Computational protocol: The Properties of Outer Retinal Band Three Investigated With Adaptive-Optics Optical Coherence Tomography

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Protocol publication

[…] The adaptive optics OCT system consisted of two subsystems: a spectral-domain (SD)-OCT subsystem and an AO subsystem based upon a Shack-Hartmann wavefront sensor (SHWS) and voice-coil based deformable mirror (DM).The spectral-domain OCT subsystem consisted of a light source (λ = 783 nm, Δλ = 47 nm; Broadlighter; Superlum, Inc., County Cork, Ireland; only one superluminescent diode of three was used), and a Michaelson interferometer consisting of a 80/20 fiber beam splitter that sent 80% and 20% 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, and the components of the AO system. The reference channel consisted of a collimating lens, five planar mirrors to fold the beam into a compact footprint, a water vial designed to match chromatic dispersion induced by the eye, and a 50-mm lens focusing the beam onto a planar reference mirror. Reflected light from the two interferometer arms was combined again in the fiber coupler and sent to the spectrometer, which consisted of a fiber collimator, transmissive holographic diffraction grating, F-theta lens, and a complementary metal–oxide–semiconductor (CMOS) line scan detector (4096 pixels × 2 lines [736 × 2 used], with 12 bits per pixel, Sprint spL4096-140km; Basler AG, Ahrensburg, Germany). The optical coherence tomography system acquired 250,000 lines/sec. The theoretical axial resolution was 5.7 μm in air and 4.2 μm in the eye (n = 1.38). Axial sampling of the OCT was measured to be 3.46 μm/pixel using a calibration procedure described previously. The optical coherence tomography design employed here is slightly different from that used earlier, with the main goal being increased speed and reduced eye movement artifacts.The adaptive optics subsystem incorporated a SHWS consisting of a lenslet array (diameter 10 mm; lenslet diameter 500 μm; f = 30 mm; Northrop Grumman Corp., Arlington, VA, USA) and an sCMOS camera (2048 × 2048 pixel, Ace acA2040-180km; Basler AG), and a high-speed, high-stroke DM (DM-97-15; ALPAO SAS, Montbonnot-Saint-Martin, France). The adaptive optics system operated at 20 Hz using the full 4 megapixel spots image. The pupil diameter was 6.75 mm, 13.5 mm, and 10 mm at the eye, DM, and SHWS, respectively. Diffraction-limited imaging (wavefront RMS < λ/14, according to the Maréchal criterion) was achieved for all subjects, corresponding to a theoretical lateral resolution of 2.5 μm in the retina (3.4 μm in the air, validated using a 1951 United States Air Force resolution test chart).Custom software controlled the AO and OCT subsystems, written in C++ and Python/Cython/Numpy, respectively. 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 this system is described in detail elsewhere. [...] Analysis of OCT (Heidelberg Engineering) images consisted of measurements of band 3 thickness and the separation between bands 2 and 3. Analysis of AO-OCT images consisted of axial and lateral characterization of band 3. All analysis was conducted on linear scale images using open source tools available in the Python/Numpy/Scipy stack. […]

Pipeline specifications

Software tools Numpy, SciPy
Applications Miscellaneous, WGS analysis