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.
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.
1. System Configuration
2. System Alignment
3. In vivo Multimodal Retinal Imaging
Figure 2 shows the 2-D fundus images of SD-OCT and PAOM acquired simultaneously in an albino rat (A and B) and a pigmented rat (C and D), respectively. In the SD-OCT fundus images (Figures 2A and 2C), retinal vessels have dark appearance due to the hemoglobin absorption of probing light. In addition to retinal vessels (RV in Figure 2B), PAOM visualizes the choroidal vasculatures (CV in Figure 2B) in albino eye because of the lacking RPE melanin. Because pigmented eye has high melanin concentration, PAOM images RPE (Figure 2D) with high contrast in addition to the retinal vessels. In all retinal imaging, the maximum scanning angle is 26 degrees and the imaging acquisition takes ~2.7 sec. To demonstrate the three-dimensional imaging capability of PAOM, a volumetric rendering of the data shown in Figure 2b is given in Figure 3.
Figure 1. Photograph of the five-axis animal holder. The arrows 1-5 highlight the five adjustable freedoms and the arrow 6 highlights the animal restrainer.
Figure 2. Simultaneously acquired SD-OCT (A and C) and PAOM (B and D) fundus images. A) and B) are acquired from an albino rat, and C) and D) are acquired from a pigmented rat. RV: retinal vessel; CV: choroidal vessel; RPE: retinal pigment epithelium. Bar: 500 μm.
Figure 3. Volumetric visualization of PAOM in an albino rat retina.
Here, we present a detailed instruction on simultaneous in vivo retinal imaging of rat eyes using PAOM combined with SD-OCT. Optical-scattering-based SD-OCT is, perhaps, the clinical “gold standard” for retinal imaging3; however, it is not sensitive to detect the optical absorption in the retina. The newly-developed PAOM is the only optical-absorption-based ophthalmic imaging modality that provides optical absorption properties of the retina6. Because hemoglobin and melanin are endogenously strong optical absorbing pigments, PAOM enables the investigating of the anatomy and functions of the retinal/choroidal vessels and the RPE without resorting to additional contrast agents.
In PAOM, the unfocused ultrasonic transducer has a limited sensitivity region (~2.8×2.8 mm2)10 due to its finite active element, which causes a decayed detection sensitivity of PA signals toward the periphery of the field of view (FOV). Thus, the tilting angle of the transducer should be carefully adjusted to achieve a homogeneous retinal FOV. A potential substitution for the traditional piezoelectric transducer is to apply micro-ring resonator, which has lower noise equivalent pressure values and wider detection directivity 14, which may provide a more homogeneous retinal image with better SNR in PAOM. Comparing with SD-OCT, PAOM has similar lateral resolution (~20 μm) but much worse axial resolution (~23 μm) due to the currently limited ultrasonic bandwidth6. The axial resolution of PAOM can potentially be improved by employing novel ultrasonic detector as well. The calibration method of PAOM resolutions was previous reported 6,15 .
In summary, the integrated PAOM and SD-OCT imaging system offers more comprehensive anatomical and functional evaluation of the retina, and, therefore, holds great promises in the future clinical diagnosis and managements of many ocular disorders.
The authors have nothing to disclose.
We thank the generous support from the National Science Foundation (CAREER CBET-1055379) and the National Institutes of Health (1RC4EY021357, 1R01EY019951). We also acknowledge the support from the China Scholarship Council to Wei Song.