dalga plakaları (HWP) yönünü çevirin. referans ve nesne kiriş hem içine bir ekran koyarak bu kontrol edin. Referans ışınının kutuplaşma polarizasyon duyarlı uzaysal ışık modülatörü (SLM) sığacak şekilde PBS yönünü seçin. </li><li> İki kiriş içine referans ışını bölmek için referans ışınına bir ışın ayırıcı (BS) koyun. BS1 gelen bu iki profil, sırasıyla OM1 ve OM2 geçebilir optik modülatörleri (OM) yerleştirin. &lt;/ Li&gt; <li> İki ayna istihdam BS2 de OM1 ve OM2 geçen iki kirişler birleştirin. Her iki kirişler mekansal ayrılır, böylece ışın bölücülerin ve aynaları ayarlayınız. </li><li> Dikkatle her iki kiriş insidansı yönü ilk başta piksel SLM düzlemi, görmezden BS3 ve BS4 dik olmasını sağlamak için BS5 aynı hizaya getirin. İlk başta, hiçbir şey, yani SLM görüntülenir, bu (2. adımda boyunca) kalibrasyon sonuna kadar bir ayna gibi davranır. </li><li> konumu ve iki lens (L) arasındaki mesafeyi tamamlayıcı metal oksit yarı iletken (CMOS) kamerada SLM uçağın keskin bir görüntü elde etmek için Kepler teleskobu oluşturan ayarlayın. sapmaları en aza indirmek için L1 ve L2 doğru yönünü (düz kenarlar birbirine bakacak) izleyin. </li></ol></li><li> Uzak taraf hazırlanması <ol><li> İki kiriş içine nesne ışını bölmek ve iki aynalar kullanılarak BS8 onları birleştirmek için BS7 kullanın. Yine, kiriş bölücülerin ve mil ayarlamakHer iki profil uzamsal olarak ayrılmış şekilde rrors. </li><li> mikroskop objektif (OBJ) bunları hedefliyoruz BS9 kullanarak her iki gözünüzü almaması. modlu fiber (MMF) uzak ucunda obj2 Odak. L3 ve şarj çiftli aygıt (CCD) kamera kullanılarak MMF geri yansıma gözlemleyerek odağı kontrol edin. </li></ol></li><li> Bağlama yakın ve uzak yan <ol><li> MMF kullanan obj1 çıkan nesne kiriş ışık .birleştirecek. </li><li> ilk başta doğrusal polarize (LP) görmezden BS6 kullanarak nesne ışın bölün. Referans BS3 de kiriş ve BS4 bir ayna kullanılarak hem hem nesne kirişler birleştirin. kiriş bölücülerin ve aynaları ayarlayın, böylece küçük bir açı (en az 1 °) ile kesişen SLM de referans ve nesne ışın örtüşme her çift. </li><li> Referans gücü ve nesne ışın, hwp yönünü çevirerek 1.1.2 adıma göre yaklaşık eşit olduğundan emin olun. </li><li> girişim p kontrolattern CMOS kamera (hologram eksen-off) ve buna göre kesişme açısını ayarlayın. girişim saçak aralığı kabaca CMOS kamera iki piksel boyutunu eşit oluncaya kadar, açı arttırın. </li><li> Kamera görüntüsü farklı saçaklar gösterir, böylece CMOS kamera görüntüsünde girişim deseninin bir maksimum kontrast elde etmek için nesne ve referans ışınının kutuplaşmayı maç LP yönünü ayarlayın. </li></ol></li></ol> 2. Sistem kalibrasyonu <ol><li> SLM ve CMOS arasındaki piksel ilişkisini kalibre <ol><li> Sadece referans kirişlerin birini kullanarak tüm SLM Aydınlatmak ve blok diğer başvuru ve kirişler nesneleri. </li><li> CMOS kamera ile SLM bir görüntü yakalamak. </li><li> Grafik yazılımı ve PC fare imleci kullanarak CMOS kamera görüntüsünde SLM sol üst köşesinde, örneğin koordinatlarını alın. kökenli noktası olarak bu piksel koordinatlarını kullanınSLM ilgili. </li><li> Tüm ışın blokları kaldırmak. </li></ol></li><li> Sinyal yollarını Kalibre <ol><li> Her iki referans ışını 2 ve nesne ışını 2 engelleyin. </li><li> CMOS kamera ile hologramın bir görüntü yakalamak. Açısal spektrum yöntemi 19 kullanılarak kaydedilen hologram fazı değerlendirin. kirişin 1 karşılık gelen bölgede ters faz hesaplamak. </li><li> Eski ışın blokları kaldırmak ve şimdi hem referans ışını 1 ve nesne ışını 1 blok. </li><li> CMOS kamera ile hologramın bir görüntü yakalamak. Yine açısal spektrum yöntemi kullanılarak kaydedilen hologram fazı ölçün. kiriş 2, ilgili bölgede ters faz hesaplamak. </li><li> Tüm ışın blokları kaldırmak. </li></ol></li></ol> 3. Sinyalleri Verici <ol><li> Nesne ışını engelleyin. </li><li> birlikte kiriş 1 ve 2&#39;ye karşılık gelen bölgelerde hesaplanan ters faz görüntüleri Dikiş ve ekranSLM üzerindeki tüm görüntü, genellikle bilgisayar grafik bağlantı noktasını kullanarak. </li><li> OM1 ve OM2 etkinleştirerek giriş sinyalleri 1 ve 2 modülasyonunu başlatın. </li><li> CCD kamera çıkış sinyalleri 1 ve 2 gözlemleyin. </li></ol>

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The authors have nothing to disclose.

deneysel kurulum (protokolünde aşama 1) montajı birbirine göre optik bileşenleri ayrıntılı bir hizalama gerektirir. en önemli yönü yüksek bir PBR sağlamak amacıyla SLM üzerine referans ışınlarının dikdörtgen insidansı. ikiden fazla iletilen sinyallere setup artırmak amacıyla, ek bir ışın ayırma kullanılabilir. Bir alternatif olarak, elyaf bazlı bir uygulama daha kompakt ve sistem Biophotonics in situ araştırmalarda taşınabilir olmasını sağlayan s...

Bir çok modlu fiber (MMF) üzerinden birden ışık sinyallerinin iletimi iletişim mühendisliği 1 ve Biophotonics 2 belirgindir. haberleşme mühendisliği, uzay-bölmeli çoklama (SDM) çoklu tek modlu liflere kıyasla sınırlı alanlarda daha yüksek bir kullanımından yararlanan gelecekteki veri transferi uygulamaları için fiber optik iletim kapasitesini artırmak amacıyla kalıcı bir çözüm olduğuna inanılmaktadır 3.. Biophotonics, biyolojik numuneler bir MMF endoskop 4 ile ışık verici tarafından manipüle edilir. Örneğin, MMF endoskoplar kullanılarak bireysel nöronların bağımsız optik kontrol beyinde 5 nöron ağları araştırmak için optogenetics ilgi olduğunu. Ancak, MMF giriş faset üzerine yansıtılan ışık Outpu yayılması sırasında bozulma nedeniyle mod karıştırma ve dağılma tabidirMMF t faset. Bunun bir sonucu olarak, ışık yayılma sinyali iletim güçleştirmektedir, değiştirilir. Wavefront şekillendirme yöntemleri 6, 7 uzaysal ışık modülatörlerinin (SLM) kullanarak medya saçılma uygulanan ve bağlı hafif yayılması 8 sırasında saçılma bozulma için tazminat imkan verirler. Bir optik geribildirim 9 kullanarak çıktıyı optimize iteratif yaklaşım vardır. Bu yaklaşımlar yerine zaman, çünkü çok sayıda tekrarlamalar için gerekliliği ve özgürlük yüksek derecede tüketen modülatör elemanlarının çok sayıda gelen bulunmaktadır. Başka bir yaklaşım tamamen iletim matrisinin 10 tarafından açıklanan MMF içinde bozulma tespit etmektir. iletilecek modları sayısı büyükse, bu da zaman alıcı olacaktır. Bunun aksine, dijital optik faz konjugasyon (DOPC) olarak kabul edilirSadece birkaç odak noktalar beri, hızlı ve burada avantajlı MMF çıkış faset oluşturulan gerekmektedir. Faz konjugasyon yaklaşımları da biyolojik doku 12, 13, 14 ile odaklama veya görüntüleme için gösterilmiştir. Şimdiye kadar, DOPC, tek bir saat sinyali sadece 15, 16 kullanılmıştır, ve MMF 17 ışığın iletimi için uygulanmıştır. Birden fazla bağımsız sinyaller için bir DOPC yaklaşım başarılı olmamıştır. Biz bir tek faz-sadece SLM 18 istihdam her sinyal için şekillendirme bireysel wavefront kullanarak birden fazla ışık sinyallerinin bağımsız iletimini sağlayan gelişmiş bir DOPC yöntem geliştirdik. Bu amaçla, SLM Her bir sinyal için iletilecek bölgeler halinde bölünür. Önerilen deney düzeneği Şekil 1 &#39;de gösterilmektedirKalibrasyon gerçek iletim öncesi), bir gerçekleştirilir burada, b) &#39;de oluşur. <img alt="Şekil 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> Şekil 1: Deney düzeneği. BS = ışın ayırıcı, CCD = şarj çiftli aygıt, OM = optik modülatör, CMOS = tamamlayıcı metal oksit yarı iletken, HWP = yarım dalga plakası, L = objektif, LP = doğrusal polarize, MMF = modlu fiber, OBJ = mikroskop objektif, PBS = ışın ayırıcı, SLM = uzaysal ışık modülatörü polarize (faz için) - tasvir edilmektedir: (a) kalibrasyon ve (b) iletimi için sadece ilgili kirişler <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>spatial light modulator</td>
			<td>Holoeye</td>
			<td>PLUTO&#8208;VIS&#8208;016</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Mikrotron</td>
			<td>MC4082</td>
			<td></td>
		</tr>
		<tr>
			<td>diode&#8208;pumped solid state laser</td>
			<td>Laser Quantum</td>
			<td>torus 532</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>IDS</td>
			<td>U3&#8208;3482LE&#8208;M</td>
			<td>CMOS camera; suitable as well</td>
		</tr>
		<tr>
			<td>lens 1</td>
			<td>Qioptiq</td>
			<td>G063204000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 2</td>
			<td>Qioptiq</td>
			<td>G063203000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 3</td>
			<td>Thorlabs</td>
			<td>AC508&#8208;180&#8208;A&#8208;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>multimode fiber</td>
			<td>Thorlabs</td>
			<td>M14L02</td>
			<td></td>
		</tr>
		<tr>
			<td>beam splitters</td>
			<td>Thorlabs</td>
			<td>BS013</td>
			<td>9x</td>
		</tr>
		<tr>
			<td>polarizing beam splitters</td>
			<td>Thorlabs</td>
			<td>PBS251</td>
			<td></td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Thorlabs</td>
			<td>PF10&#8208;03&#8208;P01</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>microscope objectives</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>half wave plates</td>
			<td>Thorlabs</td>
			<td>WPH10M&#8208;532</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear polarizer</td>
			<td>Thorlabs</td>
			<td>LPVISB050&#8208;MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>optical modulators</td>
			<td>Thorlabs</td>
			<td>MC2000B&#8208;EC</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear and rotation stage for CMOS camera</td>
			<td>Thorlabs</td>
			<td>XYR1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>fiber connector</td>
			<td>Thorlabs</td>
			<td>S120&#8208;SMA</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>reducing ring for microscope objectives</td>
			<td>Qioptiq</td>
			<td>G061621000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>xy adjustment for objective adapters</td>
			<td>Qioptiq</td>
			<td>G061025000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>z translation mount for fiber adapter</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>rods for fiber alignment to objectives</td>
			<td>Qioptiq</td>
			<td>G061210000</td>
			<td>8x</td>
		</tr>
		<tr>
			<td>mounts for lenses 1 and 2 plus two phantom mounts</td>
			<td>Qioptiq</td>
			<td>G061047000</td>
			<td>4x</td>
		</tr>
		<tr>
			<td>rail carriers for objective and lens mounts</td>
			<td>Qioptiq</td>
			<td>G061372000</td>
			<td>6x</td>
		</tr>
		<tr>
			<td>rail for rail carriers</td>
			<td>Qioptiq</td>
			<td>G061359000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td colspan="2">adapter for CCD camera to 1 post</td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>adapter for laser to 4 posts</td>
			<td></td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>mount for lens 3</td>
			<td>Thorlabs</td>
			<td>LMR2/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for half wave plates</td>
			<td>Thorlabs</td>
			<td>RSP1D/M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>mounts for mirrors</td>
			<td>Thorlabs</td>
			<td>KM100</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>mount for linear polarizer</td>
			<td>Thorlabs</td>
			<td>RSP05/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>KM100PM/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>clamping arms for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>PM4/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>posts for mounts, rail carriers and adapters</td>
			<td>Thorlabs</td>
			<td>TR75/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>holders for posts</td>
			<td>Thorlabs</td>
			<td>PH50/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>pedestals for holders</td>
			<td>Thorlabs</td>
			<td>BE1/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>clamping forks for pedestals</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td>29x</td>
		</tr>
	</tbody>
</table>

transmission of multiple signals through an optical fiber using wavefront shaping

Bir çok modlu fiber üzerinden birden fazla bağımsız optik sinyallerin iletim fiber içinde yayılması sırasında ışık bozulmayı telafi etmek için dalga cephesi şekillendirme kullanılarak gerçekleştirilir. Metodoloji, optik dalga cephesi tek modülatör farklı bölgelerinde modüle edilir, yalnızca tek bir uzaysal ışık modülatörü, ışık sinyalinin her bir bölge kullanılarak bir dijital optik faz konjügasyonu dayanır. Dijital optik faz konjugasyon yaklaşımları (örneğin) fiber dalga yayılımı davranış tam tayini gerçekleştirilir, diğer dalga cephesi şekillendirme yaklaşımlar, daha hızlı olduğu kabul edilir. sadece ışık sinyali başına bir kalibrasyon gerektirir çünkü aksine, sunulan yaklaşım zaman etkilidir. Önerilen yöntem haberleşme mühendisliği mekansal bölmeli çoklama için potansiyel uygundur. Ayrıntılı uygulama alanları özellikle o da, Biophotonics endoskopik ışık teslim edilirbiyolojik doku tek hücre olan ptogenetics, seçici yüksek uzaysal ve zamansal çözünürlüğe sahip aydınlatılmaya.

<html> 2 m uzunluğunda elyaf uzak tarafında tipik çıkış sinyalleri, Şekil 2&#39;de tasvir edilmiştir. İstenen odak nokta (zirve) prensip olarak DOPC kusurları nedeniyle istenmeyen bir benek deseni (arka plan), eşlik unutmayın. İlgili tepeden-arka oranı (PBR) 53 tutarında, 36 sırasıyla burada (hem sinyaller 1 ve 2 &#39;açık&#39; olan) (sadece sinyal 2 &#39;açık&#39;) ve 20 (sadece sinyal 1 &#39;açık&#39; olduğunu) . (: 1710, şu anda) kullanıldığ...

Watch this Scientific Journal Video about Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping at JoVE.com

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

Wavefront Şekillendirme kullanarak bir Fiber Optik aracılığıyla Çoklu Sinyalleri İletim

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

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9.7K Görüntüleme.  TU Dresden. We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

Video: Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

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

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

Silan Kaplin Ajanlar kullanarak Silika Optik Biyosensörler için Biyolojik Probları takılması

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

Düşük yoğunluklu Ultrason Kullanımı Yapışma bağımlı Sinyalleri indüksiyonu

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Yerli, Fibröz Uzun Aralığı ve Bölümlere Uzun Aralığı Kollajen in vitro sentezi olarak

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Microtoroid Optik Rezonatörler Kullanarak Etiket serbest Tek Molekül Algılama

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Damlacık Mikroakiskan için renkli Floresan Algılama Optik Elyaf kullanma

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Bir Ayarlanabilir Nanopore aracılığıyla Nanopartikül Translokasyon Hızları aracılığıyla Zeta Potansiyeli Belirlenmesi: Bir Örnek olarak DNA modifiye Parçacıklar kullanma

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

İnsan Serumda kardiyak biyolojik ölçmek için olağanüstü optik iletim kullanma

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Etkileşimli, hastanın özel modelleri kullanarak derin beyin stimülasyonu tedavisi için nöronal Fiber yolları hedefleme

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Faj Görüntüleme Peptid Kütüphanesi Kullanılarak Fibroblast Büyüme Faktörü Reseptörü 2'yi Hedefleyen Küçük Peptidlerin Taranması ve Tanımlanması

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...