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

1. Montagem do Setup Experimental <ol><li> Preparando o lado proximal <ol><li> Colocar e fixar o laser fornecer um feixe de luz colimada - ou usar um laser de fibra-acoplado com óptica de colimação na faceta de saída da fibra. </li><li> Coloque o divisor de feixe de polarização (PBS) para dividir o feixe de laser para referência e feixe do objeto. Virar a orientação das placas de meia onda (HWP) rodando o HWP na sua rotação de montagem até que a energia de feixe de referência e feixe de objecto (no lado distal) é aproximadamente a mesma. Verifique isso colocando uma tela em ambos os de referência e feixe do objeto. Escolha da orientação do PBS, de forma que a polarização do feixe de referência se encaixa o modulador de luz espacial sensível à polarização (SLM). </li><li> Coloque um divisor de feixe (BS) para o feixe de referência para dividir o feixe de referência em dois feixes. Coloque os moduladores ópticos (OM) de tal forma que estes dois feixes provenientes de BS1 podem passar OM1 e OM2, respectivamente. &lt;/ Li&gt; <li> Combine os dois feixes que passam OM1 e OM2 em BS2 empregando dois espelhos. Ajuste os divisores de feixe e espelhos para que ambos os feixes são separados espacialmente. </li><li> alinhar cuidadosamente BS5 para assegurar que a direcção de incidência de ambas as vigas é perpendicular ao plano de pixel da SLM, ignorando BS3 e BS4 em primeiro lugar. Em primeiro lugar, nada é apresentado na SLM, isto é, actua como um espelho, até ao final da calibração (em todo o passo 2). </li><li> Ajustar a posição e a distância entre as duas lentes (L), que constitui um telescópio Kepler, a fim de obter uma imagem nítida do plano SLM na câmara complementar semicondutor de óxido metálico (CMOS). Assista a orientação correta de L1 e L2 (lados planos estão enfrentando uns aos outros) para minimizar aberrações. </li></ol></li><li> Preparação do lado distal <ol><li> Use BS7 para dividir o feixe do objeto em dois feixes e combiná-los em BS8 empregando dois espelhos. Mais uma vez, ajuste os divisores de feixe e mirrors de modo que ambos os feixes estão espacialmente separadas. </li><li> Desviar ambas as vigas usando BS9 para apontar-los para a objetiva do microscópio (OBJ). Focagem obj2 na extremidade distal da fibra multimodo (MMF). Verifique o foco observando o reflexo de volta do MMF empregando L3 e um dispositivo de câmara de carga acoplada (CCD). </li></ol></li><li> Conexão proximal e distal <ol><li> Colimar a luz do feixe do objeto sair do MMF empregando Obj1. </li><li> Dividir o feixe do objeto usando BS6, ignorar o polarizador linear (LP) em primeiro lugar. Combinar as duas vigas de objeto com as duas vigas de referência em BS3 e BS4 empregando um espelho. Ajustar os espelhos divisores de feixe e de modo que cada par de referência e feixe de objecto sobreposição na SLM, cruzando com um pequeno ângulo (inferior a 1 °). </li><li> Assegure-se que a potência de referência e o feixe de objecto são aproximadamente iguais, rodando a orientação do HWP, de acordo com o passo 1.1.2. </li><li> Verifique a interferência pattern (fora do eixo holograma) para a câmera CMOS e ajustar o ângulo de cruzamento em conformidade. Aumentar o ângulo, até que o espaçamento franja de interferência aproximadamente igual ao tamanho de dois pixels na câmara CMOS. </li><li> Ajustar a orientação do LP para coincidir com a polarização do objecto e feixe de referência, a fim de obter um máximo contraste do padrão de interferência na imagem da câmara CMOS, de modo que a imagem da câmara mostra franjas distintas. </li></ol></li></ol> 2. Calibração do Sistema <ol><li> Calibrar a relação de pixel entre SLM e CMOS <ol><li> Iluminar toda a SLM utilizando apenas um dos feixes de referência e bloquear a outra referência e objetos vigas. </li><li> Capturar uma imagem do SLM com a câmera CMOS. </li><li> Obter as coordenadas do canto superior esquerdo da SLM na imagem da câmara CMOS, por exemplo, usando o software de gráficos e o cursor do mouse no PC. Use essas coordenadas de pixel como o ponto de origemsobre a SLM. </li><li> Remova todos os blocos de feixe. </li></ol></li><li> Calibrar os caminhos de sinal <ol><li> Bloquear tanto de referência da viga 2 e objeto feixe 2. </li><li> Capturar uma imagem do holograma com a câmara CMOS. Avaliar a fase no holograma gravado usando o método espectro angular 19. Calcula-se a fase invertida na região correspondente do feixe 1. </li><li> Retirar os antigos blocos de feixe e agora bloquear tanto de referência da viga 1 e objeto feixe 1. </li><li> Capturar uma imagem do holograma com a câmara CMOS. Medir a fase no holograma gravada usando o método espectro angular novamente. Calcula-se a fase invertida na região correspondente da travessa 2. </li><li> Remova todos os blocos de feixe. </li></ol></li></ol> 3. transmitir os sinais <ol><li> Bloquear o feixe objeto. </li><li> Costurar as imagens de fase invertida calculados às regiões correspondentes do feixe 1 e 2 juntos e exibir otoda imagem na SLM, geralmente usando a porta de computação gráfica. </li><li> Comece a modulação dos sinais de entrada 1 e 2, ativando OM1 e OM2. </li><li> Observe os sinais de saída 1 e 2 na câmara CCD. </li></ol>

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The authors have nothing to disclose.

A montagem do dispositivo experimental (passo 1 no protocolo) requer um alinhamento completa dos componentes ópticos em relação uns aos outros. O aspecto mais importante é a incidência rectangular dos feixes de referência para o SLM, a fim de assegurar um elevado PBR. A fim de aumentar a configuração de mais de dois sinais transmitidos, pode ser utilizado divisores de feixe adicionais. Como uma alternativa, uma aplicação à base de fibras seria mais compacta e robusta permitindo qu...

A transmissão de sinais múltiplos de luz através de uma fibra multimodo (MMF) é evidente em comunicação de engenharia 1 e 2 biofotónica. Na engenharia de comunicações, acredita-se que a multiplexação por divisão de espaço (SDM) como uma solução viável para melhorar a capacidade de transmissão de fibras ópticas para aplicações de transferência de dados futuras que beneficiam de uma maior utilização do espaço limitado, em comparação com várias fibras monomodo 3. Em biophotonics, amostras biológicas são manipulados pela transmissão da luz através de um endoscópio MMF 4. Por exemplo, o dispositivo de controlo óptico independente de neurónios individuais usando endoscópios MMF é de interesse para Optogenetics, a fim de estudar as redes neuronais no cérebro 5. No entanto, a luz projetada sobre a faceta de entrada MMF está sujeito a distorção devido à mistura de modo e dispersão durante a propagação para o Output faceta do MMF. Como resultado, a propagação da luz é alterada, o que torna a transmissão de sinal desafiador. Wavefront moldar métodos 6, 7 são aplicados na dispersão mídia usando moduladores de luz espacial (SLM) e permitir a compensação para a distorção devido à dispersão durante a propagação da luz 8. Existem abordagens iterativos que otimizam a saída usando uma realimentação óptica 9. Estas abordagens são bastante demorado devido à necessidade de numerosas iterações e o elevado grau de liberdade, correspondendo a um grande número de elementos de modulador. Outra abordagem é para determinar completamente a distorção dentro do MMF descrito por sua matriz de transmissão 10. Se o número de modos a serem transmitidos é grande, este irá ser demorado bem. Em contraste, a conjugação de fase digital óptico (DOPC) é considerado como sendorápido e vantajoso aqui, uma vez que apenas alguns pontos focais têm que ser gerado no faceta do MMF saída. Abordagens conjugação de fase também foram demonstradas para a focagem de imagem ou através do tecido biológico 12, 13, 14. Até agora, DOPC foi empregue para um único sinal de tempo de apenas 15, 16, e foi aplicada para a transmissão de luz através de um MMF 17. Uma abordagem DOPC para vários sinais independentes não foi realizado. Desenvolvemos um método de DOPC melhorada proporcionando o transporte independente de sinais de luz múltiplas utilizando frente de onda individuais que constituem para cada sinal que emprega uma única fase apenas SLM 18. Para este objectivo, o SLM é segmentado em regiões, um para cada sinal a ser transmitido. A configuração experimental proposto é mostrado na Figura 1, Onde uma calibração é realizado em a), antes da transmissão efectiva acontece em b). <img alt="figura 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> Figura 1: Instalação experimental. BS = divisor de feixe, CCD = charge-coupled device, OM = modulador óptico, CMOS = complementar semicondutor de óxido de metal, HWP = meia placa onda, L = lente, LP = polarizador linear, MMF = fibra multimodo, objetiva obj = microscópio, PBS = polarização divisor de feixe, SLM = modulador de luz espacial (fase única) - apenas vigas relevantes para (a) a calibração e (b) a transmissão são representados <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">por favor clique aqui para ver uma versão maior desta 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

A transmissão de sinais ópticos múltiplos independentes, através de uma fibra multimodo é realizado utilizando moldagem de frente de onda, a fim de compensar a distorção durante a propagação da luz no interior da fibra. Nossa metodologia é baseada na conjugação de fase digital óptico empregando apenas um único modulador de luz espacial, onde a frente de onda óptico é modulado individualmente em diferentes regiões do modulador, uma região por sinal luminoso. abordagens de conjugação de fase óptica digitais são considerados para ser mais rápido do que outras abordagens de frente de onda moldar, onde (por exemplo) é realizada uma determinação completa do comportamento de propagação da onda da fibra. Em contraste, a abordagem apresentada é rápida e eficiente uma vez que requer apenas uma calibração por cada sinal de luz. O método proposto é potencialmente adequado para a multiplexação por divisão espacial em engenharia de comunicações. Outros campos de aplicação são a entrega de luz endoscópica em biophotonics, especialmente em optogenetics, onde as células individuais no tecido biológico tem que ser iluminada seletivamente com alta resolução espacial e temporal.

<html> Sinais de saída típica no lado distal da fibra longa de 2 m estão descritos na Figura 2. Note-se que o ponto focal desejado (pico) é acompanhado por um padrão de manchas indesejadas (background), o que é devido à imperfeição da DOPC como uma questão de princípio. O correspondente rácio de pico-a-fundo (PBR) ascende a 53 (apenas um sinal 1 é &#39;on&#39;), 36 (apenas o sinal 2 é &#39;ligado&#39;) e 20 (ambos os sinais 1 e 2 são &#39;on&#39;) aqui, res...

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.

Transmissão de múltiplos sinais através de uma fibra óptica Usando Wavefront 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 visualizações.  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.

Vídeo: 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 Optical resolução Microscopia Fotoacústica

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

Bioengenharia

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.

Colocar Sondas Biológicas para Biossensores ópticos de sílica usando agentes de acoplamento 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.

Indução de Sinais de adesão dependentes Usando ultra-som de baixa intensidade

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.

Em síntese in vitro de Nativo, Espaçamento fibroso Long e Colágeno Espaçamento segmentar Longo

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.

Único Detecção de Moléculas livre-Label Usando Microtoroid ópticos Resonators

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 detecção de fluorescência para Gota microfluídica Utilizando 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.

Determinação do Potencial Zeta de nanopartículas de translocação através de velocidades por meio de um Tunable Nanopore: Utilizando partículas modificadas-ADN como um Exemplo

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).

Usando transmissão óptica extraordinária para quantificar biomarcadores cardíacos no soro 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.

Um protocolo para múltiplo knockout de genes em ratos Organoids Intestinal Pequeno Usando um 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.

Segmentação Neuronal fibra folhetos para a terapia de estimulação profunda do cérebro usando modelos interativos, paciente específico

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