Os autores gostariam de reconhecer o apoio financeiro da Fundação Nacional de Ciência Natural da China (61822505; 11774101; 61627827; 81630046), O Projeto de Planejamento científico e tecnológico da província de Guangdong, China (2015B020233016) e o Programa de Ciência e Tecnologia de Guangzhou (Nº 2019020001).

Todos os experimentos em animais foram realizados em conformidade com as diretrizes fornecidas pelo comitê institucional de cuidados e uso de animais da Universidade Normal do Sul da China, Guangzhou, China.
1. Configuração do sistema
<ol><li>Caminho óptico(Figura 1)<ol>
<li>Use um laser de pulso de 532 nm como fonte de laser do sistema. Defina a taxa de repetição do laser para 10 kHz, a energia de saída para 100% e a configuração do gatilho externo para o gatilho externo usando um programa definido pelo usuário.</li>
		<li>Acople o raio laser à fibra de modo único (SMF) através de um acoalador de fibra óptica (FC1). Cobrem o raio laser usando um collimador de fibra óptica (FC2) em um estágio motorizado bidimensional (Motor, velocidade máxima: 20 mm/s).</li>
		<li>Desvie o raio laser usando um scanner galvanômetro de dois eixos (Galva). Use um espelho movevel (M1) para refletir o feixe. Concentre o feixe através de uma lente objetiva de 4× (OL, abertura numérica: 0,1).</li>
		<li>Use um suporte de tradutor XY (TM) para corrigir o transdutor ultrassônico oco auto-fabricado (UT, frequência central: 25 MHz; Largura de banda: mais de 90%; Orifício central: 3 mm) na parte inferior do OL12. Passe o feixe focado através do buraco central do transdutor ultrassônico.</li>
	</ol>
</li>
	<li>Caminho de digitalização<ol>
<li>Bloqueie a Galva usando uma matriz de portão programável de campo (FPGA 2) durante o WIM. Defina o intervalo de varredura apropriado e a velocidade de digitalização por um programa definido pelo usuário.</li>
		<li>Bloqueie o motor usando uma matriz de portão programável de campo (FPGA 1) durante o RIM. Defina a frequência de digitalização e o número de pontos de varredura usando FPGA 2. Use um programa definido pelo usuário para controlar o início e parar a varredura.</li>
	</ol>
</li>
	<li>Aquisição de dados<ol>
<li>Use um amplificador de 50 dB (AMP) para amplificar o sinal PA. Digitalizar o sinal pelo cartão de aquisição de dados (DAQ). Obtenha o sinal de gatilho através de FPGA 1 ou FPGA 2.</li>
		<li>Use uma unidade de processamento gráfica (GPU) para processar dados e exibir imagens em paralelo13.</li>
	</ol>
</li>
	<li>Sistema de imagem CCD<ol>
<li>Use um LED branco em forma de anel (Temperatura da cor: 6500 K; Illuminance: 40000 lux; Diâmetro: 7,5 cm) como fonte de iluminação. Remova M1, use um espelho fixo (M2) para refletir a luz.</li>
		<li>Registo as imagens usando uma câmera CCD (6,3 milhões de pixels) no sistema de imagem PA. Exibir as imagens com um software de exibição.</li>
	</ol>
</li>
</ol>2. Alinhamento do sistema
<ol><li>Selecione um reservatório de água (10 cm × 10 cm × 4,4 cm; janela inferior: 3 cm × 3 cm). Cubra todo o reservatório de água usando uma membrana de polietileno (membrana grossa: 10 μm). Adicione água ultrauso suficiente.</li>
	<li>Coloque o reservatório de água na etapa de trabalho.</li>
	<li>Ligue o interruptor laser. Selecione o programa de controle a laser. Pré-aqueça por 5 minutos. Pressione o botão "ON" no interruptor de bombeamento. Defina os parâmetros de laser conforme a etapa 1.1.1. Abra o defletor do laser.</li>
	<li>Selecione o programa coletado da linha A. Pressione o botão "Iniciar" para capturar o sinal de ponto único e exibir amplitude e espectro do sinal atual da linha A.</li>
	<li>Coloque uma lâmina no fundo do reservatório de água. Mergulhe a parte inferior da UT no reservatório de água para acoplamento acústico. Evite bolhas na parte inferior da UT.</li>
	<li>Ajuste a posição de Galva, ajuste o tradutor XY entre UT e OL para evitar sinal de oscilação e certifique-se de que isso seja confocal.</li>
	<li>Ajuste a altura do estágio de trabalho para maximizar a amplitude do sinal e determine a posição de foco.</li>
</ol>3. Experimento animal
<ol><li>Use um mouse BALB/c de 5\u20126 semanas de idade com um peso corporal de 20\u201230 g.</li>
	<li>Anestesiar o animal usando uretano (1 g/kg) injetado intraperitoneally antes do experimento.</li>
	<li>Realizar transição entre WIM e RIM.<ol>
<li>Use um transdutor ultrassônico planar. Raspe a pele na parte de trás do mouse usando um aparador e creme depilatório. Coloque o mouse no suporte (8 cm × 2,8 cm × 2 cm) na posição propensa.</li>
		<li>Permita que a região de imagem esteja em contato com a membrana de polietileno usando gel de ultrassom. Evite bolhas na parte de contato.</li>
		<li>Coloque o suporte no estágio de trabalho para acoplamento acústico. Siga os passos 2.3\u20122.4 para iniciar o laser e coletar o sinal de linha A. Siga os passos 2.6\u20122.7 para alinhar. Pressione "Pare" para terminar a coleção após o alinhamento.</li>
		<li>Selecione o programa WIM. Nomeie a pasta recém-criada. Defina o parâmetro de digitalização em 20 mm/s na guia "Velocidade de varredura", "20 mm*20 mm" na guia "Área de varredura" e "20" na guia "Passo". Clique no botão Coletar para iniciar a varredura.</li>
		<li>Clique no botão Parar para finalizar a digitalização após a aquisição. Clique em Devolver a Zero para trazer o Motor a zero. Feche o defletor do laser. Ajuste a configuração do gatilho para o gatilho interno. Pressione o botão OFF para o interruptor de bombeamento.</li>
		<li>Substitua o gatilho WIM como gatilho RIM e conecte-o ao gatilho externo do laser. Pressione o botão ON para o interruptor de bombeamento. Ajuste a configuração do gatilho para o gatilho externo. Clique no botão Sair para sair do programa WIM.</li>
		<li>Use a etapa 2.4 para coletar o sinal da linha A. Abra o defletor do laser. Siga os passos 2.6\u20122.7 para alinhar. Pressione Pare para finalizar a coleta após o alinhamento.</li>
		<li>Selecione o programa RIM. Nomeie a pasta recém-criada. Clique no botão Coletar para iniciar a varredura.</li>
		<li>Clique no botão Parar para finalizar a digitalização após concluir a aquisição. Clique no botão Sair para sair do programa RIM.</li>
		<li>Eutanize o animal usando luxação cervical na conclusão da imagem.</li>
	</ol></li>
	<li>Conduzir WIM de visualização vascular.<ol>
<li>Use um transdutor ultrassônico focado (frequência central: 25 MHz; Largura de banda: mais de 90%; Distância focal: 8 mm). Remova o cabelo da orelha ou couro cabeludo dos ratos.<ol>
<li>Use um bisturi para fazer uma pequena incisão no lado lateral da parte superior temporal craniana do mouse (profundidade para o crânio). Use uma tesoura oftálmica para começar a partir desta incisão. Corte o couro cabeludo ao redor do lado externo do crânio. Comprimir o ponto de sangramento para parar de sangrar. Lave a ferida com soro fisiológico normal. Coloque o mouse no suporte.</li>
		</ol>
</li>
		<li>Permita que a região de imagem esteja em contato com a membrana de polietileno usando gel de ultrassom. Evite bolhas na região de contato(Figura Complementar 1).</li>
		<li>Coloque o suporte no palco de trabalho para acoplamento acústico. Use as etapas 2.3\u20122.4 para abrir o laser e coletar sinal de linha A. Use a etapa 2.6\u20122.7 para alinhar. Pressione Pare para finalizar a coleta após o alinhamento.</li>
		<li>Selecione o programa WIM. Nomeie a pasta recém-criada. Defina o parâmetro de digitalização como "10 mm/s" na guia "Velocidade de varredura", "10 mm*10 mm" sob a guia "Área de varredura" e "10" na guia "Passo". Clique no botão Coletar para iniciar a varredura.</li>
		<li>Clique no botão Parar para finalizar a digitalização após concluir a aquisição. Clique no Return to Zero para fazer o Motor voltar a zero. Clique no botão Sair para sair do programa WIM.</li>
		<li>Eutanize o animal na conclusão do procedimento enquanto o animal ainda está sob anestesia.</li>
	</ol></li>
	<li>Conduzir RIM para monitoramento dinâmico de animais de pequeno porte.<ol>
<li>Raspe o cabelo do abdômen do rato. Coloque o mouse sobre o suporte na posição supina.</li>
		<li>Permita que a região de imagem esteja em contato com a membrana de polietileno usando gel de ultrassom. Evite bolhas na região de contato.</li>
		<li>Coloque o suporte no palco de trabalho para acoplamento acústico. Execute as etapas 2.3\u20122.4 para iniciar o laser e coletar o sinal de linha A. Execute a etapa 2.6\u20122.7 para alinhar. Pressione Pare para finalizar a coleta após o alinhamento.</li>
		<li>Selecione o programa RIM. Nomeie a pasta recém-criada. Clique no botão Coletar para iniciar a varredura.</li>
		<li>Clique no botão Parar para finalizar a digitalização após concluir a aquisição. Clique no botão Sair para sair do programa RIM.</li>
	</ol></li>
	<li>Use os dados RIM para reconstrução da projeção de amplitude máxima (MAP) ao longo da direção de profundidade pelo programa definido pelo usuário. Observe as mudanças dinâmicas no animal.</li>
</ol>

<ol>
	<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-impulse+panoramic+photoacoustic+computed+tomography+of+small-animal+whole-body+dynamics+at+high+spatiotemporal+resolution.">Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution.</a> Nature Biomedical Engineering. 1 (5), 0071 (2017).</li><li>Jeon, S., Kim, J., Lee, D., Baik, J. W., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+practical+photoacoustic+microscopy.">Review on practical photoacoustic microscopy.</a> Photoacoustics. 15, 100141 (2019).</li><li>Baik, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super+wide-field+photoacoustic+microscopy+of+animal+and+humans+in+vivo.">Super wide-field photoacoustic microscopy of animal and humans in vivo.</a> IEEE Transactions on Medical Imaging. 39 (4), 975-984 (2019).</li><li>Omar, M., Aguirre, J., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optoacoustic+mesoscopy+for+biomedicine.">Optoacoustic mesoscopy for biomedicine.</a> Nature Biomedical Engineering. 3 (5), 354-370 (2019).</li><li>Lin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-breath-hold+photoacoustic+computed+tomography+of+the+breast.">Single-breath-hold photoacoustic computed tomography of the breast.</a> Nature Communications. 9 (1), 2352 (2018).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+monitoring+and+real-time+local+recoding+microvascular+networks+on+small+animals+with+a+dual-raster-scanned+photoacoustic+microscope.">Wide-field monitoring and real-time local recoding microvascular networks on small animals with a dual-raster-scanned photoacoustic microscope.</a> Journal of Biophotonics. 13 (6), 202000022 (2020).</li><li>Sun, J., Zhou, Q., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+photoacoustic+imaging+guided+sclerotherapy+for+vascular+malformations:+a+feasibility+study.">Label-free photoacoustic imaging guided sclerotherapy for vascular malformations: a feasibility study.</a> Optics Express. 26 (4), 4967-4978 (2018).</li><li>Xu, D., Yang, S., Wang, Y., Gu, Y., Xing, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+and+high-resolving+photoacoustic+dermoscopy+of+human+skin.">Noninvasive and high-resolving photoacoustic dermoscopy of human skin.</a> Biomedical Optics Express. 7 (6), 2095-2102 (2016).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+photoacoustic+probe+for+in+vivo+imaging+of+subcutaneous+microvessels+within+human+skin.">Miniaturized photoacoustic probe for in vivo imaging of subcutaneous microvessels within human skin.</a> Quantitative Imaging in Medicine and Surgery. 9 (5), 807-814 (2019).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultracompact+high-resolution+photoacoustic+microscopy.">Ultracompact high-resolution photoacoustic microscopy.</a> Optics Letters. 43 (7), 1615-1618 (2018).</li><li>Lan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+widefield+photoacoustic+microscopy+of+small-animal+hemodynamics.">High-speed widefield photoacoustic microscopy of small-animal hemodynamics.</a> Biomedical Optics Express. 9, 4689-4700 (2018).</li><li>Ma, H., Yang, S., Cheng, Z., Xing, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+confocal+dermoscope+with+a+waterless+coupling+and+impedance+matching+opto-sono+probe.">Photoacoustic confocal dermoscope with a waterless coupling and impedance matching opto-sono probe.</a> Optics Letters. 42 (12), 2342-2345 (2017).</li><li>Kang, H., Lee, S. W., Lee, E. S., Kim, S. H., Lee, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+GPU-accelerated+processing+and+volumetric+display+for+wide-field+laser-scanning+optical-resolution+photoacoustic+microscopy.">Real-time GPU-accelerated processing and volumetric display for wide-field laser-scanning optical-resolution photoacoustic microscopy.</a> Biomedical Optics Express. 6 (12), 4650-4660 (2015).</li></ol>

Todos os experimentos em animais foram realizados de acordo com as diretrizes e regulamentos aprovados do Comitê Institucional de Cuidado e Uso de Animais. Os autores não têm interesses financeiros relevantes no manuscrito e nenhum outro potencial conflito de interesse para divulgar.

Aqui apresentamos um biomedusor fotoacâmico de pequeno animal para visualização vascular não invasiva que foi projetado e desenvolvido para capturar a estrutura da vasculatura e a mudança dinâmica relacionada do sangue. A vantagem do DRS-PAI é que ele integra o WIM e o RIM em um sistema, o que facilita o estudo da estrutura de rede vascular dinâmica e vascular de pequenos animais. O sistema pode fornecer visualização vascular de campo amplo de alta resolução e dinâmica sanguínea em tempo real.
<p class=...

No campo biomédico básico, pequenos animais podem simular a função fisiológica humana. Portanto, a imagem de pequenos animais desempenha um papel importante na orientação da pesquisa de doenças homólogas humanas e na busca de um tratamento eficaz1. A imagem fotoacâmica (PAI) é uma técnica de imagem não invasiva que combina as vantagens da imagem óptica e da imagem de ultrassom2. A microscopia fotoacústica (PAM) é um valioso método de imagem para pesquisa básica de animais de pequeno porte3. Pam pode facilmente obter imagens de alta resolução, penetração profunda, alta especificidade e alto contraste com base na excitação óptica e detecção de ultrassom4.
Um laser de pulso com um comprimento de onda específico é absorvido por cromóforos endógenos de tecidos. Posteriormente, a temperatura do tecido sobe, o que resulta na produção de ondas ultrassônicas induzidas por foto. As ondas ultrassônicas podem ser detectadas por um transdutor ultrassônico. Após aquisição de sinal e reconstrução de imagem, a distribuição espacial do absorvente pode ser obtida5. Por um lado, a visualização da rede vascular de órgãos inteiros requer um amplo campo de visão. O processo de varredura em larga área geralmente leva muito tempo para garantir alta resolução6,7,8. Por outro lado, observar as atividades hemodinâmicas de pequenos animais requer imagens rápidas em tempo real. A imagem em tempo real é benéfica para estudar os sinais vitais de pequenos animais em tempo real9,10,11. O campo de visão da imagem em tempo real geralmente é suficientemente pequeno para garantir uma alta taxa de atualização. Assim, muitas vezes há uma troca entre alcançar um amplo campo de visão e imagens em tempo real. Anteriormente, dois sistemas diferentes eram usados para imagens em larga campo ou imagens em tempo real, separadamente.
Este trabalho relata um imager fotoacústico de varredura dupla (DRS-PAI), que integrou imagens de campo largo com base em um estágio de tradução motorizada bidimensional e imagens em tempo real baseadas em um scanner galvanômetro de dois eixos. O modo de imagem de campo largo (WIM) é realizado para mostrar morfologia vascular. Para o modo de imagem em tempo real (RIM), existem atualmente duas funções. Primeiro, o RIM pode fornecer imagens de varredura B em tempo real. Medindo o deslocamento da vasculatura ao longo da direção de profundidade, as características da respiração ou pulso podem ser reveladas. Em segundo lugar, o RIM pode medir quantitativamente a área específica na imagem WIM. Ao fornecer imagens comparáveis das regiões locais de WIM, os detalhes da mudança local podem ser revelados com precisão. O sistema projeta uma transição flexível entre imagens de campo amplo de visualização vascular e imagens em tempo real da dinâmica local. O sistema é desejável em pesquisas biomédicas básicas onde há necessidade de imagens de pequenos animais.

<table>
	<tbody>
		<tr>
			<td>12 bit multi-purpose digitizer</td>
			<td>Spectrum</td>
			<td>M3i.3221</td>
			<td>Data acquisition card</td>
		</tr>
		<tr>
			<td>A-line collected program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>RF Bay</td>
			<td>LNA-650</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Veet</td>
			<td>33-II</td>
			<td>Animal depilatory</td>
		</tr>
		<tr>
			<td>Fiberport Coupler</td>
			<td>Thorlab</td>
			<td>PAF-X-7-A</td>
			<td>Fiber Coupler</td>
		</tr>
		<tr>
			<td>Field Programmable Gate Array</td>
			<td>Altera</td>
			<td>Cyclone IV</td>
			<td>Trigger Control</td>
		</tr>
		<tr>
			<td>Fixed Focus Collomation Packages</td>
			<td>Thorlabs</td>
			<td>F240FC-532</td>
			<td>Fiber Collimator</td>
		</tr>
		<tr>
			<td>Foused ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics Processing Unit</td>
			<td>NVIDIA</td>
			<td>GeForce GTX 1060</td>
			<td>Processing data</td>
		</tr>
		<tr>
			<td>Holder</td>
			<td>Self-made</td>
			<td></td>
			<td>Animal fixation</td>
		</tr>
		<tr>
			<td>Laser control program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Guangdong Medical Laboratory Animal Center</td>
			<td>BALB/c</td>
			<td>Animal Model</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Mshot</td>
			<td>MS60</td>
			<td>CCD camera</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Daheng Optics</td>
			<td>GCO-2111</td>
			<td>Objective Lens</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Daheng Optics</td>
			<td>GCC-1011</td>
			<td>Moveable/Fixed Mirror</td>
		</tr>
		<tr>
			<td>Moving Magnet Capacitive Detector Galvanometer Scanner</td>
			<td>Century Sunny</td>
			<td>S8107</td>
			<td>real-time scanner</td>
		</tr>
		<tr>
			<td>Mshot image analysis system</td>
			<td>Mshot</td>
			<td></td>
			<td>Display software</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CR DOUBLE-CRANE</td>
			<td>H34023609</td>
			<td>Normal Saline</td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>SUJIE</td>
			<td></td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Planar ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>HJSJLSL</td>
			<td></td>
			<td>Polyethylene Membrane</td>
		</tr>
		<tr>
			<td>Program Control Software</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined Program</td>
		</tr>
		<tr>
			<td>Pulsed Q-swithched Laser</td>
			<td>Laser-export</td>
			<td>DTL-314QT</td>
			<td>532-nm pulse Laser</td>
		</tr>
		<tr>
			<td>Real-time imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Ring-shaped white LED</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Codos</td>
			<td>CP-9200</td>
			<td>Animal Shaver</td>
		</tr>
		<tr>
			<td>Single-Mode Fibers</td>
			<td>Nufern</td>
			<td>460-HP</td>
			<td>Single-mode fiber</td>
		</tr>
		<tr>
			<td>Surgical Blade</td>
			<td>SUJIE</td>
			<td>11</td>
			<td>Blade</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>SUJIE</td>
			<td>7</td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Translation Stage</td>
			<td>Jiancheng Optics</td>
			<td>LS2-25T</td>
			<td>wide-field scanning stage</td>
		</tr>
		<tr>
			<td>Ultrasonic Transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>GUANGGONG PAI</td>
			<td>ZC4252418</td>
			<td>Acoustic Coupling</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Tokyo Chemical Industry</td>
			<td>C0028</td>
			<td>Animal Anestheized</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>XY Translator Mount</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dual raster-scanning photoacoustic small-animal imager for vascular visualization

A imagem de redes vasculares em animais de pequeno porte tem desempenhado um papel importante na pesquisa biomédica básica. A tecnologia de imagem fotoacoustic tem grande potencial para aplicação na imagemologia de pequenos animais. A imagem fotoacâmica de campo largo de pequenos animais pode fornecer imagens com alta resolução espástica, penetração profunda e múltiplos contrastes. Além disso, o sistema de imagem fotoacâmica em tempo real é desejável observar as atividades hemodinâmicas da vasculatura de pequenos animais, que podem ser usadas para pesquisar o monitoramento dinâmico de características fisiológicas de pequenos animais. Aqui, um imager fotoacoustic de varredura dupla é apresentado, apresentando uma função de imagem de modo duplo comutavel. A imagem de campo largo é conduzida por um estágio de tradução motorizada bidimensional, enquanto a imagem em tempo real é realizada com galvanômetros. Ao definir diferentes parâmetros e modos de imagem, a visualização in vivo da rede vascular de pequenos animais pode ser realizada. A imagem em tempo real pode ser usada para observar a mudança de pulso e a mudança de fluxo sanguíneo de induzida por drogas, etc. A imagem de campo largo pode ser usada para acompanhar a mudança de crescimento da vasculatura tumoral. São fáceis de serem adotados em diversas áreas da pesquisa básica de biomedicina.

O esquema do DRS-PAI é mostrado na Figura 1. O sistema permite uma comutação flexível e repetível entre WIM e RIM. O sinal PA adquirido é processado rapidamente para gerar imagens PA B-Scan e MAP. A câmera CCD pode fornecer fotografias de amostras.
Todos os componentes do DRS-PAI são integrados e montados em uma configuração de imager(Figura 2),facilitando a montagem e opere. No WIM, é utilizada a varredura contínua de ras...

Watch this Scientific Journal Video about Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization at JoVE.com

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

Foi projetado um imager fotoacoustic de varredura dupla rastering, que integrou imagens de campo amplo e imagens em tempo real.

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 ...

dual-raster-scanning-photoacoustic-small-animal-imager-for-vascular

Research

JoVE Journal

Bioengineering

4.0K visualizações.  South China Normal University. Foi projetado um imager fotoacoustic de varredura dupla rastering, que integrou imagens de campo amplo e imagens em tempo real.

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

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Detecção e isolamento de células de melanoma circulantes usando Fluxometria Fotoacústica

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Oftalmoscopia Fotoacústica integrada e Spectral-domínio Tomografia de Coerência Óptica

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Cistografia fotoacústica

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Usando impedância Cell-substrato e imagens de células vivas para medir as mudanças em tempo real no celular Adesão e De-adesão Induzidas por Matrix Modificação

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

The Loop arteriovenosa (AV) em um modelo animal pequeno para estudar angiogénese e Engenharia de tecido vascularizado

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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 ...

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

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

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

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

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 ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Cultura de células de hidrogel ultrafinos Porated elástico como uma membrana do porão biomimético para Dual

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

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

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