Tan Liu

Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.

Combined photoacoustic microscopy and optical coherence tomography can measure metabolic rate of oxygen.

We proposed to measure the metabolic rate of oxygen (MRO2) in small animals in vivo using a multimodal imaging system that combines laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) and spectral-domain optical coherence tomography (SD-OCT). We first tested the capability of the multimodal system to measure flow rate in a phantom made of two capillary tubes of different diameters. We then demonstrated the capability of measuring MRO2 by imaging two parallel vessels selected from the ear of a Swiss Webster mouse. The hemoglobin oxygen saturation (sO2) and the vessel diameter were measured by the LSOR-PAM and the blood flow velocity was measured by the SD-OCT, from which blood flow rate and MRO2 were further calculated. The measured blood flow rates in the two vessels agreed with each other.

A combined method to quantify the retinal metabolic rate of oxygen using photoacoustic ophthalmoscopy and optical coherence tomography

Quantitatively determining physiological parameters at a microscopic level in the retina furthers the understanding of the molecular pathways of blinding diseases, such as diabetic retinopathy and glaucoma. An essential parameter, which has yet to be quantified noninvasively, is the retinal oxygen metabolic rate (rMRO2). Quantifying rMRO2 is challenging because two parameters, the blood flow rate and hemoglobin oxygen saturation (sO2), must be measured together. We combined photoacoustic ophthalmoscopy (PAOM) with spectral domain-optical coherence tomography (SD-OCT) to tackle this challenge, in which PAOM measured the sO2 and SD-OCT mapped the blood flow rate. We tested the integrated system on normal wild-type rats, in which the measured rMRO2 was 297.86 ± 70.23 nl/minute. This quantitative method may shed new light on both fundamental research and clinical care in ophthalmology in the future.

Collecting back-reflected photons in photoacoustic microscopy

Since the photoacoustic effect relies only on the absorbed optical energy, the back-reflected photons from samples in optical-resolution photoacoustic microscopy are usually discarded. By employing a 2 × 2 single-mode fiber optical coupler in a laser-scanning optical-resolution photoacoustic microscope for delivering the illuminating laser light and collecting the back reflected photons, a fiber-optic confocal microscope is integrated with the photoacoustic microscope. Thus, simultaneous multimodal imaging can be achieved with a single light source and images from the two modalities are intrinsically registered. Such capabilities are demonstrated in imaging both phantoms and small animals in vivo.

Photoacoustic generation by multiple picosecond pulse excitation

PURPOSE The purpose of this work is to demonstrate that higher amplitude of ultrashort laser induced photoacoustic signal can be achieved by multiple-pulse excitation when the temporal duration of the pulse train is less than the minimum of the mediums thermal relaxation time and stress relaxation time. Thus, improved signal-to-noise ratio can thus be attained through multiple-pulse excitation while minimizing the energy of each pulse. METHODS The authors used a Michelson interferometer together with a picoseconds laser system to introduce two 6 ps pulses separated by a controllable delay by introducing a path length difference between the two arms of the interferometer. The authors then employed a series of three interferometers to create a pulse train consisting of eight pulses. The average pulse energy was 11 nJ and the temporal span of the pulse train was less than 1 ns. RESULTS The detected peak-to-peak amplitude of the multiple-pulse induced photoacoustic waves were linearly dependent on the number of pulses in the pulse train and such a linearity held for different optical absorption coefficients. The signal-to-noise ratio improved when the number of pulses increased. Moreover, nonlinear effects were not detected and no photoacoustic saturation effect was observed. CONCLUSIONS The authors have shown that multiple-pulse excitation improves the signal-to-noise ratio through an accumulated energy deposition effect. This method is invaluable for photoacoustic measurements that require ultrashort laser pulses with minimized pulse energy to avoid laser damage.

Saturation effect in functional photoacoustic imaging

We investigate the saturation effect, which describes the violation of the linearity between the measured photoacoustic amplitude and the objects optical absorption coefficient in functional photoacoustic imaging when the optical absorption in the object increases. We model the optical energy deposition and photoacoustic signal generation and detection in a semi-infinite optical absorbing object. Experiments are carried out by measuring photoacoustic signals generated from an ink-filled plastic tube. The saturation effect is studied by varying the optical absorption coefficient in the model and the ink concentration in the photoacoustic experiments. By changing the center frequency of the ultrasonic detector, the requirement to minimize the saturation effect in functional photoacoustic imaging is established.

Image chorioretinal vasculature in albino rats using photoacoustic ophthalmoscopy.

We imaged the microvascular network in both the retina and the choroid in an albino rat eye using photoacoustic ophthalmoscopy guided by optical coherence tomography. Relying on optical absorption and ultrasonic detection, photoacoustic ophthalmoscopy can image both retinal and choroidal vessel networks with high contrast.

Fundus Camera Guided Photoacoustic Ophthalmoscopy

Abstract Purpose: To demonstrate the feasibility of fundus camera guided photoacoustic ophthalmoscopy (PAOM) system and its multimodal imaging capabilities. Methods: We integrated PAOM and a fundus camera consisting of a white-light illuminator and a high-sensitivity, high-speed CCD. The fundus camera captures both retinal anatomy and PAOM illumination at the same time to provide a real-time feedback when we position the PAOM illuminating light. We applied the integrated system to image rat eyes in vivo and used full-spectrum, visible (VIS), and near infrared (NIR) illuminations in fundus photography. Results: Both albino and pigmented rat eyes were imaged in vivo. During alignment, different trajectories of PAOM laser scanning were successfully visualized by the fundus camera, which reduced the PAOM alignment time from several minutes to 30 s. In albino eyes, in addition to retinal vessels, main choroidal vessels were observed using VIS-illumination, which is similar to PAOM images. In pigmented eyes, the radial striations of retinal nerve fiber layer were visualized by fundus photography using full-spectrum illumination; meanwhile, PAOM imaged both retinal vessels and the retinal pigmented epithelium melanin distribution. Conclusions: The results demonstrated that PAOM can be well-integrated with fundus camera without affecting its functionality. The fundus camera guidance is faster and easier comparing with our previous work. The integrated system also set the stage for the next-step verification between oximetry methods based on PAOM and fundus photography.

Near-infrared light photoacoustic ophthalmoscopy

We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.

Automatic retinal vessel segmentation based on active contours method in Doppler spectral-domain optical coherence tomography.

Abstract. We achieved fast and automatic retinal vessel segmentation by employing the active contours method in Doppler spectral-domain optical coherence tomography (SD-OCT). In a typical OCT B-scan image, we first extracted the phase variations between adjacent A-lines and removed bulk motion. Then we set the initial contour as the boundary of the whole image and iterated until all of the segmented vessel contours became stabilized. Using a typical office computer, the whole segmentation took no more than 50 s, making real-time retinal vessel segmentation possible. We tested the active contours method segmentation in both controlled phantom and in vivo rodent eye images.