The OCT system consisted of a light source (
λ = 840 nm,
Δλ = 112 nm; Broadlighter, Superlum Inc., Carrigtwohill, Co. Cork, Ireland), and a Michelson interferometer consisting of a 90/10 fiber beam splitter that sent 90% and 10% of the source's output, respectively, to the reference and sample arms. The sample arm contained
x- and
y-direction galvanometers for scanning the beam over the retina, a custom achromatizing lens to cancel longitudinal chromatic aberration due to the eye,
30 and the components of the AO system. The reference channel consisted of a planar mirror and a water cuvette designed to match chromatic dispersion induced by the eye. Reflected and back-scattered light from the two interferometer arms was combined again in the fiber coupler and sent to the spectrometer, consisting of a fiber collimator, transmissive holographic diffraction grating, F-theta lens, and a CMOS linescan detector (4096 pixel ×2 lines [2048 × 2 used]; Sprint spL4096-140 km; Basler AG, Ahrensburg, Germany). The OCT system acquired 125,000 lines/sec. The theoretical axial resolution was 2.8 μm in air and 2.0 μm in the eye (
n = 1.38).
The AO system incorporated a Shack-Hartmann wavefront sensor (SHWS) consisting of a lenslet array (Darray = 10 mm, Dlenslet = 0.5 mm, f = 30 mm; Northrop Grumman Corp., Falls Church, VA, USA) and sCMOS detector (2048x2048 pixel; Ace acA2040-180 km; Basler AG), and a high-speed, high-stroke deformable mirror (DM; DM-97-15; ALPAO SAS, Montbonnot, France). The AO system was limited by centroid computation time, and ran at 25 Hz using the full 4 megapixel spots image. The pupil diameter was 6.75, 13.5, and 10 mm at the eye, DM, and SHWS, respectively. Diffraction-limited imaging (σZernike ≤ 60 nm) was achieved for all subjects, corresponding to a theoretical lateral resolution (Rx) of 2.5 μm in the retina (3.4 μm in air, validated using a 1951 United States Air Force resolution test chart).
Custom software was developed for both instrumentation systems. The OCT software was written in C++, with specialized graphics processing unit (GPU)-based OCT post processing, which permitted MHz-rate A-scan computation and real-time volume display.
31 The AO software was written in Python/NumPy, and implemented a novel pupil scaling technique and a far-field camera, which provided real-time images of the double-pass lateral point spread function (PSF). The AO system provided real-time wavefront reconstruction and residual error feedback, informing operators when the correction was diffraction limited. The AO system also provided a method for dynamically adding positive or negative defocus, by modifying the SHWS reference coordinates. As such, it was capable of shifting the plane of best focus in the retina.
The sample channel of the system was designed to correct for beam distortions and astigmatism that accumulate as the imaging light is relayed by the multiple off-axis spherical mirrors in the sample channel. The out-of-plane design of the new system is described in detail elsewhere.
32
Because the hypotheses investigated in this study involve measurement of axial morphology, it was critical to calibrate the depth scale of the OCT. We performed two independent calibration procedures, one in the spectral domain and one in the spatial domain (see Appendix for details). From these calibration steps we determined that the depth scale of the OCT was 2.55 μm/pixel in air and 1.85 μm in the retina. Morphometric measurements presented here employ this depth scale.