A los autores les gustaría reconocer el apoyo financiero de la Fundación Nacional de Ciencias Naturales de China (61822505; 11774101; 61627827; 81630046), El Proyecto de Planificación científica y tecnológica de la provincia de Guangdong, China (2015B020233016) y El Programa de Ciencia y Tecnología de Guangzhou (Nº 2019020001).

Todos los experimentos con animales se realizaron de conformidad con las directrices proporcionadas por el comité institucional de cuidado y uso de animales de la Universidad Normal del Sur de China, Guangzhou, China.
1. Configuración del sistema
<ol><li>Trayectoria óptica (Figura 1)<ol>
<li>Utilice un láser de pulso de 532 nm como fuente láser del sistema. Establezca la tasa de repetición del láser en 10 kHz, la energía de salida en 100% y la configuración del disparador en disparador externo mediante un programa definido por el usuario.</li>
		<li>Acopla el rayo láser a la fibra monomodo (SMF) a través de un acoplador de fibra óptica (FC1). Coteja el rayo láser utilizando un colimador de fibra óptica (FC2) en una etapa motorizada bidimensional (Motor, velocidad máxima: 20 mm/s).</li>
		<li>Desvíe el rayo láser utilizando un escáner de galvanómetro de dos ejes (Galva). Utilice un espejo movible (M1) para reflejar la viga. Enfoque el haz a través de una lente objetiva de 4× (OL, apertura numérica: 0.1).</li>
		<li>Utilice un soporte de traductor XY (TM) para fijar el transductor ultrasónico hueco hecho a sí mismo (UT, frecuencia central: 25 MHz; Ancho de banda: más del 90%; Orificio central: 3 mm) en la parte inferior del OL12. Pase el haz enfocado a través del agujero central del transductor ultrasónico.</li>
	</ol>
</li>
	<li>Ruta de escaneo<ol>
<li>Bloquee el Galva utilizando una matriz de compuerta programable en campo (FPGA 2) durante WIM. Establezca el rango de escaneo y la velocidad de escaneo adecuados mediante un programa definido por el usuario.</li>
		<li>Bloquee el motor utilizando una matriz de compuerta programable en campo (FPGA 1) durante RIM. Establezca la frecuencia de escaneo y el número de puntos de escaneo mediante FPGA 2. Utilice un programa definido por el usuario para controlar el inicio y detener el análisis.</li>
	</ol>
</li>
	<li>Adquisición de datos<ol>
<li>Utilice un amplificador de 50 dB (AMP) para amplificar la señal PA. Digitalice la señal por la tarjeta de adquisición de datos (DAQ). Obtenga la señal del gatillo a través de FPGA 1 o FPGA 2.</li>
		<li>Utilice una unidad de procesamiento de gráficos (GPU) para procesar datos y mostrar imágenes en paralelo13.</li>
	</ol>
</li>
	<li>Sistema de imágenes CCD<ol>
<li>Utilice un LED blanco en forma de anillo (Temperatura de color: 6500 K; Iluminancia: 40000 lux; Diámetro: 7,5 cm) como fuente de iluminación. Retire M1, utilice un espejo fijo (M2) para reflejar la luz.</li>
		<li>Grabe las imágenes con una cámara CCD (6,3 millones de píxeles) en el sistema de imágenes pa. Visualice las imágenes con un software de visualización.</li>
	</ol>
</li>
</ol>2. Alineación del sistema
<ol><li>Seleccione un depósito de agua (10 cm × 10 cm × 4,4 cm; ventana inferior: 3 cm × 3 cm). Cubra todo el depósito de agua utilizando una membrana de polietileno (membrana gruesa: 10 μm). Agregue suficiente agua ultrapura.</li>
	<li>Coloque el tanque de agua en el escenario de trabajo.</li>
	<li>Encienda el interruptor láser. Seleccione el programa de control láser. Precalentamiento durante 5 minutos. Pulse el botón "ON" del interruptor de bombeo. Establezca los parámetros láser según el paso 1.1.1. Abra el deflector del láser.</li>
	<li>Seleccione el programa recopilado de línea A. Pulse el botón "Inicio" para capturar la señal de un solo punto y mostrar la amplitud y el espectro de la señal de línea A actual.</li>
	<li>Coloque una cuchilla en la parte inferior del tanque de agua. Sumerja la parte inferior de UT en el tanque de agua para el acoplamiento acústico. Evite burbujas en la parte inferior de UT.</li>
	<li>Ajuste la posición de Galva, ajuste el traductor XY entre UT y OL para evitar la señal de oscilación, y asegúrese de que esto es confocal.</li>
	<li>Ajuste la altura de la etapa de trabajo para maximizar la amplitud de la señal y determine la posición de enfoque.</li>
</ol>3. Experimento con animales
<ol><li>Utilice un ratón BALB/c de 5\u20126 semanas de edad con un peso corporal de 20\u201230 g.</li>
	<li>Anestesiar al animal usando uretano (1 g/kg) inyectado por vía intraperitoneally antes del experimento.</li>
	<li>Realice la transición entre WIM y RIM.<ol>
<li>Utilice un transductor ultrasónico plano. Afeita el pelaje en la parte posterior del ratón con una recortadora y una crema depilatoria. Coloque el ratón sobre el soporte (8 cm × 2,8 cm × 2 cm) en posición propensa.</li>
		<li>Permita que la región de imágenes esté en contacto con la membrana del polietileno mediante gel de ultrasonido. Evite burbujas en la parte de contacto.</li>
		<li>Coloque el soporte en el escenario de trabajo para el acoplamiento acústico. Siga los pasos 2.3\u20122.4 para iniciar el láser y recoger la señal de línea A. Siga los pasos 2.6\u20122.7 para alinear. Pulse "Detener" para finalizar la colección después de la alineación.</li>
		<li>Seleccione el programa WIM. Asigne un nombre a la carpeta recién creada. Establezca el parámetro de escaneo en 20 mm/s en la pestaña "Velocidad de escaneo", "20 mm * 20 mm" en la pestaña "Área de escaneo" y "20" en la pestaña "Paso". Haga clic en el botón Recopilar para iniciar la exploración.</li>
		<li>Haga clic en el botón Detener para finalizar la exploración después de la adquisición. Haga clic en Volver a cero para poner el motor a cero. Cierre el desconcierto del láser. Establezca la configuración del desencadenador en desencadenador interno. Pulse el botón OFF para el interruptor de bombeo.</li>
		<li>Reemplace el disparador WIM como disparador RIM y conéctelo al disparador láser externo. Pulse el botón ON para el interruptor de bombeo. Establezca la configuración del desencadenador en el desencadenador externo. Haga clic en el botón Salir para salir del programa WIM.</li>
		<li>Utilice el paso 2.4 para recoger la señal de línea A. Abra el deflector láser. Siga los pasos 2.6\u20122.7 para alinear. Pulse Detener para finalizar la recopilación después de la alineación.</li>
		<li>Seleccione el programa RIM. Asigne un nombre a la carpeta recién creada. Haga clic en el botón Recopilar para iniciar la exploración.</li>
		<li>Haga clic en el botón Detener para finalizar la exploración después de completar la adquisición. Haga clic en el botón Salir para salir del programa RIM.</li>
		<li>Eutanasiar al animal usando dislocación cervical al final de la toma de imágenes.</li>
	</ol></li>
	<li>Realizar WIM de visualización vascular.<ol>
<li>Utilice un transductor ultrasónico enfocado (frecuencia central: 25 MHz; Ancho de banda: más del 90%; Distancia focal: 8 mm). Retire el cabello de ratones oreja o cuero cabelludo.<ol>
<li>Utilice un bisturí para hacer una pequeña incisión en el lado lateral de la parte superior temporal craneal del ratón (profundidad al cráneo). Utilice tijeras oftálmicas para comenzar a partir de esta incisión. Corta el cuero cabelludo alrededor del lado externo del cráneo. Comprima el punto de sangrado para detener el sangrado. Lave la herida con solución salina normal. Coloque el ratón en el soporte.</li>
		</ol>
</li>
		<li>Permita que la región de imágenes esté en contacto con la membrana del polietileno mediante gel de ultrasonido. Evite burbujas en la región de contacto(Figura suplementaria 1).</li>
		<li>Coloque el soporte en el escenario de trabajo para el acoplamiento acústico. Utilice los pasos 2.3\u20122.4 para abrir el láser y recoger la señal de línea A. Utilice el paso 2.6\u20122.7 para alinear. Pulse Detener para finalizar la recopilación después de la alineación.</li>
		<li>Seleccione programa WIM. Asigne un nombre a la carpeta recién creada. Establezca el parámetro de escaneo en "10 mm/s" en la pestaña "Velocidad de escaneo", "10 mm * 10 mm" bajo la pestaña "Área de escaneo" y "10" en la pestaña "Paso". Haga clic en el botón Recopilar para iniciar la exploración.</li>
		<li>Haga clic en el botón Detener para finalizar la exploración después de completar la adquisición. Haga clic en El retorno a cero para hacer que el motor vuelva a cero. Haga clic en el botón Salir para salir del programa WIM.</li>
		<li>Eutanasiar al animal al final del procedimiento mientras el animal todavía está bajo anestesia.</li>
	</ol></li>
	<li>Lleve a cabo RIM para el monitoreo dinámico de animales pequeños.<ol>
<li>Afeita el cabello del abdomen del ratón. Coloque el ratón en el soporte en posición supina.</li>
		<li>Permita que la región de imágenes esté en contacto con la membrana del polietileno mediante gel de ultrasonido. Evite burbujas en la región de contacto.</li>
		<li>Coloque el soporte en el escenario de trabajo para el acoplamiento acústico. Realice los pasos 2.3\u20122.4 para iniciar el láser y recoger la señal de línea A. Realice el paso 2.6\u20122.7 para alinear. Pulse Detener para finalizar la recopilación después de la alineación.</li>
		<li>Seleccione el programa RIM. Asigne un nombre a la carpeta recién creada. Haga clic en el botón Recopilar para iniciar la exploración.</li>
		<li>Haga clic en el botón Detener para finalizar la exploración después de completar la adquisición. Haga clic en el botón Salir para salir del programa RIM.</li>
	</ol></li>
	<li>Utilice los datos rim para la reconstrucción de la proyección de amplitud máxima (MAP) a lo largo de la dirección de profundidad por programa definido por el usuario. Observe los cambios dinámicos en el 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 los experimentos con animales se realizaron de acuerdo con las directrices y reglamentos aprobados del Comité Institucional de Cuidado y Uso de Animales. Los autores no tienen intereses financieros relevantes en el manuscrito ni otros conflictos potenciales de interés que revelar.

Aquí presentamos un imager fotoacústico de animales pequeños de doble escaneo ráster para la visualización vascular no invasiva que fue diseñado y desarrollado para capturar la estructura de la vasculatura y el cambio dinámico relacionado de la sangre. La ventaja de DRS-PAI es que integra el WIM y el RIM en un solo sistema, lo que facilita el estudio de la estructura de red vascular dinámica y vascular de los animales pequeños. El sistema puede proporcionar visualización vascular de campo amplio de alta resoluc...

En el campo biomédico básico, los animales pequeños pueden simular la función fisiológica humana. Por lo tanto, la imagen de los animales pequeños desempeña un papel importante en la orientación de la investigación de las enfermedades homólogas humanas y la búsqueda de un tratamiento eficaz1. La imagen fotoacústica (PAI) es una técnica de imagen no invasiva que combina las ventajas de las imágenes ópticas y las imágenes por ultrasonido2. La microscopía fotoacústica (PAM) es un valioso método de imagen para la investigación básica de animales pequeños3. PAM puede obtener fácilmente imágenes de alta resolución, penetración profunda, alta especificidad y alto contraste basadas en excitación óptica y detección de ultrasonido4.
Un láser de pulso con una longitud de onda específica es absorbido por cromoforos endógenos de los tejidos. Posteriormente, la temperatura del tejido aumenta, lo que resulta en la producción de ondas ultrasónicas inducidas por foto. Las ondas ultrasónicas pueden ser detectadas por un transductor ultrasónico. Después de la adquisición de la señal y la reconstrucción de la imagen, la distribución espacial del absorbente se puede obtener5. Por un lado, la visualización de la red vascular de órgano entero requiere un amplio campo de visión. El proceso de escaneo de campo ancho suele tardar mucho tiempo en garantizar la alta resolución6,7,8. Por otro lado, observar las actividades hemodinámicas de los animales pequeños requiere imágenes rápidas en tiempo real. La imagen en tiempo real es beneficiosa para estudiar los signos vitales de los animales pequeños en tiempo real9,10,11. El campo de visión de las imágenes en tiempo real suele ser lo suficientemente pequeño como para garantizar una alta tasa de actualización. Por lo tanto, a menudo hay un equilibrio entre lograr un amplio campo de visión y imágenes en tiempo real. Anteriormente, se utilizaban dos sistemas diferentes para imágenes de campo amplio o imágenes en tiempo real, por separado.
Este trabajo informa de un imager fotoacústico de escaneo ráster dual (DRS-PAI), que integró imágenes de campo amplio basadas en una etapa de traducción motorizada bidimensional e imágenes en tiempo real basadas en un escáner de galvanómetros de dos ejes. El modo de imagen de campo ancho (WIM) se realiza para mostrar morfología vascular. Para el modo de imagen en tiempo real (RIM), actualmente hay dos funciones. En primer lugar, RIM puede proporcionar imágenes de escaneo B en tiempo real. Al medir el desplazamiento de la vasculatura a lo largo de la dirección de profundidad, se pueden revelar las características de la respiración o el pulso. En segundo lugar, el RIM puede medir cuantitativamente el área específica de la imagen WIM. Al proporcionar imágenes comparables de las regiones WIM locales, los detalles del cambio local se pueden revelar con precisión. El sistema diseña una transición flexible entre imágenes de campo amplio de visualización vascular e imágenes en tiempo real de la dinámica local. El sistema es deseable en la investigación biomédica básica donde hay una necesidad de imágenes de animales pequeños.

<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

La toma de imágenes de redes vasculares en animales pequeños ha desempeñado un papel importante en la investigación biomédica básica. La tecnología de imágenes fotoacústicas tiene un gran potencial de aplicación en la imagenología de animales pequeños. Las imágenes fotoacústicas de campo ancho de animales pequeños pueden proporcionar imágenes con alta resolución espaciotemporal, penetración profunda y múltiples contrastes. Además, el sistema de imágenes fotoacústicas en tiempo real es deseable para observar las actividades hemodinámicas de la vasculatura de animales pequeños, que se puede utilizar para investigar el monitoreo dinámico de las características fisiológicas de los animales pequeños. Aquí, se presenta un imager fotoacústico de escaneo ráster dual, con una función de imagen en doble modo conmutable. La imagen de campo ancho es impulsada por una etapa de traducción motorizada bidimensional, mientras que las imágenes en tiempo real se realizan con galvanómetros. Al establecer diferentes parámetros y modos de imagen, se puede realizar una visualización in vivo de la red vascular de animales pequeños. Las imágenes en tiempo real se pueden utilizar para observar el cambio de pulso y el cambio de flujo sanguíneo de inducido por drogas, etc. Las imágenes de campo amplio se pueden utilizar para realizar un seguimiento del cambio de crecimiento de la vasculatura tumoral. Estos son fáciles de adoptar en varias áreas de la investigación básica de biomedicina.

El esquema del DRS-PAI se muestra en la figura 1. El sistema permite una conmutación flexible y repetible entre WIM y RIM. La señal PA adquirida se procesa rápidamente para generar imágenes PA B-Scan y MAP. La cámara CCD puede proporcionar fotografías de muestras.
Todos los componentes del DRS-PAI se integran y ensamblan en una configuración de imager(Figura 2),lo que facilita el montaje y el funcionamiento. En el WIM, se utili...

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

Se diseñó un imager fotoacústico de escaneo ráster dual, que integró imágenes de campo amplio e imágenes en tiempo real.

Imagen fotoacústica fotoacústica dual para visualización 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 vistas.  South China Normal University. Se diseñó un imager fotoacústico de escaneo ráster dual, que integró imágenes de campo amplio e imágenes en tiempo real.

Video: 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 óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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.

La detección y aislamiento de las células de melanoma circulantes mediante Flujometría 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.

La oftalmoscopia integrado fotoacústica y Spectral-tomografía de coherencia óptica de dominio

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.

Cistografía 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.

Utilizando Impedancia Cell-sustrato y imágenes de células vivas para medir los cambios en tiempo real en la adhesión celular y De-adhesión inducidas por Matrix Modificación

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.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

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.

El asa arteriovenosa (AV) en un modelo de pequeños animales para estudiar la angiogénesis e Ingeniería tejido 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.

Microscopía fotoacústica de resolución óptica y acústica conmutable para<em&gt; In Vivo</em&gt; Imágenes de vasos sanguíneos de pequeños animales

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.

Un sistema de tomografía fotoacústica compacta de alto rendimiento para<em&gt; In Vivo</em&gt; Imágenes de cerebro para animales pequeños

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.

Hidrogeles ultrafino Porated elástico como una membrana del sótano biomiméticos para doble cultura de la célula

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

Imagen fotoacústica fotoacústica dual para visualización vascular

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