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

1. 실험 설정 조립 <ol><li> 전방 측 준비 <ol><li> 두고 평행 광선을 제공하는 레이저를 수정 - 또는 섬유의 출사면에서 시준 된 광학 섬유 결합 레이저를 사용한다. </li><li> 기준 및 대상 빔으로 레이저 빔을 분할하는 편광 빔 스플리터 (PBS)를 넣는다. 기준 빔 (선단 측) 오브젝트 빔의 파워가 대략 동일 마운트까지의 회전에 HWP을 회전시킴으로써 반 파장 판 (HWP)의 방향을 설정. 참조 및 대상 빔 모두에 화면을 넣어이 옵션을 선택합니다. 기준 빔의 편광은 편광 성 공간 광 변조기 (SLM)에 맞도록 PBS의 방향을 선택한다. </li><li> 두 개의 빔으로 참조광을 분할 기준 빔으로 빔 스플리터 (BS)를 넣는다. BS1에서 오는이 두 빔이 각각 OM1 및 OM2을 통과 할 수 있도록 광학 변조기 (OM)을 놓습니다. &lt;/ 리&gt; <li> 두 개의 거울을 사용 BS2에서 OM1 및 OM2을 통과하는 두 개의 빔을 결합합니다. 두 빔이 공간적으로 분리되도록 빔 스플리터와 거울을 조정합니다. </li><li> 조심스럽게 두 빔의 입사 방향은 처음 픽셀은 SLM의 평면 무시 BS3 및 BS4에 수직하도록 정렬 BS5. 처음에는 아무것도 즉, SLM 상에 디스플레이되지 않으며, 그것은 (2 단계에 걸쳐) 교정 끝까지 거울처럼 동작한다. </li><li> 위치 및 두 개의 렌즈 (L) 사이의 거리 상보 금속 산화물 반도체 (CMOS) 카메라의 SLM 평면의 선명한 이미지를 얻기 위해 케플러 망원경 구성을 조정한다. 수차를 최소화하기위한 L1 및 L2의 올바른 방향 (평탄면은 서로 대향된다)를 손목 시계. </li></ol></li><li> 말단 측 준비 <ol><li> 두 개의 빔으로 물체 광을 분할하여 두 개의 거울을 이용하는 BS8 그들을 결합 BS7를 사용한다. 또, 빔 스플리터 및 MI를 조정두 빔들이 공간적으로 분리되도록 널 오 류. </li><li> 현미경 목표 (OBJ)로 목표로 BS9를 사용하여 두 빔을 편향. 다중 모드 광섬유 (MMF)의 선단에 집중 OBJ2. L3 및 전하 결합 소자 (CCD) 카메라를 채용 MMF에서 다시 반사를 관찰함으로써 포커스를 확인한다. </li></ol></li><li> 연결 근위 및 원위 측면 <ol><li> MMF를 채용 OBJ1 나오는 대상 빔으로부터의 광을 시준. </li><li> 우선 선형 편광자 (LP)을 무시 BS6를 사용하여 물체 광을 분할. 참조 BS3에서 보와 BS4는 거울을 사용하는 모두 모두 대상 빔을 결합합니다. 빔 스플리터와 거울을 조정 있도록 작은 각도 (이하 1 °)와 교차하는 SLM의 기준 및 대상 빔 중첩의 각 쌍. </li><li> 기준의 힘과 물체 빔의 HWP의 방향을 돌려 1.1.2 단계에있어서, 대략 동일한 지 확인합니다. </li><li> 간섭 페이지를 확인attern 모스 카메라 (홀로그램 축 OFF)를 따라 교차 각도를 조정한다. 간섭 무늬의 간격은 대략 CMOS 카메라에 두 개의 화소의 크기에 달할 때까지의 각도를 증가시킨다. </li><li> 카메라 이미지 뚜렷한 줄무늬 표시되도록 상기 CMOS 카메라 화상에서의 간섭 패턴의 최대 콘트라스트를 얻기 위해 객체 및 기준 빔의 편광과 일치하도록 LP의 방향을 조정한다. </li></ol></li></ol> 2. 시스템 보정 <ol><li> SLM 및 CMOS 픽셀 간의 관계를 보정 <ol><li> 전용 기준 빔 중 하나를 사용하여 전체 SLM을 조명 블록은 다른 기준 빔과 오브젝트. </li><li> 모스 카메라로 SLM의 이미지를 캡처. </li><li> 그래픽 소프트웨어 및 PC의 마우스 커서를 이용하여 CMOS 카메라 화상에서의 SLM의 좌측 상단 모서리 등의 좌표를 얻을. 원점 이러한 픽셀 좌표를 사용하여은 SLM에. </li><li> 모든 빔 블록을 제거합니다. </li></ol></li><li> 신호 경로 보정 <ol><li> 모두 참조 빔 (2)와 객체 빔 (2)를 차단합니다. </li><li> 모스 카메라 홀로그램 이미지를 캡처. 각 스펙트럼에있어서 (19)를 사용하여 기록 된 홀로그램의 위상을 평가한다. 빔 (1)의 해당 영역에서의 반전 된 위상을 계산한다. </li><li> 전자 빔 블록을 제거하고 지금은 모두 참조 빔 1 개체 빔 1을 차단합니다. </li><li> 모스 카메라 홀로그램 이미지를 캡처. 다시 각 스펙트럼 방법을 사용하여 기록 된 홀로그램의 위상을 측정한다. 빔 (2)의 해당 영역에서 반전 된 위상을 계산합니다. </li><li> 모든 빔 블록을 제거합니다. </li></ol></li></ol> 3. 신호를 전송 <ol><li> 대상 빔 차단. </li><li> 함께 빔 (1)과 (2)의 대응하는 영역에서 산출 된 위상 반전 이미지 스티칭 및 표시는 SLM에 전체 이미지는 일반적으로 컴퓨터 그래픽 포트를 사용. </li><li> OM1 및 OM2를 활성화함으로써 입력 신호 (1) 및 (2)의 변조를 시작한다. </li><li> CCD 카메라의 출력 신호들 (1)과 (2)를 관찰한다. </li></ol>

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The authors have nothing to disclose.

실험 장치 (프로토콜 단계 1)의 조립은 서로에 대한 상기 광학 구성 요소의 완전한 정렬을 필요로한다. 가장 중요한 목적은 높은 PBR을 보장하기 위해서 SLM 상으로 기준 빔의 입사 직사각형이다. 두 개 이상의 송신 된 신호에 대한 설정을 향상시키기 위하여, 추가적인 빔 스플리터가 사용될 수있다. 대안으로, 섬유 기반의 구현은보다 컴팩트 한 시스템은 바이오 포토닉스 시...

다중 모드 광섬유 (MMF)을 통한 다중 광 신호의 전송은 통신 기술 한 바이오 포토닉스 및 2에서 분명하다. 통신 공학, 공간 분할 다중화 (SDM)는 다수의 단일 모드 광섬유와 비교하여 제한된 공간의 활용도 혜택 장래 데이터 전송 애플리케이션을위한 광섬유의 전송 용량을 향상시키기 위해 가능한 해결책이 될 것으로 여겨진다 3. 바이오 포토닉스에서, 생물학적 시료는 MMF 내시경 (4)를 통해 광 투과에 의해 조작된다. 예를 들어, MMF 내시경을 이용하여 각각의 뉴런의 독립적 제어 광은 뇌 5 신경 네트워크를 연구하기 위해 optogenetics 대한 관심사이다. 그러나, MMF 입력면에 투사 된 빛이 outpu에 전파하는 동안 왜곡으로 인해 모드 혼합과 분산이 적용됩니다MMF를의 t 패싯. 결과적으로, 빛의 전파를 신호 전송 도전하게하는 변경된다. 파면을 형성하는 방법 (6), (7) 공간 광 변조기 (SLM)를 사용하여 미디어를 산란인가 의한 광의 전파 중에 8 산란으로 왜곡 보상을 가능하게한다. 광 피드백 9를 이용하여 출력을 최적화하는 반복적 인 방법이있다. 이러한 접근은 다소 시간이 있기 때문에 다수의 반복을위한 필요성과 높은 자유도 걸리는 모듈레이터 많은 요소에 대응한다. 또 다른 방법은 완전히 전송 행렬 (10)에 의해 설명 된 MMF 내의 왜곡을 결정하는 것이다. 전송하고자하는 모드의 개수가 큰 경우,이뿐만 아니라 시간이 소모 될 것이다. 반대로, 디지털 광 위상 공액 (DOPC)가되는 것으로 간주된다단지 소수의 초점 스팟 보낸 빠르고 여기 유리는 MMF의 출력면에서 발생해야한다. 위상 공액 접근법은 또한 생체 조직 (12, 13), (14)를 통해 포커싱 또는 이미징 입증되었다. 지금까지, DOPC 단일 시보 15, 16을 사용 하였다 및 MMF (17)을 통해 빛의 전송에 적용되었다. 다중 독립 신호용 DOPC 방식은 달성되지 않았다. 우리는 단일 위상 한정 SLM (18)를 이용하는 각각의 신호를 정형 개별 파면을 사용하여 다중 광 신호의 독립 송신을 제공하는 향상된 DOPC 방법을 개발 하였다. 이를 위해 상기 SLM은 각각의 신호에 대해 하나가 송신되는 영역들로 분할된다. 제안 된 실험 장치는 그림 1에 도시되어있다보정은 실제 전송에 앞서)가 수행된다) (B)에서 발생한다. <img alt="그림 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> 그림 1 : 실험 설정. BS = 빔 스플리터, CCD = 전하 결합 소자, OM은 = 광 변조기, CMOS는 = 상보 형 금속 산화 반도체, HWP는 = 반파 장판, L = 렌즈, LP = 선형 편광자 MMF = 다중 모드 광섬유, OBJ = 현미경 대물, PBS = 빔 스플리터, SLM = 공간 광 변조기 편광 (위상 만 해당) - 도시되어있다 (a)는 교정 및 (b)에 전송에만 해당 빔 <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

다중 모드 광섬유를 통해 여러 개의 독립적 인 광 신호의 전송은 섬유 내의 전파 중에 광 왜곡을 보상하기 위해 파면 정형을 이용하여 달성된다. 우리의 방법은, 광 파면 개별적 변조기의 서로 다른 영역에서 변조 된 단일 공간 광 변조기, 광 신호 당 하나의 영역을 채용하는 디지털 광 위상 공액에 기초한다. 디지털 광 위상 공액 접근법 (예를 들어) 광섬유의 전파 동작의 완전한 결정이 수행되는 다른 파면을 형성하는 방법,보다 빠른 것으로 간주된다. 에만 광 신호 당 하나의 교정을 필요로하기 때문에 이와 대조적으로, 제시된 방법은 시간이 효과적이다. 제안 된 방법은 통신 공학 공간 분할 다중화 잠재적으로 적합하다. 또한 응용 분야, 특히 O를, 바이오 포토닉스에서 내시경 빛을 전달하다생체 조직에서 하나의 세포가 ptogenetics은 선택적으로 높은 공간 및 시간 해상도 조명한다.

<html> 2 m 길이의 섬유의 선단 측의 일반적인 출력 신호는도 2에 도시된다. 원하는 초점 지점 (피크) 원칙의 문제로 DOPC의 불완전 성 때문이다 원하지 않는 스펙 클 패턴 (배경), 동반합니다. 대응하는 피크 - 투 - 배경 비율 (PBR)이 53 금액, (36) 각각 여기에 (두 신호 1, 2 &#39;에&#39;있다 ()으로 만 신호 2 &#39;에&#39;있다), 20 (단독 신호 1 &#39;에&#39;입니다) . (: 1710 현재)을...

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.

파면 정형을 이용하여 광섬유를 통해 다중 신호 전송

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 조회수.  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.

비디오: 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.

입체 광학 해상도 Photoacoustic 현미경

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

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생체공학

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.

실란 커플링 에이전트를 사용하여 실리카 광 Biosensors에 생물 학적 프로브를 부착

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.

낮은 강도의 초음파를 사용하여 접착 종속 신호의 유도

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.

기본, 섬유 긴 간격 및 Segmental 긴 간격 콜라겐의 체외 합성에

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 광 공진기를 사용하여 라벨이없는 단일 분자 검출

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.

물방울 미세 유체에 대한 여러 가지 빛깔의 형광 검출 광학 섬유를 사용하여

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.

조정 나노 기공을 통해 나노 입자 전좌 속도를 통해 제타 전위의 측정 : 예를 들어 DNA 변성 입자를 사용하여

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).

특별 한 광학 전송 인간의 혈 청에 심장 Biomarkers 척도를 사용 하 여

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

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPR-concatemer를 이용한 마우스 작은 창자 organoids에서 다중 유전자 녹아웃을위한 프로토콜

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

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.

대화형, 환자 전용 모델을 사용 하 여 깊은 두뇌 자극 치료를 위한 신경 섬유 책자를 대상으로

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