The overall goal of this procedure is to demonstrate a switchable acoustic and optical resolution photoacoustic microscopy system for in vivo small-animal blood vasculature imaging. Photoacoustic microscopy is a fast-growing in vivo imaging modality that combines optics and ultrasound, providing imaging depth beyond optical path with high resolution. This work present is switchable acoustic optical resolution photo acoustic microscopic system capable of higher-resolution imaging at shallow depth as well as lower-resolution deep tissue imaging of the same sample in vivo.
Demonstrating the procedure will be Dr.Mohesh Moothanchery, a research fellow from my laboratory, and Arunima Sharma, a PhD student from my lab. First, construct a nanosecond, tunable laser system from a diode-pumped, solid-state Nd:YAG laser and a ditunable laser with a range of 559 to 576 nanometers. Set the ditunable laser to 570 nan.
Mount on an optical table an acrylic tank with a seven-centimeter polyethylene imaging window in the floor of the tank. Fix an imaging stage to an optical post beneath the imaging window. Over the tank, mount an optical cage containing the AR-OR-PAM imaging system on a switching plate fastened to a computer-controlled three-axis motorized translation stage.
To configure the system for AR-PAM, use a right angel prism mounted on a motorized continuous rotational stage to direct the beam through a variable neutral-density filter to a multi-mode fiber cable with a 25 NA fiber coupler. Connect the fiber output to an XY translation stage in the imaging system. Direct the beam through a plano convex lens 25 millimeters from the output, and then through a conical lens to create a ring shaped beam.
Focus the ring shaped beam on an optical condenser around a 50 megahertz ultrasonic transducer with an acoustic lens mounted at the transducer output. Next, to configure the system for OR-PAM, rotate the first right angle prism by 90 degrees to direct the laser beam through an iris, a variable neutral-density filter, a condenser lens, and a pinhole set at 75 millimeters from the condenser lens. Use a 1 NA single-mode fiber coupler and a single-mode fiber to send the beam to the Z translation stage in the imaging system.
Direct the beam through an achromatic doublet lens mounted 50 millimeters from the fiber output. Divert the beam with a kinematic, controllable, elliptical mirror to a second achromatic doublet lens in a lens tube. Focus the beam on a right-angle prism separated by a layer of silicon oil from a rhomboid prism, bearing an acoustic lens and a 50 megahertz ultrasonic transducer, which forms the OR-PAM scanning head.
Prior to beginning the system alignment, fill the acrylic tanks with degassed water. Then use the three-axis motorized stage to move the scanning assembly over the tank. Lower the assembly until the acoustic lenses of both systems are submerged.
Turn on the laser. Connect the ultrasound transducers to two 25-decibel, fixed-gain amplifiers. Position the AR-PAM scanning head over the imaging window.
Place a glass slide wrapped in black electrical tape on the imaging stage and raise the stage to bring the slide into contact with the imaging window. Adjust the conical lens until the photoacoustic signal amplitude generated from the slide reaches a maximum, indicating that the optical and acoustic lenses are confocal. Then manually switch to the OR-PAM system.
Adjust the achromatic doublet lens until the optical and acoustic lenses are confocal, as indicated by maximizing the photoacoustic signal amplitude from the tape-covered slide. To determine the lateral resolution of each system, first place 1 milliliter of a dilute solution of 100 nanometer gold nanoparticles in water on a a cover slip. Place the slide on the imaging stage.
and raise the stage until the nanoparticle solution contacts the imaging window. Switch the laser and the scanning head to the AR-PAM system. Configure the instrument software for an AR-PAM scan and perform a single raster scan.
Repeat this process for the OR-PAM system. Then remove the slide and clean the imaging window with an alcohol swab. Fit the point spread functions determined from the acquired images to a Gaussian curve.
The full width at half maximum is the lateral resolution for the corresponding scanning system. Next, to determine the maximum imaging depth in tissue, first insert a sharp metal plate wrapped in black tape into a small section of chicken tissue at a shallow angle. Place the tissue in the water tank under the scanner head.
Obtain a single B-scan image for each system. Measure the depth beneath the tissue surface at which the black tape is no longer clearly discernible. Prior to image, ensure that the polyethylene imaging window and the animal imaging stage are both clean.
Then obtain a 25-gram, four-week-old female mouse for the imaging procedure after anesthetizing the mouse. Remove hair from the ear surface with depilatory cream. Apply sterile ocular ointment to the mouse's eyes to prevent dryness and block scattered laser beams.
Place the mouse on an imaging stage with the plate for positioning the ear to me imaged. Monitor the mouse's physiological condition with a pulse oximeter clamped to the mouses tail or leg. Apply ultrasound gel to the ear to be imaged.
Slowly raise the imaging stage to gently bring the ear into contact with the polyethylene imaging window. Acquire AR-PAM and OR-PAM raster scans of the mouse's ear, monitoring the mouse's physiological status throughout. Allow the mouse to recover fully after imaging.
The blood vasculature system of a mouse here was imaged in vivo with a switchable AR-OR-PAM system. Blood vessels thicker than 45 micrometers were clearly visible in the AR-PAM image. Single capillaries of about five micrometers wide were resolved with OR-PAM imaging.
The combined AR-OR-PAM system has an approximately four micron lateral resolution. And at point, four mm imaging for the OR-PAM and an approximately 45 micrometer lateral resolution and 7.8 mm imaging for the AR-PAM. The switchable combined AR-OR-PAM system allows imaging without moving the sample between different imaging systems.
The developed system can be used for pre-clinical imaging. Major pre-clinical applications include imaging of androgenesis, tumor micro-environments, microcirculations, drug response, brain function, bio-markers, and gene activities.
