Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

The overall goal of this novel imaging setup and operating procedure is to perform photoacoustic microscopy and optical coherence tomography dual-modality for non-invasive, label-free chorioretinal imaging of the eyes of larger mammals, such as rabbits. This method can help answer key questions in ophthalmic imaging about the imaging depth, resolution, and clinical translation required to discern oxygen saturation, blood distribution, and melanin in larger animals. The main advantage of this technique is that it allows noninvasive visualization of ocular tissue for the acquisition of photoacoustic microscopy`and optical coherence tomography dual-modality images.

To begin, select an optical parametric oscillator laser pumped by a diode pump solid state laser as the light source for the photoacoustic microscope and use a beam splitter to divide the column aided beam with a split ratio of 19 reflection to 10 transmission. Successively deflect the reflected portion by the M3 mirror and dichroic mirror and use a two-dimensional galvanometer to raster scan the reflection. Deliver the scanned beam through a telescope composed of a scanned lens with a focal length of 36 millimeters and an ophthalmic lens with a focal length of 10 millimeters and focus the beam on the fundus by the rabbit eye optics.

Before beginning the imaging procedure, please a water circulating heating blanket on the body support and set the temperature of the circulating water to 38 degree Celsius. Record the pre procedure vitals and confirm the appropriate level of sedation by the rabbit's heart and respiratory rates. Dilate the rabbit pupils with one drop each of tropicamide 1%ophthalmic and phenylephrine hydrochloride 2.5%ophthalmic.

Use an ultrasonic amplifier to amplify the signal and filter the signal by a low pass filter. Place a power meter above the rabbit eye and measure the laser pulse energy on the rabbit cornea to keep it below the American National Standards Institute safety limit. To enable the optical coherence tomography or OCT system to image the posterior segment of the rabbit eye, add an ophthalmic lens after the scan lens.

Use a zooming housing tube to adjust the length of the reference arm to confirm its match with the optical path length of the sample arm. For spectral domain OCT imaging, place the rabbit on to the imaging platform of a clinical fundus camera and obtain 50 degree fundus red free and autofluoroscence images. Next, transfer the animal to the platform of the OCT system with one eye under the ophthalmic lens.

Illuminate the eye using the LED light and open the OCT software. Check the CCD camera image of the fundus and draw a straight line in the software to represent the OCT B scan of interest. Begin scanning adjusting the reference arm length to visualize the OCT image and to optimize the dispersion compensation factor in the OCT software to get the sharpest images.

Then set data, the appropriate data acquisition parameters and save the images. For photoacoustic microscope imaging, turn on the optical parametric oscillator laser. Start the photoacoustic microscope control software and tune the lase wavelength to one of the absorption peaks of the targeted chromaforce.

Initialize the position of the galvanometer and monitor the laser energy before the rabbit cornea to confirm that it is below the American National Standard Institute Safety Limit. Map the ultrasonic transducer on a three dimensional translation stage, and place the transducer tip in contact with the rabbit conjunctiva so that the tip is pointing to the fundus. Use a drop of eye lubricant to better couple the transducer tip in the rabbit conjunctiva and turn on the LED illumination light.

Visualize the rabbit fundus through the MatLab software and set the scanning region of interest including the center and the physical size of the region. Open the laser shutter and start B scan of the beam observing the detected photoacoustic signal on the oscilloscope and finally tuning the transducer position to maximize the signal intensity along the entire B scan as necessary. Then set the data acquisition parameters process the raw data and visualize the photoacoustic microscope image in two dimensions.

At the end of the imaging session transfer the rabbit back to the fundus camera to examine the fundus for any post imaging morphological changes and rinse the rabbit cornea with eye wash. Apply flurbiprofen ophthalmic, neomycin and polymyxin b sulfates and dexamethasone ophthalmic ointment to the cornea and close the eye. Then transfer the rabbit and the water circulating blanket into a recovery chamber with monitoring until full recovery.

In this representative fundus photograph, the choroidal vessels are spread over most parts of the rabbit funds, while the retinal vessels are confined within the medullary ray. Here a typical photoacoustic microscopy image of the choroidal vasculature within the fundus is shown with the choroidal vessels blood vessels delineated at high lateral resolution. In this OCT B scan image, the retina, choroid, and sclera are visualized with a high axial resolution with the choroidal vessels observed below the retinal pigment epithelium layer.

Using the dual-modality imaging system 2D maximum intensity projection and 3D volumetric rendering of retinal vessels can be obtained by photoacoustic imaging. Orthogonal slices of the 3D image demonstrate how photoacoustic microscopy can also be used to visualize individual retinal vessels which lie above the retinal pigment epithelium layer and to confirm the different depths at which the retinal and choroidal vessels are located. Here an OCT B scan image shows individual retinal vessels.

And the nerve fiber layer confirming the photoacoustic microscopy imaging results. Once mastered, this technique can be completed in approximately one hour if it is performed properly via to remember to set the laser irradiance at low ANLI setting limits to revive lubricants once eye rinse to avoid dehydration and to monitor the rabbit condition every 15 minutes. Following the procedure, automated slide immunohistochemistry can be performed to answer additional question about confirming choroidal neovascularization development and to amplify the volume of the neovascularization in rabbit.

After its development this technique paved the way for researchers in the field of ophthalmology to explore choroidal diseases in rabbit bearing disease models including choroidal neovascularization models. After watching this video, you should have a good understanding of how to detect single chorioretinal blood vessels in rabbit eyes using a novel multimodal molecular imaging system for early stage diagnosis of wet age related macular degeneration. Don't forget this.

Working with glass fall laser and animal can be extremely hazardous and that precaution such as wearing goggle and lab coats should always be taken while performing this procedure.