La investigación está respaldada por la subvención de Nivel 2 financiada por el Ministerio de Educación de Singapur (ARC2 / 15: M4020238) y el Consejo Nacional de Investigación Médica del Ministerio de Salud de Singapur (NMRC / OFIRG / 0005/2016: M4062012). Los autores desean agradecer al Sr. Chow Wai Hoong Bobby por la ayuda de la tienda de maquinaria.

Todos los experimentos con animales se realizaron de acuerdo con las directrices y reglamentos aprobados por el Comité Institucional de Cuidado y Uso de Animales de la Universidad Tecnológica de Nanyang, Singapur (Número de Protocolo Animal ARF-SBS / NIE-A0263). 1. Descripción del sistema <ol><li> Monte el PLD dentro del escáner circular, como se muestra en la Figura 1a . Conecte el PLD a la unidad de control del láser (LDU). NOTA: El PLD proporciona impulsos ~ 136-ns a una longitud de onda de ~ 803 nm, con una energía de impulso máxima de ~ 1,42 mJ y una tasa de repetición de hasta 7 kHz. La unidad de accionamiento de láser (LDU) incluye: un controlador de temperatura, una fuente de alimentación variable, una fuente de alimentación (12 V) y un generador de funciones; Ver la Tabla de Materiales. La fuente de alimentación variable se utiliza para controlar la potencia del láser, y el generador de funciones se utiliza para cambiar la tasa de repetición del PLD. </li><li> Encienda el láser PLD. Establezca la tasa de repetición de PLD en &quot;7,000&quot; HZ utilizando el generador de funciones en la LDU. Aumentar la energía del pulso a 1,42 mJ ajustando la tensión de la fuente de alimentación variable a &quot;3,1&quot; V. </li><li> Monte el difusor óptico (OD) delante de la ventana de salida PLD para hacer el haz de salida homogéneo, como se muestra en la Figura 1a . NOTA: Utilice un difusor con un grano fino ( es decir, pulimento de grano 1.500). </li><li> Monte la UST enfocada en el soporte UST, de tal manera que se encuentre en el centro del área de exploración, como se muestra en la Figura 1a . NOTA: La frecuencia central de la UST es 2,25 MHz, y la distancia focal es 1,9 pulg. </li><li> Coloque el detector de ultrasonidos dentro del tanque de acrílico, como se muestra en la Figura 1a . Llene el tanque con agua de modo que el UST esté totalmente sumergido. NOTA: Se utiliza un medio acuoso para acoplar la señal fotoacústica del cerebro (muestra) a la UST. Un tanque de agua de acrílico (WT, véase la tabla de materiales) era costumbre-designado para imágenes de animales pequeños. El esquema del diseño del tanque de agua se muestra en la Figura 1b . </li><li> Compruebe la señal de PA de la muestra usando una unidad de pulsador / receptor (PRU, vea la Tabla de Materiales ). NOTA: Estas señales fueron digitalizadas por una tarjeta DAQ de 12 bits (ver la Tabla de Materiales ) a una velocidad de muestreo de 100 MS / s y se guardaron en una computadora. </li></ol> 2. Preparación de animales para imágenes cerebrales de ratas NOTA: Se demostró el sistema PLD-PAT descrito anteriormente para la formación de imágenes de cerebros de animales pequeños. Para estos experimentos, se utilizaron ratas hembra sanas (véase la Tabla de Materiales ). <ol><li> Anestesiar al animal inyectando intraperitonealmente un cóctel de 2 ml de ketamina, 1 ml de xilazina y 1 ml de solución salina (dosificación de 0,2 ml / 100 g). </li><li> Quitar la piel en el cuero cabelludo del animal con una podadora de pelo. Aplicar suavemente la crema depilatoria en el afeitadoPara el agotamiento de la piel. <ol><li> Retire la crema aplicada después de 4-5 min usando un hisopo de algodón. </li><li> Aplicar ungüento desgarrante artificial a los ojos del animal para evitar la sequedad debido a la anestesia y la iluminación láser. </li></ol></li><li> Montar el soporte para animales (ver la Tabla de Materiales ) equipado con una máscara respiratoria (ver la Tabla de Materiales ) en un gato de laboratorio. </li><li> Coloque el animal en posición prona sobre el soporte. Asegúrelo al soporte usando cinta quirúrgica para evitar el movimiento del animal durante la formación de imágenes. </li><li> Asegúrese de que la máscara respiratoria cubre la nariz y la boca de la rata para administrar anestésico inhalado. </li></ol> 3. In Vivo Rat Brain Imaging <ol><li> Conecte la máscara de respiración a la máquina de anestesia. Encienda la máquina de anestesia y colóquela para suministrar 1,0 L / min de oxígeno con isoflurano al 0,75%. <ol><li> Sujete el oxímetro de pulsoS para controlar la condición fisiológica del animal. </li></ol></li><li> Aplicar una capa de gel de ultrasonido incoloro en el cuero cabelludo de la rata. Ajuste la posición del gato de laboratorio al centro del escáner. La máscara de respiración se personaliza para adaptarse a la ventana de imagen. El 10% del cono de la nariz comercialmente disponible se corta y luego se conecta a un trozo de guante. <ol><li> Ajuste la altura del gato de laboratorio manualmente para que el plano de imagen esté en el foco de la UST. </li></ol></li><li> Ajuste los parámetros en el software de adquisición de datos (consulte la Tabla de Materiales) según sea necesario. Ejecute el programa de adquisición de datos para iniciar la adquisición ( es decir, imágenes). NOTA: El programa se utiliza para girar el UST y recoger las señales PA de una línea. Las líneas A recogidas se guardarán en el ordenador. </li><li> Observe al animal durante todo el período de formación de imágenes y proceda a la reconstrucción PAT después de completar la imagen. </li><li> Una vez finalizada la adquisición de datos,Reconstruir la imagen del cerebro transversal de las líneas A utilizando el programa de software de reconstrucción. </li><li> Apague el sistema de anestesia, retire al animal del escenario, devuélvalo a su jaula, y vigírelo hasta que recupere la conciencia. NOTA: Por ejemplo, si el UST se gira durante 5 s, el PLD entrega 35.000 pulsos (= 5 x 7.000) y el UST recoge 35.000 líneas A. Las 35.000 líneas A se reducen a 500 en un promedio de más de 70 señales (después de promediar líneas A = 35.000 / 70 = 500). La figura 1c ilustra la iluminación de los pulsos de láser y la recopilación de líneas A. Se debe utilizar un programa de reconstrucción basado en el algoritmo de retroproyección de demora y suma. </li></ol><img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55811/55811fig1.jpg" /> Figura 1: Esquemas del sistema PLD-PAT. (A) Esquema del PLD-PAT. PLD: diodo láser pulsado, CSP: circuUnidad de impulsión del láser, unidad de pulsador / receptor, UST: transductor de ultrasonido, WT: depósito de agua, PF: película de polímero y DAQ: Tarjeta de adquisición de datos. ( B ) Esquema del tanque de agua, vista superior (1) y vista en sección transversal (2) para imágenes de cerebro de animal pequeño in vivo . A: tornillo métrico, B: placa anular de acrílico, C: anillo de silicona &quot;O&quot;, D: 100 μm de espesor, cubierta de polietileno transparente. El tanque tenıa un orificio de 9 cm de diámetro en su fondo y se selló con una membrana de polietileno de 100 μm de grosor ultrasónica y ópticamente transparente. ( C ) Esquema de la iluminación de pulsos láser de las líneas PLD y A, recogida en un tiempo de exploración continua de 5 s. <a href="http://ecsource.jove.com/files/ftp_upload/55811/55811fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

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Los autores no tienen intereses financieros relevantes en el manuscrito y no hay otros posibles conflictos de interés que revelar.

Este trabajo presenta un protocolo para la realización de imágenes cerebrales in vivo en ratas utilizando un sistema PLD-PAT. El protocolo incluye una descripción detallada del sistema de imagen y su alineación, así como una ilustración de imágenes cerebrales en ratas. Los sistemas de PAT existentes basados ​​en OPO son caros y voluminosos y pueden proporcionar una imagen en sección transversal en 5-10 min. El sistema PLD-PAT es compacto, portátil y de bajo costo y puede proporcionar imág...

<html> La tomografía fotoacústica (PAT) es una modalidad de imagen híbrida que tiene muchas aplicaciones tanto en estudios clínicos como preclínicos 1 , 2 , 3 , 4 , 5 . En PAT, los pulsos láser de nanosecondos irradian tejido biológico. La absorción de la luz incidente por los cromóforos del tejido conduce a un aumento local de la temperatura, que luego produce ondas de presión emitidas en forma de ondas sonoras. Un detector de ultrasonidos recoge las señales fotoacústicas en varias posiciones alrededor de la muestra. Las señales fotoacústicas (PA) se reconstruyen utilizando varios algoritmos (como un algoritmo de retardo y suma) 6 para generar la imagen fotoacústica. Esta modalidad de imagen híbrida ofrece alta resolución, imágenes de tejidos profundos y contraste de absorción óptica alta 7 ,Class = &quot;xref&quot;&gt; 8. Recientemente, se consiguió una profundidad de formación de imágenes de ~ 12 cm en el tejido del pecho de pollo con la ayuda de una longitud de onda más larga (~ 1.064 nm) y un agente de contraste exógeno llamado ftalocianina de fósforo. Esta sensibilidad a la profundidad es mucho mayor que la sensibilidad a la profundidad de otros métodos ópticos, como la microscopía confocal de fluorescencia, la microscopía de fluorescencia de dos fotones, la tomografía de coherencia óptica 11 , etc. Utilizando más de una longitud de onda, PAT puede demostrar cambios estructurales y funcionales en los órganos . Para muchas enfermedades humanas, los modelos de animales pequeños han sido bien establecidos 12 , 13 , 14 , 15 . Para la imagen de animales pequeños, se han demostrado varias modalidades. De todos estos enfoques, la imagen de PA ha ganado atención con bastante rapidez debido a las ventajas mencionadas anteriormente. PensilvaniaT ha demostrado su potencial para la imagen de los vasos sanguíneos en los tejidos y órganos ( es decir, el corazón, los pulmones, el hígado, los ojos, el bazo, el cerebro, la piel, la médula espinal, el riñón, etc ) de los animales pequeños 4 , 16 , 17 , 18 . PAT es una modalidad bien establecida para la imagen de cerebro de animales pequeños. PA ondas se producen debido a la absorción de luz por los cromóforos, por lo que de múltiples longitudes de onda PAT permite la cartografía de la concentración total de hemoglobina (HbT) y saturación de oxígeno (SO 2 ) 19 , 20 , 21 , 22 . La imagen neurovascular del cerebro se logró con la ayuda de agentes de contraste exógenos 12 , 23 , 24 . La modalidad PA puede ayudar a dar una mejor comprensión de la salud del cerebroProporcionando información a nivel molecular y genético. Para la obtención de imágenes de animales pequeños, los láseres Nd: YAG / OPO son ampliamente utilizados como fuentes de excitación PAT. Estos láseres suministran ~ 5 ns pulsos de infrarrojo cercano con energía (~ 100 mJ en la ventana de salida OPO) a una frecuencia de repetición de ~ 10 Hz 25 . El sistema de PA equipado con tales láseres es costoso y voluminoso y permite la formación de imágenes a baja velocidad con transductores de ultrasonido de un solo elemento (UST) debido a la baja tasa de repetición de la fuente láser. Un tiempo típico de adquisición de línea A en tales sistemas de PA es ~ 5 min por sección transversal 25 . Un sistema de imágenes con un tiempo de medición tan largo no es ideal para la formación de imágenes de animales pequeños, ya que es difícil controlar los parámetros fisiológicos para la imagen de todo el cuerpo, imágenes temporales resueltas en el tiempo, etc. , O un láser de alta frecuencia de repetición, es posible aumentar la velocidad de formación de imágenes de PASistemas. El uso de un solo UST de un solo elemento para recoger todas las señales PA alrededor de la muestra limitará la velocidad de imagen del sistema. Múltiples UST de un solo elemento dispuestos en geometría circular o semicircular se demuestran para técnicas de imagen de alta velocidad y altamente sensibles. Los UST 26 basados ​​en matrices, tales como arrays lineales, semicirculares, circulares y volumétricos, se han utilizado con éxito para la obtención de imágenes en tiempo real 1 . Estos USTs basados ​​en array incrementarán la velocidad de imagen y reducirán la sensibilidad de medición, pero son caros. Sin embargo, la velocidad de formación de imágenes de los sistemas de PA que usan USTs basados ​​en array está todavía limitada por la velocidad de repetición del láser. La tecnología de láser pulsado avanzó para producir diodos láser pulsado de alta frecuencia (PLD). 7.000 imágenes / s de imagen B-scan fotoacústica se demostró con PLDs utilizando una plataforma clínica de ultrasonido [ 27] . Tales PLD pueden mejorar la velocidad deE PAT, incluso con la geometría de exploración circular UST de un solo elemento. Los UST de un solo elemento son menos costosos y altamente sensibles, a diferencia de los UST basados ​​en array. Durante la última década, se informó poca investigación sobre el uso de PLDs de alta tasa de repetición como la fuente de excitación para la imagenología PA. Una fibra basada en el infrarrojo cercano-PLD se demostró para la imagen de PA fantasmas [ 28] . La imagen in vivo de los vasos sanguíneos a una profundidad de ~ 1 mm por debajo de la piel humana se demostró utilizando PLD de baja energía [ 29] . Se informó de un microscopio fotoacústico de resolución óptica basado en PLD (ORPAM). Utilizando PLDs, se demostró una imagen de 1,5 cm de profundidad a una velocidad de fotogramas de 0,43 Hz 30 . Muy recientemente, se informó de un sistema PLD-PAT que proporcionó imágenes en tan corto como ~ 3 sy a ~ 2 cm de profundidad de imagen en el tejido biológico [ 25 , 31] . Este estudio demostró que un sistema compacto de bajo costo puede proporcionarIncluso a altas velocidades. El sistema PLD-PAT puede utilizarse para la imagen fotoacústica de alta velocidad de fotogramas (7,000 fps), imágenes de vasos sanguíneos superficiales, imágenes de las articulaciones de los dedos, imágenes de tejidos de 2 cm de profundidad, imágenes cerebrales de animales pequeños, etc. Los pulsos de energía de pulsos bajos de PLD limitan su aplicación a imágenes multi-espectrales y de tejidos profundos. Se han llevado a cabo experimentos en animales pequeños utilizando el mismo sistema PLD-PAT usado para aplicaciones preclínicas. El propósito de este trabajo es proporcionar la demostración experimental visualizada del sistema PLD-PAT para la obtención de imágenes en vivo en 2D en vivo de animales pequeños.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Pulsed laser diode</td>
			<td style="width:195px;">Quantel, France</td>
			<td style="width:327px;">QD-Q1910-SA-TEC</td>
			<td style="width:449px;">It is the excitation laser source with specifications 803 nm, 1.4mJ per pulse, 136 ns pulse, 7kHz maximum, dimentions : 11.0 x 6.0 x 3.6 cm, weight: ~150 gm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Stepper motor with gearbox</td>
			<td style="width:195px;">LIN Engineering (Servo Dynamics)</td>
			<td style="width:327px;">Motor: CO-5718-01P, Gearbox: DPL64/1, I = 10 for NEMA 23; power supply PW100-48</td>
			<td style="width:449px;">To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasonic pulser/receiver</td>
			<td>Olympus</td>
			<td>5072PR</td>
			<td style="width:449px;">To receive, filter and ampligy the PA signal from UST. Its bandwidth is 35MHz, and gain is &#177;59 dB.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasound Transducer</td>
			<td>Olympus</td>
			<td>V306-SU-NK-CF1.9IN/Q4200069</td>
			<td style="width:449px;">Ultrasonic sensors used for photoacoustic detection. Central freqency 2.25 MHz, 0.5 in, Cylindrical focus 1.9 inch</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCIe DAQ (Data acquisition) Card</td>
			<td>GaGe</td>
			<td>CSE4227/ A6000610/B0E00610</td>
			<td style="width:449px;">12 bit, 100 Ms/s, 2 channels, 1 Gs on board memory, PCIe x16 interface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Rats</td>
			<td>In Vivos Pte Ltd, Singapore</td>
			<td style="width:327px;">NTac:SD, Sprague Dawley / SD</td>
			<td>Female, weight 100&#177;10g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Acrylic water tank </td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td style="width:449px;">It contains the water that acts as an acoustic coupling medium between brain and detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Circular Scanner </td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Scanner is made out of Alluminum </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Anesthetic Machine</td>
			<td>medical plus pte ltd</td>
			<td style="width:327px;">Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td>
			<td>Supplies oxygen and isoflurane to animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Pulse Oxymeter portable</td>
			<td>Medtronic</td>
			<td style="width:327px;">PM10N with veterinary sensor</td>
			<td>Monitors the pulse oxymetry of the animal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasound gel</td>
			<td style="width:195px;">Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Clear ultrasound gel</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Data acqusison software</td>
			<td style="width:195px;">National Instruments Corporation,Austin,TX,USA)</td>
			<td style="width:327px;">NI LabVIEW 2015 SP1</td>
			<td style="width:449px;">LabVIEW based program was developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Data processing software</td>
			<td style="width:195px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:327px;">Matlab R2012b</td>
			<td style="width:449px;">Matlab code for reconstruction of PA images was developed in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller </td>
			<td>LaridTech, MO,USA</td>
			<td>MTTC1410</td>
			<td>It will constantly control temperature of the PLD </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 V power supply </td>
			<td>Voltcraft </td>
			<td style="width:327px;">PPS-11810</td>
			<td>To supply operating voltage for PLD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable power supply </td>
			<td>BASETech</td>
			<td>BT-153</td>
			<td>To change the laser output power</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Funtion generator </td>
			<td style="width:195px;">Funktionsgenerator</td>
			<td>FG250D</td>
			<td style="width:449px;">To change the repetetion rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Animal distributor</td>
			<td style="width:195px;">In Vivos Pte Ltd, Singapore</td>
			<td style="width:327px;"></td>
			<td>Animal distributor that supplies small animals for research purpose.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Animal holder</td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Used for holding the animal on its abdomen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Breathing mask</td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Used along with animal holder to supply anesthesia mixture to the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Pentobarbital sodium</td>
			<td style="width:195px;">Valabarb</td>
			<td style="width:327px;"></td>
			<td>Used for euthanizing the animal after the expeirment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Optical diffuser</td>
			<td style="width:195px;">Thorlabs</td>
			<td style="width:327px;">DG10-1500</td>
			<td>Used to to make the laser beam homogeneous</td>
		</tr>
	</tbody>
</table>

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

La formación de imágenes de animales pequeños in vivo tiene un papel importante que desempeñar en los estudios preclínicos. La tomografía fotoacústica (PAT) es una modalidad emergente de imágenes híbridas que muestra un gran potencial tanto para aplicaciones preclínicas como clínicas. Los sistemas PAT (OPO-PAT) basados ​​en osciladores paramétricos ópticos convencionales son voluminosos y caros y no pueden proporcionar imágenes de alta velocidad. Recientemente, diodos láser pulsado (PLDs) han sido demostrados con éxito como una fuente de excitación alternativa para PAT. El diodo láser pulsado PAT (PLD-PAT) ha sido demostrado con éxito para imágenes de alta velocidad en fantasmas fotoacústicos y tejidos biológicos. Este trabajo proporciona un protocolo experimental visualizado para la imagenología cerebral in vivo utilizando PLD-PAT. El protocolo incluye la configuración compacta del sistema PLD-PAT y su descripción, la preparación de los animales para la obtención de imágenes cerebrales y un procedimiento experimental típico para la obtención de imágenes en 2D de cerebro de rata en sección transversal. El sistema PLD-PAT es compacto y económicoY puede proporcionar alta velocidad, imágenes de alta calidad. Se presentan imágenes cerebrales recogidas in vivo a diversas velocidades de exploración.

En esta sección se presentan los resultados in vivo de la obtención de imágenes cerebrales que demuestran las capacidades del sistema PLD-PAT descrito. Para demostrar las capacidades de formación de imágenes a alta velocidad del sistema PLD-PAT, se realizó la formación de imágenes en vivo de dos ratas sanas diferentes en vivo . La Figura 2 muestra las imágenes cerebrales de una rata hembra (93 g) a diversas velocidades de ...

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

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

Se demuestra un sistema compacto de tomografía fotoacústica por diodos láser pulsado (PLD-PAT) para la obtención de imágenes cerebrales in vivo de alta velocidad en animales pequeños.

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 ...

a-high-performance-compact-photoacoustic-tomography-system-for-vivo

Research

JoVE Journal

Bioengineering

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

10.4K vistas.  Nanyang Technological University.  Se demuestra un sistema compacto de tomografía fotoacústica por diodos láser pulsado (PLD-PAT) para la obtención de imágenes cerebrales in vivo de alta velocidad en animales pequeños. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional 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

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Las nanopartículas virales para<em&gt; En vivo</em&gt; Imágenes de tumores

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Las reconstrucciones anatómicas del sistema venoso cardíaco humana usando tomografía computarizada con contraste de las muestras de perfusión fijos

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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 ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

Posicionamiento adecuado y la contención de una rata de extremidades para la proyección de imagen enfocada de alta resolución del hueso utilizando la microarquitectura In Vivo Micro-tomografía computada

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

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

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

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

Fotoacústica clínica mano sistema de imagen por imagen no invasiva en tiempo real de Animal pequeño

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

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Rígida fijación de fijo y manchados, todo, escala milimétrica muestras para histología sin sección 3D de tomografía Micro computada

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In Vivo 2-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...