The overall goal of this procedure is to demonstrate the integration of Photoacoustic Ophthalmoscopy with spectral domain optical coherence tomography for simultaneous in vivo imaging of the retina in small animals. This is accomplished by first preparing an anesthetized animal by dilating the pupil and paralyzing the iris sphincter muscle. Then real-time S-D-O-C-T cross-section imaging is used to locate the retinal region of interest and to optimize the optical parameters.
A needle ultrasonic transducer placed in contact with the animal's eyelid detects the laser induced photoacoustic signals, and optimizes the position of the ultrasonic transducer for maximal signal amplitude. The final steps are to set the scanning parameters and acquire S-D-O-C-T and PAOM images simultaneously. Ultimately, the optical absorption contrast and optical scattering contrast from the collected images can reveal minute functional variations in the retinal circulation and pigment epithelium together with detailed retinal anatomy.
Forte of ophthalmoscope is a newly developed ophthalmic imaging modernity. This technology can reveal the optical absorption contrast of the retina, and thus has a potential advantage over existing mod in imaging. The retinal choroidal muscular network and the retinal pigment epithelium Spectral domain optical coherence tomography, S-D-O-C-T relates on bank reflected photons to form a volumetric image of the retina and is widely used in clinics.
The integration of PAOM with S-D-O-A-T allows us to acquire more comprehensive anatomic and the functional information of the retina. The photoacoustic oscopy subsystem includes an N-D-Y-A-G laser to serve as an illumination source. The output laser set at 1064 nanometers is frequency doubled to 532 nanometers by a beta barium bore.
Eight crystal. A laser line mirror splits this beam. Then the 532 nanometer light is delivered through a single mode optical fiber, and the 1064 nanometer laser is recorded by a photo diode, which triggers photoacoustic signal acquisition.
The laser light coming out of the single mode, optical fiber is delivered into the retina by a galvanometer and a telescope configuration. An unfocused needle transducer is placed in contact with the eyelid to detect the photoacoustic signals generated from the retina. Ultrasonic gel is applied between the transducer tip and the animal's eyelid for good acoustic coupling.
The photoacoustic signal is amplified by two amplifiers and then digitized by a data acquisition board. The spectral domain optical coherence tomography subsystem or SD OCT uses a low coherence light source, a broadband super luminescent diode, which determines the axial resolution to six micrometers. The near infrared light is split by a 50 by 50 commercial single mode fiber coupler, which deliver light to the sample arm and to the reference arm.
The beam delivered to the OCT sample arm is combined with the PAOM illuminating light by a hot mirror. The sample arm shares the same scanning and delivery optics as the PAOM. Lastly, a home-built spectrometer is used to record the S-D-O-C-T interference signals where a line scan CCD camera allows an A line rate of 24 kilohertz.
There are a few steps needed to align the two subsystems. First, maximize the frequency doubling efficiency of the BBO crystal. Second, collate the fiber output laser of the PAOM to 2.0 millimeters in diameter.
Third, align the combined illumination lights of the PAOM and the S-D-O-C-T, so they are coaxial. Lastly, set the PAOM excitation light at about 40 nano joules per pulse and set the S-D-O-C-T probing light at about 0.8 milliwatts. Both of these settings are reported to be eye safe after anesthetizing a rat.
Using a standard ISO fluorine protocol, restrain the rat in a homemade holder with five axes of adjustable freedom. To maintain the rat's body temperature near 37 degrees Celsius, use a heating pad to maintain the anesthesia. Provide the rat 1%ISO fluorine at a flow of 1.5 liters per minute throughout the experiment.
Now, remove the eyelash from the eyelid using a small surgical scissor, then dilate the pupils using a 1%tropic aide ophthalmic solution, followed by paralyzing the iris sphincter muscle using 0.5%TETRACAINE hydrochloride ophthalmic solution. Throughout the procedure, it is critical to apply artificial teardrops to the rat's eye every other minute. Next, turn on SDO CT Illuminating light, and check that the probing light is about 0.8 milliwatts.
Now activate the galvanometer scanning. Align the S-D-O-C-T irradiation light onto the rat retina, and identify the retinal region of interest by adjusting the five axis animal holder and center of the field of view on this region. The optic disc is viewed in this example.
Further adjust the animal holder to optimize the retinal cross-section imaging in one scanning direction. Then switch the raster scanning direction and continue making adjustments until the best focal point is found. Now, prepare the needle transducer attached to a five axis adjustable platform.
Apply a drop of ultrasonic gel to the transducer tip and gently contact the transducer tip to the animal's eyelid. Set the PAOM laser to the external trigger mode. Start the galvanometer scanning and activate the realtime display of PAOM.
Cross-sectional images. Carefully adjust the transducer orientation for good overlap of its sensitivity region with the scanning region of the PAOM laser. Stop when the PAOM image has the best signal to noise ratio and evenly distributed PA amplitude patterns in both scanning directions.
Now set the driven voltages of the two galvanometer scanners according to the size of the field of interest and conduct the scan. When the scan is triggered. A fast 2D raster scan by the GALVANOMETER is controlled by an analog output board, which also triggers both the PAOM laser firing and the signal acquisition of the OCT spectrometer.
Hence, synchronizing the two subsystems. A photo DDE Recording the PAOM laser sequence then triggers the PAOM data acquisition. After the experiment, turn off the S-D-O-C-T probing light and immediately remove the animal from the holder.
Keep the animal warm and in the dark until it wakes up naturally. Then turn off the heating and keep it in the dark for an extra hour later, reconstruct the three dimensional images offline using a freely available MATLAB based visualization program called Vue Construct, the 3D volumetric images and 2D fundus images from 256 B Bcan images. 2D fundus images of S-D-O-C-T and PAOM were acquired simultaneously in an albino rat and in a pigmented rat.
The maximum scanning angle was 26 degrees, and the image acquisition time was about 2.7 seconds. In the S-D-O-C-T fundus images, retinal vessels have a dark appearance due to the hemoglobin absorption of the probing light. Because the pigmented eye has high melanin concentration, PAOM images show the retinal pigment epithelium or RPE with high contrast in addition to the retinal vessels, because the albino eye lacks RPE melanin here, PAOM visualizes retinal vessels and choroidal vasculature To demonstrate the three dimensional imaging capability of P-A-O-M-A volumetric rendering was made of this image.
Once mastered, this imaging process can be completed within 10 minutes. We anticipate that POM will play an important role in both the fundamental understanding and clinical diagnosis of several blinding diseases such as diabetic retinopathy and retinal degeneration.
