The overall goal of this procedure is to demonstrate in vivo non-ionizing photoacoustic cystography using near infrared optical absorbance as non-toxic optical turbid tracers. This is accomplished by first anesthetizing the experimental animal. The second step is to prepare the animal for imaging by removing the hair in the abdominal region, positioning the rat atop of a custom made animal holder, inserting a catheter and applying ultrasound gel to the imaging region.
Next images of the abdominal area are obtained using a photoacoustic cystography system before and after injection of optical absorbance into the bladder. Ultimately, this process allows for the imaging of a rat bladder using a non-ionizing and non-invasive photoacoustic cystography system providing no radiation exposure and no long-term heavy agent accumulation For acoustic tomography has become a premier biomedical imaging modality because of a strong optical absorption contrast, high ultrasound spatial resolution in biological tissues. The key advantage of photo tomography are that it is completely free from ionizing radiation, the injected agent to is not accumulated in the bladder and it is fast and costly effective.
For this procedure, use a Q switched neodymium dope atrium aluminum garnet laser that has been frequency doubled to generate light at 532 nanometers. The laser output is passed through an optical parametric oscillator or OPO with a wavelength tunable range of 680 to 2, 500 nanometers. Start by setting the OPO pulse duration to five nanoseconds for each laser shot and set the repetition rate to 10 hertz.
Then set the optical wavelength to 667 nanometers for use with the methylene blue contrast agent. This wavelength may need to be modified if another contrast agent is used. Deliver the light coming out of the OPO through a right angle prism to a homemade spherical conical lens.
The conical lens is made from a 2.5 centimeter diameter BK seven lens with a cone angle of 152 degrees. Next, redirect the diverging donut shaped light beam through a 2.5 centimeter thick optimal condenser made of transparent acrylic. The diameters of the top and bottom surfaces are 6.1 and 4.8 centimeters respectively.
Make sure the beam pattern is a perfect ring shape. If the donut shaped beam pattern is not properly generated, the photoacoustic signals originating from the skin surface will be dominant. Thus, it will be difficult to achieve deep tissue imaging.
Then prepare a small water container to boost acoustic coupling between the animal and the laser. The bottom opening is wrapped with a clear thin polyethylene film, which is optically and acoustically transparent. Using a piece of black electrical tape, coaxially aligned the line shaped light with the ultrasound focal zone.
If these are not well aligned, the system will suffer from low signal to noise ratio. Next, Mount Spherically focused ultrasound transducer to detect the photoacoustic waves. For this a V 3 0 8 Olympus NDT immersion transducer is used.
It collects data at five megahertz and has transverse and axial resolutions of 590 and 144 micrometers respectively. Then connect the transducer to a broadband ultrasonic pulse or receiver, which will amplify the photoacoustic waves and connect this to an oscilloscope. Once the system has been set up, obtain one dimensional time resolved images called ALINE images by measuring the arrival times of the photoacoustic waves through the oscilloscope.
Then mechanically control a linear raster stage using lab view to acquire two dimensional B scans and three dimensional photoacoustic images. The three dimensional imaging takes approximately 25 minutes per sample for a field 2.5 centimeters by 2.4 centimeters by 1.5 centimeter in size to acquire 3D images. Set up the lab view program to control the stage.
Acquire 125 samples in the X direction with a step size of 0.2 millimeters, 60 samples along the Y direction with the step size of 0.4 millimeters and 500 data points with a 50 megahertz sampling rate along the Z direction. Finally, set up a MathWorks MATLAB software system to acquire, convert, and process the raw data into a sequence of cross-sectional bcan images that represent the volumetric data. The recorded images are then displayed as 2D maximum amplitude projection images.
To prepare rats for imaging, anesthetize a female sprague dolly rat with a weight between 200 and 250 grams by intraperitoneal injection of a mixture of ketamine and xylazine. Then remove the hairs in a five centimeter square around the abdominal area using hair removal lotion. Next, position the rat atop of custom made animal holder.
Then prepare a 22 gauge catheter for insertion by coating it with a lubricant and insert the distal end of the catheter horizontally into the urethra until the hub of the catheter reaches the opening and urine begins to void via the catheter. Finally, position the rat so that it is on top of the animal holder and below the water container in the photoacoustic cystography system. To begin imaging fully anesthetize the rat using vaporized iso fluorine during the in vivo photoacoustic imaging experiments.
Then apply ultrasound gel between the animal skin surface and plastic membrane to improve acoustic coupling. Next, run the prepared MathWorks mat lab program to obtain a control three dimensional photoacoustic image prior to injection of contrast agents. Once the control image is obtained, use a one milliliter syringe to introduce an aqueous solution of methylene blue to the bladder via the catheter following injection of methylene blue.
Acquire a series of in vivo photoacoustic images every 12 hours until 48 hours post-injection. Next, sacrifice the rat by injecting an overdose using 150 milligrams per kilogram of pentobarbital. Then remove the bladder and kidney and place them on a glass plate.
The bladder and kidney from an unin injected animal should be used as a control for ex vivo imaging. Position the glass plate below the water container in the photoacoustic cystography system and apply ultrasound gel between the excised organs and plastic membrane to improve acoustic coupling during imaging. The image shown here is a two dimensional XY abdominal scan of a rat imaged with 667 nanometer laser light prem methylene blue injection.
Here, the blood vessels can be easily seen as blood absorbs light to generate a photoacoustic signal, but there is no visible bladder due to the lack of contrast agent. When imaged 12 hours after injection of the methylene blue contrast agent, the bladder becomes easily visible with very strong amplitude throughout the bladder. When measured again at 24 and 48 hours, the contrast agent has left the body and is no longer visible by changing the optical wavelength to 850 nanometers with the optical parametric oscillator, the methylene blue contrast agent no longer absorbs the energy and becomes invisible.
This makes the bladder effectively disappear and acts as another negative control for the system. Depth resolved images obtained from the Z plane along the dotted lines shown here, provide additional depth data along areas of interest as shown here or can be performed for the entire image to describe the area in three dimensions. In this video, we demonstrated the physi ability of no and non-ionizing process per bladder imaging in vivo called Photoacoustic oscopy.
Using our photoacoustic imaging system, we have successfully imaged our left bladder filled with an injection of optical effect tracer in lead In the future. This technique may also be applied to monitoring urological problems such as bisco reflux in pediatric patients as a low radiation dose. Portability and inexpensive nature of this procedure are optimal for this type of application.
