Name: a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging
Uploaded: 2017-06-21T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

A pesquisa é apoiada pelo subsídio de Nível 2 financiado pelo Ministério da Educação em Cingapura (ARC2 / 15: M4020238) e pelo Conselho Nacional de Pesquisa Médica do Ministério da Saúde de Singapura (NMRC / OFIRG / 0005/2016: M4062012). Os autores agradecem ao Sr. Chow Wai Hoong Bobby pela ajuda da maquina.

Todas as experiências com animais foram realizadas de acordo com as diretrizes e regulamentos aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade Tecnológica de Nanyang, em Cingapura (Número de Protocolo Animal ARF-SBS / NIE-A0263). 1. Descrição do sistema <ol><li> Monte o PLD dentro do scanner circular, como mostrado na Figura 1a . Conecte o PLD à unidade de driver a laser (LDU). NOTA: O PLD fornece pulsos ~ 136-ns a um comprimento de onda de ~ 803 nm, com uma energia de pulso máxima de ~ 1,42 mJ e uma taxa de repetição de até 7 kHz. A unidade de condução a laser (LDU) inclui: um controlador de temperatura, uma fonte de alimentação variável, uma fonte de alimentação (12 V) e um gerador de função; Veja a Tabela de Materiais. A fonte de alimentação variável é usada para controlar a potência do laser e o gerador de função é usado para alterar a taxa de repetição do PLD. </li><li> Ligue o laser PLD. Defina a taxa de repetição PLD para &quot;7.000&quot; HZ usando o gerador de função no LDU. Aumente a energia de pulso para 1,42 mJ ajustando a tensão da fonte de alimentação variável para &quot;3.1&quot; V. </li><li> Monte o difusor óptico (OD) na frente da janela de saída PLD para tornar o feixe de saída homogêneo, como mostrado na Figura 1a . NOTA: Use um difusor com um grão fino ( ou seja, um polimento de 1.500 grãos). </li><li> Monte o UST focado no suporte do UST, de modo que ele enfrente o centro da área de digitalização, como mostrado na Figura 1a . NOTA: A freqüência central do UST é 2,25 MHz e a distância focal é de 1,9 pol. </li><li> Coloque o detector de ultra-som dentro do tanque de acrílico, como mostrado na Figura 1a . Encha o tanque com água de modo que o UST esteja totalmente imerso. NOTA: Um meio de água é usado para acoplar o sinal fotoacústico do cérebro (amostra) ao UST. Um tanque de água acrílica (WT, veja a Tabela de Materiais) foi personalizado- projetado para imagens de animais pequenos. O esquema do desenho do tanque de água é mostrado na Figura 1b . </li><li> Verifique o sinal de PA da amostra usando uma unidade de pulsador / receptor (PRU, veja a Tabela de Materiais ). NOTA: esses sinais foram digitalizados por um cartão DAQ de 12 bits (veja a Tabela de Materiais ) a uma taxa de amostragem de 100 MS / s e foram salvos em um computador. </li></ol> 2. Preparação de animais para imagens de cérebro de ratos NOTA: O sistema PLD-PAT descrito acima foi demonstrado para imaginar cérebros de pequenos animais. Para estas experiências, foram utilizadas ratos fêmeas saudáveis ​​(ver Tabela de Materiais ). <ol><li> Anestesiar o animal por injeção intraperitoneal de um coquetel de 2 mL de cetamina, 1 mL de xilazina e 1 mL de solução salina (dose de 0,2 mL / 100 g). </li><li> Remova a pele no couro cabeludo do animal usando uma cortadora de cabelo. Aplique suavemente o creme de depilação ao raspadoÁrea para maior depleção do pêlo. <ol><li> Retire o creme aplicado após 4-5 min usando um cotonete de algodão. </li><li> Aplique pomada de lágrima artificial aos olhos do animal para evitar a secura devido à anestesia e à iluminação a laser. </li></ol></li><li> Monte o suporte para animais feito sob medida (veja a Tabela de Materiais ) equipado com uma máscara de respiração (veja a Tabela de Materiais ) em um laboratório. </li><li> Coloque o animal em posição inclinada no suporte. Fixe-o ao suporte usando fita cirúrgica para evitar o movimento do animal durante a imagem. </li><li> Certifique-se de que a máscara de respiração cobre o nariz e a boca do rato para administrar anestesia inalada. </li></ol> 3. In Vivo Rat Brain Imaging <ol><li> Conecte a máscara de respiração à máquina de anestesia. Ligue a máquina de anestesia e ajuste-a para fornecer 1,0 L / min de oxigênio com 0,75% de isoflurano. <ol><li> Aperte o oxímetro de pulsoS tail para monitorar a condição fisiológica do animal. </li></ol></li><li> Aplique uma camada de gel ultra-som incolor ao couro cabeludo do rato. Ajuste a posição lab-jack no centro do scanner. A máscara de respiração é personalizada para se adequar à janela de imagem. 10% do cone do nariz comercialmente disponível é cortado e conectado a um pedaço de luva. <ol><li> Ajuste a altura do lab-jack manualmente para que o plano de imagem esteja no foco da UST. </li></ol></li><li> Defina os parâmetros no software de aquisição de dados (consulte a Tabela de Materiais) conforme necessário. Execute o programa de software de aquisição de dados para iniciar a aquisição ( ou seja, imagens). NOTA: O programa é usado para girar o UST e coletar os sinais de A-line PA. As linhas A coletadas serão salvas no computador. </li><li> Observe o animal durante todo o período de imagem e proceda à reconstrução do PAT após a conclusão da imagem. </li><li> Após a conclusão da aquisição de dados,Reconstruir a imagem do cérebro transversal das linhas A usando o programa de software de reconstrução. </li><li> Desligue o sistema de anestesia, remova o animal do palco, entregue-o à sua gaiola e monitore-o até recuperar a consciência. NOTA: Por exemplo, se o UST for girado por 5 s, o PLD entrega 35.000 (= 5 x 7.000) pulsos e a UST coleta 35.000 linhas A. As 35.000 linhas A são reduzidas para 500 por média de mais de 70 sinais (após a média de linhas A = 35,000 / 70 = 500). A Figura 1c ilustra a iluminação dos pulsos de laser e da coleção de linhas A. Um programa de reconstrução baseado em algoritmo de retroprojeção de atraso e soma deve ser usado. </li></ol><img alt="figura 1" class="xfigimg" src="/files/ftp_upload/55811/55811fig1.jpg" /> Figura 1: Esquemas do sistema PLD-PAT. (A) Esquema do PLD-PAT. PLD: diodo laser pulsado, CSP: circuPlaca de varredura, AM: máquina de anestesia, M: motor, MPU: unidade de polia do motor, LDU: unidade de driver a laser, PRU: unidade de pulsador / receptor, UST: transdutor de ultra-som, WT: tanque de água, PF: filme de polímero e DAQ: Cartão de aquisição de dados. ( B ) Esquema do tanque de água, vista superior (1) e vista em corte transversal (2) para imagens cerebrais de animais pequenos in vivo . A: métrico Parafuso, B: placa anular acrílica, C: anel &quot;O&quot; de silicone, D: tampa de polietileno transparente de 100 μm de espessura. O tanque tinha um orifício de 9 cm de diâmetro na parte inferior e foi selado com uma membrana de polietileno 100 μm de espessura ultra-sônica e opticamente transparente. ( C ) Esquema da iluminação de pulsos a laser das linhas PLD e A, coleta em um tempo de varredura contínua de 5 s. <a href="http://ecsource.jove.com/files/ftp_upload/55811/55811fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

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Os autores não têm interesses financeiros relevantes no manuscrito e nenhum outro potencial conflito de interesse para divulgar.

Este trabalho apresenta um protocolo para realização de imagens cerebrais in vivo em ratos usando um sistema PLD-PAT. O protocolo inclui uma descrição detalhada do sistema de imagem e seu alinhamento, bem como uma ilustração da imagem cerebral em ratos. Os sistemas de PAT baseados em OPO existentes são caros e volumosos e podem fornecer uma imagem transversal em 5-10 min. O sistema PLD-PAT é compacto, portátil e de baixo custo e pode fornecer imagens de boa qualidade em 3 s. O desempenho do sistema foi...

<html> A tomografia fotoacústica (PAT) é uma modalidade de imagem híbrida que tem muitas aplicações em estudos clínicos e pré-clínicos 1 , 2 , 3 , 4 , 5 . Em PAT, pulsos de laser de nanosegundo irradiam tecido biológico. A absorção de luz incidente pelos cromóforos de tecido leva a um aumento de temperatura local, que então produz ondas de pressão emitidas sob a forma de ondas sonoras. Um detector de ultra-som coleta os sinais fotoacústicos em várias posições ao redor da amostra. Os sinais fotoacústicos (PA) são reconstruídos usando vários algoritmos (como um algoritmo de atraso e soma) 6 para gerar a imagem fotoacústica. Esta modalidade de imagem híbrida oferece imagens de alta resolução e de tecido profundo e alto contraste de absorção óptica 7 ,Class = &quot;xref&quot;&gt; 8. Recentemente, uma profundidade de imagem de ~ 12 cm 9 foi alcançada no tecido mamário de frango com o auxílio de um comprimento de onda mais longo (~ 1,064 nm) e um agente de contraste exógeno chamado ftalocianina de fósforo. Essa sensibilidade de profundidade é muito maior do que a sensibilidade de profundidade de outros métodos ópticos, como a microscopia de fluorescência confocal, a microscopia de fluorescência de dois fótons, a tomografia de coerência 10 , 11 , etc. Usando mais de um comprimento de onda, PAT pode demonstrar mudanças estruturais e funcionais nos órgãos . Para muitas doenças humanas, modelos de animais pequenos foram bem estabelecidos 12 , 13 , 14 , 15 . Para a imagem de animais pequenos, várias modalidades foram demonstradas. Fora de todas essas abordagens, a imagem em PA ganhou atenção com bastante rapidez devido às vantagens mencionadas acima. PAT mostrou seu potencial para imaginar vasos sanguíneos nos tecidos e órgãos ( ie coração, pulmões, fígado, olhos, baço, cérebro, pele, medula espinhal, rim, etc. ) de pequenos animais 4 , 16 , 17 , 18 . O PAT é uma modalidade bem estabelecida para imagens cerebrais de animais pequenos. As ondas PA são produzidas devido à absorção de luz pelos cromóforos, portanto, PAT de múltiplas ondas permite o mapeamento da concentração total de hemoglobina (HbT) e saturação de oxigênio (SO 2 ) 19 , 20 , 21 , 22 . A imagem neurovascular do cérebro foi conseguida com a ajuda de agentes de contraste exógenos 12 , 23 , 24 . A modalidade de PA pode ajudar a melhorar a compreensão da saúde do cérebroFornecendo informações nos níveis molecular e genético. Para imagens de animais pequenos, os láseres Nd: YAG / OPO são amplamente utilizados como fontes de excitação PAT. Esses laser fornecem pulsos de infravermelho próximo do ~ 5 ns com energia (~ 100 mJ na janela de saída OPO) com uma taxa de repetição de ~ 10 Hz 25 . O sistema de PA equipado com tais láser é caro e volumoso e permite imagens de baixa velocidade com transdutores de ultra-som de um elemento (UST) devido à baixa taxa de repetição da fonte do laser. Um tempo típico de aquisição da linha A em tais sistemas de PA é ~ 5 min por seção transversal 25 . Um sistema de imagem com um tempo de medição tão longo não é ideal para imagens de animais pequenos, pois é difícil controlar os parâmetros fisiológicos para a imagem completa do corpo, imagens funcionais resolvidas no tempo, etc. Ao adotar vários USTs de um único elemento, matriz Com base em USTs, ou um laser de alta taxa de repetição, é possível aumentar a velocidade de imagem da PASistemas. Usar apenas um UST de um único elemento para coletar todos os sinais de PA em torno da amostra irá limitar a velocidade de imagem do sistema. Múltiplos UST de um único elemento dispostos em geometria circular ou semi-circular são demonstrados para técnicas de imagem de alta velocidade e altamente sensíveis. Os USTs 26 baseados em matriz, como matrizes lineares, semi-circulares, circulares e volumétricas, foram utilizados com sucesso na imagem em tempo real 1 . Esses USTs baseados em matriz aumentarão a velocidade de imagem e reduzirão a sensibilidade de medição, mas são caros. No entanto, a velocidade de imagem dos sistemas de PA que usam USTs baseados em matriz ainda está limitada pela taxa de repetição do laser. Tecnologia de laser pulsado avançada para fazer diodos de laser pulsado de alta taxa de repetição (PLDs). 7,000 quadros / s A imagem fotoacústica B-scan foi demonstrada com PLDs usando uma plataforma de ultra-som clínica 27 . Tais PLDs podem melhorar a velocidade de imagem daE sistema PAT, mesmo com geometria de varredura circular UST de um elemento. Os UST de elemento único são menos caros e altamente sensíveis, ao contrário dos USTs baseados em matrizes. Ao longo da última década, poucas pesquisas foram relatadas sobre o uso de PLD de alta taxa de repetição como fonte de excitação para imagens de PA. Foi demonstrado um PLD de infravermelho próximo a fibra para imagem PA de fantasmas 28 . A imagem in vivo de vasos sanguíneos a uma profundidade de ~ 1 mm abaixo da pele humana foi demonstrada usando PLD de baixa energia 29 . Foi relatado um microscópio fotoacústico de resolução óptica baseado em PLD (ORPAM). Usando PLDs, foi demonstrada uma imagem de imagem de 1,5 cm de profundidade a uma taxa de quadros de 0,43 Hz 30 . Muito recentemente, um sistema PLD-PAT foi relatado que forneceu imagens em tão curto quanto ~ 3 s e a uma profundidade de imagem de ~ 2 cm em tecido biológico 25 , 31 . Este estudo revelou que um sistema tão baixo e compacto pode fornecer alta qualidadeImagens, mesmo em altas velocidades. O sistema PLD-PAT pode ser usado para imagens fotoacústicas de alta taxa de quadros (7.000 fps), imagem de vasos sanguíneos superficiais, imagem de junção de dedos, imagem de tecido de 2 cm de profundidade, imagens de cérebro de animais pequenos, etc. O único comprimento de onda e Os impulsos de pouca pulsação de energia da PLD limitam sua aplicação a imagens multi-espectrales e de tecidos profundos. Experimentos foram realizados em pequenos animais usando o mesmo sistema PLD-PAT usado para aplicações pré-clínicas. O objetivo deste trabalho é fornecer a demonstração experimental visualizada do sistema PLD-PAT para imagens cerebrais transversais 2D in vivo de animais pequenos.

<table><tbody><tr><td>Pulsed laser diode</td><td>Quantel, France</td><td>QD-Q1910-SA-TEC</td><td>It is the excitation laser source with specifications 803 nm, 1.4 mJ per pulse, 136 ns pulse, 7 kHz maximum, dimentions : 11.0 x 6.0 x 3.6 cm, weight: ~150 gm</td></tr><tr><td>Stepper motor with gearbox</td><td>LIN Engineering (Servo Dynamics)</td><td>Motor: CO-5718-01P, Gearbox: DPL64/1, I = 10 for NEMA 23; power supply PW100-48</td><td>To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td></tr><tr><td>Ultrasonic pulser/receiver</td><td>Olympus</td><td>5072PR</td><td>To receive, filter and ampligy the PA signal from UST. Its bandwidth is 35 MHz, and gain is &amp;plusmn;59 dB.</td></tr><tr><td>Ultrasound Transducer</td><td>Olympus</td><td>V306-SU-NK-CF1.9IN/Q4200069</td><td>Ultrasonic sensors used for photoacoustic detection. Central freqency 2.25 MHz, 0.5 in, Cylindrical focus 1.9 inch</td></tr><tr><td>PCIe DAQ (Data acquisition) Card</td><td>GaGe</td><td>CSE4227/ A6000610/B0E00610</td><td>12 bit, 100 Ms/s, 2 channels, 1 Gs on board memory, PCIe x16 interface</td></tr><tr><td>Rats</td><td>In Vivos Pte Ltd, Singapore</td><td>NTac:SD, Sprague Dawley / SD</td><td>Female, weight 100 &amp;plusmn;10g</td></tr><tr><td>Acrylic water tank&amp;nbsp;</td><td>NTU workshop</td><td>Custom-made</td><td>It contains the water that acts as an acoustic coupling medium between brain and detector</td></tr><tr><td>Circular Scanner&amp;nbsp;</td><td>NTU workshop</td><td>Custom-made</td><td>Scanner is made out of Alluminum&amp;nbsp;</td></tr><tr><td>Anesthetic Machine</td><td>medical plus pte ltd</td><td>Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td><td>Supplies oxygen and isoflurane to animal</td></tr><tr><td>Pulse Oxymeter portable</td><td>Medtronic</td><td>PM10N with veterinary sensor</td><td>Monitors the pulse oxymetry of the animal</td></tr><tr><td>Ultrasound gel</td><td>Progress/parker acquasonic gel</td><td>PA-GEL-CLEA-5000</td><td>Clear ultrasound gel</td></tr><tr><td>Data acqusison software</td><td>National Instruments Corporation,Austin,TX,USA)</td><td>NI LabVIEW 2015 SP1</td><td>LabVIEW based program was developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td></tr><tr><td>Data processing software</td><td>Matlab (Mathworks, Natick, MA, USA)</td><td>Matlab R2012b</td><td>Matlab code for reconstruction of PA images was developed in our lab</td></tr><tr><td>Temperature controller&amp;nbsp;</td><td>LaridTech, MO,USA</td><td>MTTC1410</td><td>It will constantly control temperature of the PLD&amp;nbsp;</td></tr><tr><td>12 V power supply&amp;nbsp;</td><td>Voltcraft&amp;nbsp;</td><td>PPS-11810</td><td>To supply operating voltage for PLD</td></tr><tr><td>Variable power supply&amp;nbsp;</td><td>BASETech</td><td>BT-153</td><td>To change the laser output power</td></tr><tr><td>Funtion generator&amp;nbsp;</td><td>Funktionsgenerator</td><td>FG250D</td><td>To change the repetetion rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td></tr><tr><td>Animal distributor</td><td>In Vivos Pte Ltd, Singapore</td><td></td><td>Animal distributor that supplies small animals for research purpose.</td></tr><tr><td>Animal holder</td><td>NTU workshop</td><td>Custom-made</td><td>Used for holding the animal on its abdomen</td></tr><tr><td>Breathing mask</td><td>NTU workshop</td><td>Custom-made</td><td>Used along with animal holder to supply anesthesia mixture to the animal</td></tr><tr><td>Pentobarbital sodium</td><td>Valabarb</td><td></td><td>Used for euthanizing the animal after the expeirment.</td></tr><tr><td>Optical diffuser</td><td>Thorlabs</td><td>DG10-1500</td><td>Used to to make the laser beam homogeneous</td></tr></tbody></table>

a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging

A imagem in vivo de animais pequenos tem um papel importante a desempenhar em estudos pré-clínicos. A tomografia fotoacústica (PAT) é uma modalidade de imagem híbrida emergente que mostra grande potencial para aplicações clínicas e pré-clínicas. Os sistemas convencionais PAT (OPO-PAT) baseados em osciladores paramétricos convencionais são volumosos e caros e não podem fornecer imagens de alta velocidade. Recentemente, os díodos a laser pulsado (PLDs) foram demonstrados com sucesso como uma fonte de excitação alternativa para PAT. O DIP de laser pulsado PAT (PLD-PAT) foi demonstrado com sucesso para imagens de alta velocidade em fantasmas fotoacústicos e tecidos biológicos. Este trabalho fornece um protocolo experimental visualizado para imagens cerebrais in vivo usando PLD-PAT. O protocolo inclui a configuração compacta do sistema PLD-PAT e sua descrição, preparação animal para imagens cerebrais e um procedimento experimental típico para imagens cerebrais de ratos 2D de seção transversal. O sistema PLD-PAT é compacto e custo-efeEficaz e pode fornecer imagens de alta velocidade e alta qualidade. Imagens de cérebro coletadas in vivo a várias velocidades de varredura são apresentadas.

Os resultados de imagens cerebrais in vivo que demonstram as capacidades do sistema PLD-PAT descrito são exibidos nesta seção. Para demonstrar as capacidades de imagem de alta velocidade do sistema PLD-PAT, realizou-se a imagem cerebral in vivo de dois ratos saudáveis ​​diferentes. A Figura 2 mostra as imagens cerebrais de um rato fêmea (93 g) a várias velocidades de varredura. As figuras 2a e b mostr...

Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

É demonstrado um sistema compacto de tomografia fotoacústica baseada em diodos a laser pulsado (PLD-PAT) para imagens cerebrais in vivo de alta velocidade em pequenos animais.

Um sistema de tomografia fotoacústica compacta de alto desempenho para<em&gt; In Vivo</em&gt; Imagem de cérebro de animais pequenos

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

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Research

JoVE Journal

Bioengineering

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

10.4K visualizações.  Nanyang Technological University.  É demonstrado um sistema compacto de tomografia fotoacústica baseada em diodos a laser pulsado (PLD-PAT) para imagens cerebrais in vivo de alta velocidade em pequenos animais. 

Vídeo: A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

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

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Tomografia Computadorizada guiada por tomografia difusa no domínio do tempo de fluorescência em pequenos animais para localização de biomarcadores de câncer

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicina

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universal de mão tridimensional Optoacoustic imagem Sonda para Deep Tissue Angiografia Humano e Estudos pré-clínicos funcionais em Tempo Real

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Parênquima não-invasivo, Vascular e Metabólica alta freqüência ultra-som e Fotoacústica Rat Cerebral Profunda Imagem

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neurociência

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Microscopia fotoacústica de resolução acústica e óptica comutável para<em&gt; In Vivo</em&gt; Imagem de Vasculatura de Sangue de Pequeno Animal

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Hand-Held fotoacústico clínico sistema de imagem para imagens de animais em tempo real não-invasiva pequenas

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

Tomografia fotoacústica de desktop com base em diodo laser pulsada para monitoramento de lavagem e lavagem de corante em vasculatura cortical de ratos

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imagem fotoacoustic de pequeno animal de varredura dupla raster-scaning para visualização vascular

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

Um sistema de neuronação estereática guiada por imagem de alta produtividade e sistema de ultrassom focado para abertura de barreira hemencefálica em roedores

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Imagem integrada não invasiva de todo o cérebro PAUSAT de acidente vascular cerebral isquêmico

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Autor em destaque: Tomografia fotoacústica, ultrassom e angiográfica integrada (PAUSAT) para imagens não invasivas de todo o cérebro de AVC isquêmico  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Fotoacústica, Ultrassonografia e Angiotomografia Integradas (PAUSAT) para Imagem Não Invasiva do Cérebro Inteiro do Acidente Vascular Cerebral Isquêmico

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

Um sistema de tomografia fotoacústica compacta de alto desempenho para

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