2.7 Integrated en face photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging system

The schematic diagram of a custom-built integrated en face PAM and OCT system is shown in Fig. 1 [22]. For PAM, laser pulses with a pulse width of 3~5 ns and a pulse repetition rate of 1 kHz were generated from an optical parametric oscillator (OPO) pumped by a diode-pumped solid-state laser (NT-242, Ekspla, Vilnius, Lithuania, tunable wavelength range 405 – 2600 nm) and served as the illumination source. The laser light output from the OPO was delivered through a half-wave plate attenuator and collimated to 2 mm in diameter by a beam collimator. The collimated light was then transmitted to a scanning head and raster-scanned by a two-dimensional galvanometer, which is a shared component with the spectral domain (SD)-OCT system. The scanned beam traveled through a telescope consisting of a scan lens (focal length 36 mm, OCT-LK3-BB, Thorlabs, Inc., Newton, NJ) and an ophthalmic lens (OL, focal length 10 mm, AC080-010-B-ML, Thorlabs) and was finally focused on the fundus by the rabbit eye optics. The laser-induced acoustic signals were detected by a custom-made 27.0 MHz needle-shaped ultrasound transducer (Optosonic Inc., Arcadia, CA, USA). The transducer was mounted in contact with the conjunctiva and aligned to enable accurate alignment with the illuminating laser light. The generated PA signal was amplified by a low-noise amplifier (gain 57 dB, AU-1647, L3 Narda-MITEQ, NY). Then, the signals were digitized and recorded using a high-speed digitizer at a sampling rate of 200 MS/s (PX1500-4, Signatec Inc., Newport Beach, CA). The recorded data was then used to reconstruct two-dimensional (2D) or three-dimensional (3D) images of the retinal blood vessels. For image reconstruction, a single laser pulse excitation at a fixed position creates the acoustic signal, which is recorded and converted into 1D depth-resolved PA image along the Z axis, referred to as A-lines. By implementing horizontal scanning lines along the x-axis on the sample, the two-dimensional depth-sensitive PA image is acquired. To obtain 3D volumetric PA image, each sample is scanned along x- and y-directions optical-scanning galvanometer with a resolution of 7.5 x 7.5 µm². For a 1.5 x 1.5 mm² field of view, the acquisition time was approximately 40 s. For quantitative evaluation, the PA amplitudes at the different region of interest (ROI) will be measured and compared [27–29]. In addition, spectroscopic PA imaging is performed at various optical wavelengths ranging from 510 to 600 nm to determine the concentration of hemoglobin, to quantify the suitable excited wavelength for detection and evaluation of photothrombosis; and to distinguish between normal to occluded vessels.

Schematic diagram of the integrated en face photoacoustic microscopy (PAM) and optical coherence tomography (OCT) for multimodal ocular imaging.

For In vivo experiments, the laser pulse energy on the eye used to obtain images was less than 80 nJ, which is half of the American National Standards Institute (ANSI) eye safety limit of 160 nJ at these settings as described previously [22]. The ultrasound transducer yields PAM lateral and axial resolutions of 4.1 and 37.0 µm, respectively. To visualize the margin of the blood vessel, 3D image reconstruction was performed using Amira software (FEI, Hillsboro, OR). Further, image segmentation was also performed to classify the positions of arteries, veins, choroidal vasculature, and neovascularization. The major retinal arteries and veins were identified from FA images (see Fig. 10). As shown in Fig. 10, delay in the appearance of the FA dye into the central retinal vessels indicate the position of retinal veins. On the other hand, slight up of dye into the central artery is completed within one or two second, which was approximately 2 to 4 seconds faster than in the retinal vein.

Spectral domain Optical Coherence Tomography (SD-OCT) was modified from a commercial OCT system (Ganymede-II-HR, Thorlabs, Newton, NJ) by adding the ocular lens after the scan lens and a dispersion compensation glass in the reference arm [22]. A combination of two superluminescent light emitting diodes with center wavelengths of 846 nm and 932nm (see Fig. 11) was used to excite the tissue. A single mode fiber delivered the light from the light source and split it into sample and reference arms. The OCT light source was coaxially aligned with the PAM system. The lateral and axial resolutions for OCT are 3.8 µm and 3 µm, respectively. A-line acquisition rate was 36 kHz. The scanning areas was 3.5 x 2.5 mm², and high-resolution OCT images were captured, where 512 x 1024 A-lines were recorded per image with an image acquisition time of 0.103 seconds. To achieve 3D OCT image, 512 x 512 B-scan were sequentially acquired. The total acquisition time was approximately 53 s.