DR reconhece o apoio da Fundação Alemã de Pesquisa (DFG) Research Grant (RA 1848/1) e do Conselho Europeu de Investigação Começando Grant. VN reconhece o apoio financeiro da avançada Investigator Award Conselho Europeu de Investigação e Inovação do BMBF em Medicina Award.

1. Optoacoustic Caracterização do detector π-FBG <ol><li> Preparação de uma esfera microscópica suspensas em agar: <ol><li> Mistura em pó de ágar (1,3% em peso) com água destilada num copo de vidro. Use uma placa quente dispositivo magnético-agitador para aquecer a solução até à temperatura de ebulição estreita e dissolve-se o pó de agar, até a solução se tornar límpida e livre de bolhas de ar. Alternativamente, a solução de agar podem ser aquecido utilizando um forno de microondas convencional, com agitação realizada manualmente, utilizando uma vareta de vidro. Despeje a solução quente em um molde de plástico, por exemplo, seringa com a ponta cortada. </li><li> Polvilhe uma pequena quantidade de esferas microscópicas sobre a solução de agar e esperar até que a solução se solidifica totalmente. Pegue o fantasma agar sólido para fora do molde, empurrando o êmbolo. </li><li> Ver o fantasma sob um microscópio estereoscópico cortar um pequeno pedaço de agar, que contém uma única esfera microscópica. </li><li> Repita o passo 1.1.1 e adicionar to da solução agar agar a peça sólida contendo o único esfera microscópica. </li><li> Após a solidificação, corte o fantasma agar sob o microscópio de tal forma que a esfera microscópica está localizado próximo à superfície do phantom. </li></ol></li><li> Medição Optoacoustic <ol><li> Utilize dois suportes de fibra de v-groove para manter a fibra firmemente em ambos os lados do π-FBG, e conectado ao seu titular um dimensional (XYZ) Tradução terceira fase operado por computador. Assegure-se que a fibra está submerso para permitir a propagação dos ultra-sons. </li><li> Localizar a posição aproximada do elemento sensor de π-FBG iluminando diferentes partes da fibra, com a maior potência nanossegundos impulso do feixe de laser. A absorção óptica do revestimento, no entanto fraca, irá criar um sinal, quando a iluminação é realizada na π-FBG. </li><li> Coloque a esfera microscópica incorporado-agar diretamente abaixo do π-FBG. A esfera microscópica deve ser visível a olho nu. </li><li> Usando a fase de tradução, execute uma varredura 2D do π-FBG no plano paralelo ao solo para encontrar o local onde o sinal da esfera microscópica é a mais forte e seu intervalo de tempo correspondente é mais curto. </li><li> Realize últimos ajustes para a iluminação para entregar potência máxima para a esfera microscópica. </li><li> Usando a fase de tradução realizar uma varredura em 3D do π-FBG e gravar o sinal para cada posição. </li><li> Para obter a resposta de freqüência espacialmente dependente do detector de ultra-som, realizar a transformada de Fourier do sinal de ultra-som no domínio do tempo gravado. </li></ol></li></ol> 2. Estimativa de Robustez e Sensibilidade de Desempenho do π-FBG Detector <ol><li> Usar dois suportes de fibra de ranhura em V para manter a fibra firmemente em ambos os lados do π-FBG e submergir o π-FBG. </li><li> Colocar uma placa escura ou um bastão de grafite resistente para enfrentar o π-FBG e iluminá-lo sagacidadeh o de alta potência nanosegundo-pulse feixe de laser para criar um campo acústico forte. </li><li> Coloque uma bomba de água no interior do tanque de água e ligá-lo, a fim de criar variações rápidas nas condições ambientais. </li><li> Para estimar a robustez do sistema, medir a saída com o circuito de bloqueio virou tanto dentro quanto fora. Quando nenhum de bloqueio é realizada, não é possível detectar com precisão o sinal de ultra-som. </li><li> Ligue a bomba de água fora. </li><li> Para estimar o benefício da sensibilidade devido ao elevado de coerência da fonte, substituir o laser de impulsos de banda larga com uma fonte de baixa coerência e repetir a medição acústica. Uma redução de mais de uma ordem de magnitude na sensibilidade é esperado quando a fonte de baixa coerência é usado. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Em conclusão, um novo método para a detecção óptica de ultra-som é introduzida, que se baseia numa combinação de um π-FBG e pulso interferometria. A técnica é especialmente adequado para aplicações de imagem optoacoustic devido à transparência do elemento sensor, que permite padrões de iluminação objeto quase arbitrárias. Em contraste, detectores de ultra-som piezoelétrico com base padrão são opacas e, assim, bloquear alguns dos caminhos ópticos ao objeto fotografado, levando a configurações de ...

Detectores de ultra-sons desempenham um papel chave em muitas aplicações de imagiologia. Convencionalmente, o ultra-som é detectada por transdutores piezelétricos, que transformam ondas de pressão em sinais de tensão 1. Na imagem optoacoustic, ultra-som é gerado através de um processo de expansão térmica, iluminando o objeto com alta potência de luz modulada 2-6. Apesar de transdutores piezoelétricos são o método de escolha em aplicações optoacoustic, seu uso muitas vezes dificulta a miniaturização principalmente porque transdutores piezoelétricos miniaturizados são frequentemente caracterizados por baixa sensibilidade. Além disso, como transdutores piezoelétricos são opticamente opaco, eles podem interferir seriamente com a entrega de luz para o objeto fotografado, limita as possibilidades de configurações de imagem utilizáveis. Luz que é back-dispersa do objeto para o transdutor pode também limitar a detecção adequada de ultra-som e complicar o projeto do sistema de imagem devido a parasit induzido opticamenteic sinaliza no transdutor 7. Detectores ópticos de ultra-som foram reconhecidos como uma possível alternativa para transdutores piezoelétricos que oferece muitos benefícios em cenários de imagem optoacoustic 8-12: Muitas vezes são transparentes e podem ser miniaturizados geralmente sem perda de sensibilidade. O princípio de funcionamento dos detectores ópticos é a detecção interferométrico da deformação minuto criada no suporte óptico devido à presença de ultra-sons. Muitas vezes, os ressoadores ópticos são utilizados para aumentar a sensibilidade de detecção por captura de luz no meio perturbado por períodos prolongados, aumentando assim o efeito de deformação sobre a fase do sinal óptico. Nesses casos, esquemas de detecção ópticos são baseados em variações de monitoramento no comprimento de onda de ressonância, que se relacionam diretamente com estruturar deformações no ressonador. Mais comumente, onda contínua estreita largura de linha (CW) técnicas são usadas em que um laser CW está sintonizado com the comprimento de onda de ressonância. Pequenas mudanças no comprimento de onda de ressonância alterar a posição relativa do comprimento de onda do laser de dentro da ressonância, causando, assim, as variações na intensidade da transmissão / reflectida a luz do laser, o qual pode ser facilmente monitorizado. No entanto, se as mudanças de ressonância são muito fortes, por exemplo, devido a grandes variações de pressão, temperatura, ou vibrações, a ressonância pode mudar completamente longe de comprimento de onda do laser, efetivamente saturar o detector 13. Pulso de interferometria 14 oferece uma solução para a limitação da saturação de sinal e permite a detecção de ultra-sons sob condições ambientais voláteis. Em contraste com a estreitar-linewidth esquemas CW, pulso interferometria emprega uma fonte de pulso de banda larga para iluminar o ressonador. Neste caso, o ressoador funciona como um filtro de banda de passagem, transmitindo apenas os comprimentos de onda que correspondem à sua frequência de ressonância, ao passo que a ressonância desloca umre detectada através da medição das variações de comprimento de onda do sinal óptico de saída do ressonador, por exemplo, utilizando um interferómetro Mach-Zehnder bloqueado para quadratura 14,15. Um circuito de reset automático é usado para restaurar imediatamente ponto de trabalho do interferômetro no caso de perda devido à variação extrema em condições ambientais. Devido à largura de banda relativamente larga da fonte, o comprimento de onda de ressonância se mantém dentro da faixa iluminada, mesmo sob fortes perturbações, permitindo a operação do detector estável, mesmo em condições ambientais desfavoráveis. O uso de uma fonte coerente para interrogatório, pulsos ópticos ou seja, facilita a detecção de baixo ruído. O sistema de interferometria de impulsos correspondente usada nas nossas experiências é mostrado na Figura 1. O laser de impulsos usado para interrogação produziu 90 pulsos FSEC uma taxa de repetição de 100 MHz com a potência de saída de 60 mW e largura espectral de mais de 100nm. O filtro óptico tinha uma largura espectral FWHM de aproximadamente 0,4 nm e foi sintonizado na freqüência da ressonância. Seguindo o filtro, um amplificador óptico foi utilizado para compensar a perda significativa na filtragem. Filtragem adicional foi aplicado após a fase de amplificação para reduzir a emissão espontânea amplificada a partir do amplificador. O ressonador utilizado em nossos experimentos é um deslocou-fase pi rede de Bragg (π-FBG) 8, fabricado pela Teraxion Inc. Particularmente para a aplicação médica de sensoriamento ultra-som, π-FBGs têm a vantagem de ser de todos os componentes de fibra, e, portanto, robusto e pequenas. Figura 2 mostra uma comparação entre as dimensões da fibra óptica utilizada neste trabalho e uma de 15 MHz de ultra-som intravascular miniaturizado (IVUS) transdutor piezoeléctrico. Algumas abordagens de detecção baseados em ressonância alternativos, tais como ressonadores micro-anel fabricado em guias de ondas planares, requerem acoplamento de fibras no componente deentrada e saída, ou levando a dispositivos mais frágeis ou dificultando a miniaturização. Em contraste, π-GBFs são em fibra de componentes, e não necessitam de acoplamento de fibra adicional. A ressonância em π-GBFs é criado pela mudança de fase pi no seu centro; luz é preso em torno do desvio de fase pi sobre a porção da fibra que é consideravelmente mais curto do que o comprimento da própria grade. Nas nossas experiências, o π-FBG tinha um comprimento de 4 mm e um coeficiente de acoplamento de κ igual a 2 mm -1 e sua sensibilidade foi distribuída de maneira não uniforme ao longo do seu comprimento, com a sensibilidade exponencialmente decrescente a partir do centro da grelha com uma taxa de κ . A largura total a metade do máximo (FWHM) de distribuição de sensibilidade (DP) era de aproximadamente 350 um. A largura de ressonância da grade é determinada por ambos o seu comprimento e o seu coeficiente de acoplamento de acordo com a seguinte equação: <img alt="Equação 1" fo:content-width="1.5in" src= &quot;Files/ftp_upload/50847/50847eq1.jpg /&quot; width = &quot;150&quot; /&gt; (1) onde λ é o comprimento de onda de ressonância e n ef é o índice de refracção efectivo do modo guiado na fibra 8. Para avaliar se o detector de π-FBG é apropriada para aplicações de imagem, a sua resposta espacialmente dependente precisa ser medido ao longo de uma faixa de freqüência de largura. No entanto, esta tarefa é extremamente difícil quando são utilizadas técnicas acústicas convencionais. Nós, portanto, empregar um método optoacoustic para detector de ultra-som caracterização 16 em que uma esfera microscópica escura embutido no agar transparente serve como uma fonte pontual optoacoustic. Na nossa experiência, a esfera microscópica tem um diâmetro de aproximadamente 100 mm e é iluminado com alto poder de nanossegundos impulsos ópticos com uma taxa de repetição de 10 Hz, a duração do pulso de cerca de 8 ns, e potência média de 200 mW. A energia óptica depositado na esf microscópicaeres gera sinais de ultra-sons de banda larga devido ao efeito optoacoustic. O detector de π-FBG é traduzido relativamente à esfera microscópica para obter a sua resposta acústica espacialmente dependente. Figura 3 mostra uma ilustração da experiência optoacoustic. Geralmente, esta técnica pode ser utilizada para caracterizar diferentes tipos de detectores de ultra-sons.

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			<td height="43" style="height:43px;width:216px;">&#960;-FBG</td>
			<td style="width:71px;">Teraxion Inc.</td>
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			<td style="width:167px;">&#160;Custom made device</td>
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			<td height="43" style="height:43px;width:216px;">microscopic sphere</td>
			<td style="width:71px;">Cospheric LLC</td>
			<td style="width:119px;">BKPMS 90-106um- 10g</td>
			<td style="width:167px;">100 &#181;m polyethylene microsphere</td>
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			<td height="64" style="height:64px;width:216px;">Femto-second pulse laser used for interrogation&#160;</td>
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			<td height="64" style="height:64px;width:216px;">Optical filter</td>
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			<td style="width:119px;">AEDFA-PM-PA-35-B-FC</td>
			<td style="width:167px;">Benchtop 35dB Gain Pre Amp Polarization Maintaining EDFA&#160;</td>
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			<td height="232" style="height:232px;width:216px;">50/50 coupler</td>
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			<td style="width:119px;">FUSED-22-1550-8/125-50/50-3S3S3S3S-3-0.5-PM</td>
			<td style="width:167px;">Fused 2x2 fiber splitter with 0.5 meter long, 3mm OD PVC jacketed 1550nm 8/125&#956; PM fiber 
			pigtails, 50/50 split ratio in the slow axes of PM fibers and with super FC/PC connectors on all 
			ports.</td>
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			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">T711/M-250</td>
			<td style="width:167px;">Metric, Post-Mountable Fiber Clamp, 250 &#181;m&#160;</td>
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			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">05039-500G</td>
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			<td style="width:119px;">VIBRANT Arrow 532 type I</td>
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			<td style="width:71px;">&#160;Faber-Castell</td>
			<td style="width:119px;">120700</td>
			<td style="width:167px;">Faber-Castell Pencil Leads - 0.7 mm</td>
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wideband optical detector of ultrasound for medical imaging applications

Os sensores ópticos de ultra-sons são uma alternativa promissora às técnicas piezoeléctricos, como foi recentemente demonstrado no campo da imagiologia optoacoustic. Em aplicações médicas, uma das principais limitações da tecnologia de detecção óptica é a sua susceptibilidade a condições ambientais, por exemplo, mudanças de pressão e temperatura, o que pode saturar a detecção. Além disso, o ambiente clínico, muitas vezes impõe limites rigorosos sobre o tamanho ea robustez do sensor. Neste trabalho, a combinação de interferometria de pulso e de detecção óptica à base de fibras é demonstrada para a detecção de ultra-sons. Interferometria de impulsos permite que o desempenho robusto do sistema de leitura, na presença de variações rápidas nas condições ambientais, ao passo que o uso de tecnologia de tudo fibra leva a um elemento sensor flexível mecanicamente compatível com altamente exigentes aplicações médicas, tais como a imagem intravascular. A fim de conseguir um curto comprimento do sensor, umafibra de fase pi deslocado Bragg é utilizado, o qual actua como um ressonador prendendo luz ao longo de um comprimento útil de 350 m. Para permitir que a largura de banda alta, o sensor é utilizado para a detecção de costado de ultra-som, que é altamente benéfica em geometrias de imagiologia, tais como imagiologia circunferenciais intravascular. Uma estrutura de imagem optoacoustic é utilizado para determinar a resposta do sensor às fontes pontuais acústicos em diferentes posições.

As Figuras 4a e 4b mostram, respectivamente, os sinais e os seus espectros correspondente da esfera microscópica, a uma distância de 1 mm a partir da fibra para três deslocamentos do centro do π-FBG. Os deslocamentos são indicados na direcção z, conforme ilustrado na Figura 3. Claramente, a sensibilidade do detector óptico de ultra-som de alta frequência (f&gt; 6 MHz) é anisotrópica e é mais elevado quando o centro do π-FBG é directamente acima da esfera m...

Watch this Scientific Journal Video about Wideband Optical Detector of Ultrasound for Medical Imaging Applications at JoVE.com

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Detecção óptica de ultra-som é impraticável em muitos cenários de imagem, porque muitas vezes requer condições ambientais estáveis. Nós demonstramos uma técnica óptica para detecção de ultra-som em ambientes voláteis, com os níveis de miniaturização e sensibilidade adequada para a imagem latente optoacoustic em cenários restritivos, por exemplo, aplicações intravasculares.

Wideband Detector óptico de ultra-som para aplicações Medical Imaging

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

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JoVE Journal

Bioengineering

10.7K visualizações.  Technical University of Munich and Helmholtz Center Munich.  Detecção óptica de ultra-som é impraticável em muitos cenários de imagem, porque muitas vezes requer condições ambientais estáveis. Nós demonstramos uma técnica óptica para detecção de ultra-som em ambientes voláteis, com os níveis de miniaturização e sensibilidade adequada para a imagem latente optoacoustic em cenários restritivos, por exemplo, aplicações intravasculares. 

Vídeo: Wideband Optical Detector of Ultrasound for Medical Imaging Applications

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

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Imagem Domínio óptico Freqüência de<em&gt; Ex vivo</em&gt; Pulmonares espécimes de ressecção: Obtenção de One to One Imagem para Histopatologia Correlação

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

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

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Preparação de Mica Apoiado Lipid Bicamadas para alta resolução Microscopia Óptica de Imagem

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

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoramento em tempo real de High Intensity Focused Ultrasound (HIFU) Ablação de<em&gt; In Vitro</em&gt; Canine Fígados Usando Movimento Harmônico Imaging para ultra-som focalizado (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

Tomografia Computadorizada e imageamento óptico de acoplamento Osteogenesis-angiogênese para avaliar a integração do osso craniano Autoenxertos e Aloenxertos

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificação de bactérias Polymers Repelente para dispositivos médicos

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal Therapy and Surgery) project. One of the aims of this project is to engineer novel, real-time image processing methods for intra-operative use in the treatment of congenital birth defects, such as spina bifida and the twin-to-twin transfusion syndrome. The developed target allows for the sterility-preserving optical distortion calibration of endoscopes within a few minutes. Good optical distortion calibration and compensation are important for mitigating undesirable effects like radial distortions, which not only hamper accurate imaging using existing endoscopic technology during fetal surgery, but also make acquired images less suitable for potentially very useful image computing applications, like real-time mosaicing. In this paper proposes a novel fabrication method to create an affordable, sterilizable calibration target suitable for use in a clinical setup. This method involves etching a calibration pattern by laser cutting a sandblasted stainless steel sheet. This target was validated using the camera calibration module provided by OpenCV, a state-of-the-art software library popular in the computer vision community.

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

Alvo esterilizável de grau médico para fetoscópio Distortion Calibração óptica imerso-Fluid

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

A longo prazo dispositivo de imagens ao vivo para melhor manipulação Experimental de larvas de Zebrafish

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Imagem óptica de miócitos ventriculares isolados de urina

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...