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

1. Montaje de la configuración experimental <ol><li> Preparación del lado proximal <ol><li> Colocar y fijar el láser emite un haz de luz colimada - o utilizar un láser de fibra acoplado con la óptica de colimación en la faceta de salida de la fibra. </li><li> Poner el divisor de haz polarizante (PBS) para dividir el haz de láser en referencia y haz objeto. Girar la orientación de las placas de media onda (PMR) por la rotación de la PMR en su rotación de montaje hasta que la potencia de haz de referencia y haz objeto (en el lado distal) es aproximadamente la misma. Marque esta poniendo una pantalla en tanto referencia y haz objeto. Elige la orientación de la PBS de manera que la polarización del haz de referencia se ajusta a la modulador espacial de luz sensible a la polarización (SLM). </li><li> Poner un divisor de haz (BS) en el haz de referencia para dividir el haz de referencia en dos haces. Coloque los moduladores ópticos (OM) tales que estos dos haces procedentes de BS1 pueden pasar OM1 y OM2, respectivamente. &lt;/ Li&gt; <li> Combinar los dos haces que pasan OM1 y OM2 en BS2 que emplea dos espejos. Ajustar los divisores de haz y espejos de manera que ambos haces están separados espacialmente. </li><li> alinear cuidadosamente BS5 para asegurar que la dirección de incidencia de los dos haces es perpendicular al plano de píxeles del SLM, BS3 ignorar y BS4 al principio. Al principio, no aparece nada en el SLM, es decir, actúa como un espejo hasta el final de la calibración (a lo largo paso 2). </li><li> Ajustar la posición y la distancia entre las dos lentes (L) que constituye un telescopio de Kepler con el fin de obtener una imagen nítida del plano de SLM en la cámara semiconductor complementario de óxido metálico (CMOS). Ver la orientación correcta de la L1 y L2 (lados planos se enfrentan entre sí) para minimizar las aberraciones. </li></ol></li><li> Preparación del lado distal <ol><li> Utilice BS7 para dividir el haz de objeto en dos haces y combinarlos en BS8 que emplea dos espejos. Una vez más, ajustar los divisores de haz y mirrors de modo que ambos haces están separados espacialmente. </li><li> Desvíe ambos vigas usando BS9 para apuntar que el objetivo del microscopio (OBJ). Enfoque OBJ2 en el extremo distal de la fibra multimodo (MMF). Comprobar el enfoque mediante la observación de la reflexión de retorno desde el MMF empleando L3 y un dispositivo de cámara de carga acoplada (CCD). </li></ol></li><li> Conexión proximal y distal <ol><li> Colimar la luz del haz de objeto que sale del MMF empleando OBJ1. </li><li> Dividir el haz objeto utilizando BS6, ignorar el polarizador lineal (LP) en un primer momento. Combinar ambos haces de objeto con ambos haces de referencia en BS3 y BS4 empleando un espejo. Ajuste los divisores de haz y espejos de manera que cada par de referencia y la superposición haz de objeto en el SLM, cruzándose con un ángulo pequeño (menos de 1 °). </li><li> Asegúrese de que el poder de la referencia y el haz objeto son aproximadamente iguales girando la orientación de la PMR, de acuerdo con el paso 1.1.2. </li><li> Compruebe la interferencia pattern (holograma fuera de eje) en el CMOS de la cámara y ajustar el ángulo de intersección en consecuencia. Aumentar el ángulo, hasta que el espaciado de franjas de interferencia más o menos es igual al tamaño de dos píxeles de la cámara CMOS. </li><li> Ajustar la orientación de la LP para que coincida con la polarización de objeto y del haz de referencia con el fin de obtener un contraste máximo de la figura de interferencia de la imagen de la cámara CMOS en, por lo que la imagen de la cámara muestra franjas distintas. </li></ol></li></ol> 2. Calibración de la cadena <ol><li> La calibración de la relación de píxeles entre el MST y CMOS <ol><li> Iluminar toda la SLM utilizando sólo uno de los haces de referencia y bloquear la otra referencia y objetos vigas. </li><li> Capturar una imagen del SLM con la cámara CMOS. </li><li> Obtener las coordenadas de la esquina superior izquierda de la SLM la imagen de cámara CMOS en, por ejemplo, utilizando software de gráficos y el cursor del ratón en el PC. Utilice estas coordenadas en píxeles como el punto de origenen relación con el MST. </li><li> Eliminar todos los bloques de la viga. </li></ol></li><li> Calibración de los caminos de la señal <ol><li> Bloquear tanto haz de referencia y el objeto 2 2 haz. </li><li> Capturar una imagen del holograma con la cámara CMOS. Evaluar la fase en el holograma registrado utilizando el método de espectro angular 19. Calcular la fase invertida en la región correspondiente de la viga 1. </li><li> Quitar los antiguos bloques de viga y ahora bloquear tanto haz de referencia 1 y el objeto viga 1. </li><li> Capturar una imagen del holograma con la cámara CMOS. Medir la fase en el holograma grabado utilizando el método angular del espectro nuevo. Calcular la fase invertida en la región correspondiente de la viga 2. </li><li> Eliminar todos los bloques de la viga. </li></ol></li></ol> 3. transmisión de las señales <ol><li> Bloquear el haz objeto. </li><li> Coser las imágenes de fase invertida calculados en las regiones correspondientes de la viga 1 y 2 juntos y mostrar latoda imagen de la SLM, por lo general mediante el puerto de gráficos por ordenador. </li><li> Iniciar la modulación de las señales de entrada 1 y 2 mediante la activación de OM1 y OM2. </li><li> Observe las señales de salida 1 y 2 de la cámara CCD. </li></ol>

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The authors have nothing to disclose.

El montaje de la configuración experimental (paso 1 en el protocolo) requiere una alineación completa de los componentes ópticos con respecto a la otra. El aspecto más importante es la incidencia rectangular de los haces de referencia en el SLM con el fin de garantizar un alto PBR. Con el fin de mejorar la configuración de más de dos señales de transmisión, se podrían utilizar divisores de haz adicionales. Como alternativa, una implementación basada en fibra sería más compacto y ...

La transmisión de múltiples señales de luz a través de una fibra multimodo (MMF) es evidente en las comunicaciones de ingeniería 1 y 2 biofotónica. En la ingeniería de las comunicaciones, la multiplexación por división espacial (SDM) se cree que es una solución viable con el fin de mejorar la capacidad de transmisión de fibras ópticas para aplicaciones de transferencia de datos futuros que se benefician de una mayor utilización de la limitación de espacio, en comparación con múltiples fibras monomodo 3. En biofotónica, las muestras biológicas son manipulados por transmisión de luz a través de un endoscopio MMF 4. Por ejemplo, el control óptico independiente de las neuronas individuales utilizando endoscopios MMF es de interés para la optogenética con el fin de estudiar las redes neuronales en el cerebro 5. Sin embargo, la luz proyectada sobre la faceta de entrada MMF está sujeta a la distorsión debido a la mezcla y el modo de dispersión durante la propagación de la Output faceta del MMF. Como resultado, la propagación de la luz se altera, lo que hace difícil la transmisión de señales. De frente de onda métodos que configuran 6, 7 se apliquen en la dispersión de los medios de comunicación que utilizan moduladores espaciales de luz (SLM) y permiten la compensación por la distorsión debida a la dispersión durante la propagación de la luz 8. Hay métodos iterativos que optimizan la salida usando una realimentación óptica 9. Estos enfoques son más bien lento debido a la necesidad de numerosas iteraciones y el alto grado de libertad, que corresponde a un gran número de elementos moduladores. Otro enfoque es determinar completamente la distorsión en el MMF se describe por su matriz de transmisión 10. Si el número de modos a transmitir es grande, esto será mucho tiempo también. En contraste, la conjugación de fase óptica digital (DOPC) se considera que esrápido y ventajoso, ya que sólo unos pocos puntos focales deberán ser aportados en la cara de salida de la MMF. Enfoques conjugación de fase también se han demostrado para el enfoque o formación de imágenes a través de tejido biológico 12, 13, 14. Hasta el momento, DOPC se empleó una única señal de tiempo de sólo el 15, 16, y se aplicó para la transmisión de luz a través de un FMM 17. Un enfoque DOPC para múltiples señales independientes no se ha logrado. Hemos desarrollado un método mejorado DOPC proporcionar la transmisión independiente de múltiples señales de luz de frente de onda utilizando individuo conformación para cada señal de empleo de una sola fase de sólo SLM 18. Para este objetivo, el SLM se segmenta en regiones, una para cada señal a transmitir. La configuración experimental propuesto se representa en la Figura 1, Donde se realiza una calibración en a) antes de la transmisión real ocurre en b). <img alt="Figura 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> Figura 1: Montaje experimental. BS = divisor de haz, CCD = dispositivo de carga acoplada, OM = modulador óptico, CMOS = semiconductor complementario de óxido metálico, PMR = la mitad de la placa de onda, L = lentes, LP = polarizador lineal, MMF = fibra multimodo, objetivo OBJ = microscopio, PBS = divisor de haz polarizante, el MST = modulador espacial de luz (fase única) - sólo vigas relevantes para (a) la calibración y (b) la transmisión están representados <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">por favor haga clic aquí para ver una versión más grande de esta figura.</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

La transmisión de múltiples señales ópticas independientes a través de una fibra multimodo se logra usando conformación del frente de onda con el fin de compensar la distorsión de luz durante la propagación dentro de la fibra. Nuestra metodología se basa en la conjugación de fase óptica digital que emplea un único modulador espacial de luz, donde el frente de onda óptica se modula de forma individual en diferentes regiones del modulador, una región por señal luminosa. enfoques conjugación de fase óptica digitales son considerados para ser más rápido que otros métodos de frente de onda, donde la formación de (por ejemplo) se lleva a cabo una determinación completa del comportamiento de la propagación de la onda de la fibra. En contraste, el enfoque presentado es eficiente en el tiempo ya que sólo requiere una calibración por señal luminosa. El método propuesto es potencialmente adecuado para la multiplexación por división espacial en la ingeniería de las comunicaciones. Otros campos de aplicación son el suministro de luz endoscópica en biofotónica, especialmente en Optogenetics, donde las células individuales en el tejido biológico tienen que estar iluminados selectivamente con alta resolución espacial y temporal.

<html> Señales de salida típica en el lado distal de la fibra larga 2 m se representan en la Figura 2. Tenga en cuenta que el punto focal deseada (pico) está acompañado por un patrón moteado no deseado (fondo), que se debe a la imperfección de la DOPC como una cuestión de principio. La correspondiente relación de pico a fondo (PBR) asciende a 53 (únicamente la señal 1 es &#39;on&#39;), 36 (únicamente la señal 2 se encuentra encendida) y 20 (ambas señales 1 y ...

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.

La transmisión de señales múltiples a través de una fibra óptica Uso de frente de onda Shaping

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 vistas.  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.

Tridimensional óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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.

Colocación de sondas biológicas de biosensores ópticos de silicio mediante agentes de acoplamiento de silano

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.

La inducción de señales de adhesión dependientes de Uso de ultrasonido de baja intensidad

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.

La síntesis in vitro de Espaciado nativo, y colágeno fibroso Long Long segmentaria Espaciado

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.

Individual Detección Molecule-Label gratis con Microtoroid ópticos Resonadores

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.

Multicolor detección por fluorescencia de la gotita de microfluídica el uso de fibras ópticas

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.

Determinación del Potencial Zeta a través de nanopartículas de translocación velocidades a través de un sintonizable nanoporos: El uso de partículas de ADN modificado como ejemplo

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).

Uso de transmisión óptica extraordinaria para cuantificar marcadores cardíacos en suero humano

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.

Un protocolo para múltiples genes Knockout en ratón Intestinal Organoids utilizando un 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 ...

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.

Dirigidos a los tractos de fibras neuronales para la terapia de estimulación cerebral profunda mediante modelos interactivos, específico para cada paciente

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