2.1.

Instrument

The OCT system (Fig. 1) is composed of four parts: a light source, reference arm, sample arm, and spectrometer. The light source is a broadband three-module superluminescent diode (T870-HP, Superlum, Ireland), which has a 188-nm bandwidth according to the manufacturer, and is centered at a wavelength of 870 nm. The power of incident light on the human eye is equal to 750 μW, as dictated by the safety standards by the American National Standards Institute. The light is split into the reference arm and sample arm by a 50:50 fiber coupler. The sample arm is interfaced to a slit-lamp microscope for convenient adjustment during imaging alignment. When retinal imaging is performed, an additional ocular lens (Volk 60D) is inserted in the sample arm. An internal fixation target displayed on a liquid crystal display monitor was employed to control the position of the subject's eye. Dispersion matching between the sample arm and reference arm is performed using a set of optical materials (BK7 glass), which are inserted in the reference light path for dispersion compensation. Dispersion matching is especially critical for imaging the posterior segment of the eye.

Fig. 1

Schematic diagram of the ultrahigh resolution spectral-domain OCT system. The retina is imaged by using an additional ocular lens (Volk 60D) in front of the eye. CL1–CL3: collimating lenses; OL1–OL3: objective lenses; PC: polarization controller.

The spectrum of the interference signal was detected by a spectrometer consisting of a collimating lens, a 1200 lines/mm volume holography transmission grating, an achromatic imaging lens with a focal length of 75 mm (Rolyn Optics, Covina, California), and a line array CCD camera (Aviiva SM2). The CCD camera has 2048 pixels with a 14-μm pixel interval and a 24-kHz data transfer rate. The acquired interference spectrum data was transferred to a computer system using a National Instrument image acquisition card (PCI 1428). Data processing was performed using custom-built software.

2.2.

Resolution

As a sort of interferometer, the axial resolution of OCT is determined by the coherence length of the light source, which is the Fourier transfer of the power spectrum of the light source. The axial resolution in air is given as²²

where λ₀ is the central wavelength and Δλ is the full width at half-maximum (FWHM) bandwidth.

A low central wavelength and a broadband light source are required to improve the axial resolution. In our experiment, a broadband SLD was employed as the low-coherence light source. As shown in Fig. 2a, the FWHM bandwidth of the SLD was 188 nm. However, the available bandwidth was narrower than this value because the bandwidth of the fiber optic components, such as the fiber coupler and attenuator, limited the full use of the broad bandwidth of the light source. Furthermore, the profile of the light spectrum was not perfectly Gaussian, so the effective axial resolution of the OCT system would not be calculated directly from Eq. 1. In order to obtain the real axial resolution, the point spread function (PSF) was measured by imaging a mirror at different positions. The FWHM of the PSF, which indicates the resolution of the OCT system, increases as the mirror position is moved farther away from the optical zero delay plane. The PSF at a path length difference of 0.2 mm was 3.1 μm in air. The corresponding axial resolution in tissue was about 2.2 μm as shown in Fig. 2b, assuming the refractive index of the tissue (cornea and retina) is 1.38.

Fig. 2

(a) Spectrum of the SLD used in the OCT system. The FWHM bandwidth was 188 nm. (b) Point-spread function obtained from a mirror at a path length difference of 0.2 mm. The measured resolution at this position is 3.1 μm in air, corresponding to 2.2 μm in tissue (refractive index: 1.38).

2.3.

Scan Depth

In SD-OCT, the spectral range detected by the line-array CCD limits the axial resolution. The spectral range should be large enough to ensure the entire bandwidth of the light source can be detected in the spectrometer. Otherwise, the axial resolution cannot be determined by Eq. 1. In order to achieve optimal resolution, the spectrometer must be designed appropriately, and the spectral resolution must be calculated. The spectral resolution of the OCT spectrometer is determined by the resolution of both the CCD and diffraction grating. The resolution of CCD indicates the wavelength range occupied by one CCD pixel, which is determined by:²³

Based on the spectral resolution of the spectrometer, the axial scan depth can be determined by

2.4.

Sensitivity

Detection sensitivity is a significant aspect of SD-OCT performance. Because most biological tissues are not highly scattering, the intensity of light backscattered or backreflected from the imaged tissues is extremely low. The sensitivity determines the image contrast and the maximum possible imaging depth. We measured the sensitivity of the OCT system by detecting the interference signal of a mirror at different depth positions. A neutral density filter with optical density (OD) of 1.5 is placed in the sample arm to reduce the signal intensity. After processing with zero padding and Fourier transformation,^{25, 26} the axial response was represented in a logarithmic scale as a function of optical path difference [Fig. 3a]. It shows that the signal peak decreased as the image depth increased, indicating the signal-to-noise ratio (SNR) of the OCT system decreased as the position moved farther away from the zero delay plane. The dynamic range of this system, which was about 65 dB at the depth of 0.2 mm, was also found from Fig. 3a.

Fig. 3

Axial response signal on a logarithmic scale (a) and sensitivity (b) at different image depth from the zero-delay plane.

The sensitivity was calculated from SNR as

3.1.

Imaging the Anterior Segment of the Human Eye

We use this SD-OCT to image the anterior and posterior segments of human eyes. The exposure time of a single line of the CCD was set to 36 μs, corresponding to a scan rate of 24,000 A-scans/s. Therefore, the imaging speed was about 12 frames/s when acquiring an image of 2048 × 2048 pixels.

Figure 4 shows an ultrahigh resolution OCT image of the anterior segment of the human eye. Custom software was used to correct the image caused by refraction at the air-cornea interface. The zero delay plane was set at the top of the image; hence, the anterior surface of the cornea was the region of highest sensitivity. In the peripheral region of the cornea, the resolution and contrast were reduced because of the curved shape of the cornea. Certain components of the cornea, such as the epithelium, Bowman's membrane, stroma, and endothelium, were clearly presented in a magnified image of the central cornea, as shown in Fig. 4b.

Fig. 4

OCT image of the anterior segment (a) and the magnified images at the apex (b). EP: epithelium; BM: Bowman's membrane; S: stroma; EN: endothelium.

The edge of a contact lens on the eye was imaged by ultrahigh resolution OCT, as shown in Fig. 5. The conjunctiva and the junction between the cornea and the sclera were visualized. A gap between the corneal epithelium and contact lens was also clear and filled with post-lens tears. This tear film was about 10 μm thick, as measured in the image.

Fig. 5

OCT image of the limbus region underneath a contact lens. The lens edge and post-lens tear film were visualized clearly.

The post-CL tear film may also play a role in CL fitting and ocular comfort. However, the relationship between ocular comfort and tear dynamics remains unclear, mainly due to the lack of an efficient tool for tracking the tear volume and tear exchange underneath the CL. The post-CL tear film is about 2-μm thick in the center and usually more than 10-μm thick in the post-CL gaps.¹⁷ The thick gaps provide an opportunity for tracking tear exchange using OCT-scattering medium to image the post-CL tear film gap (post-CL gap), as shown in Fig. 6. After the OCT-scattering media was instilled on the surface of CL, the scattering media was infiltrated from the surface into the post-CL gap, as shown in Figs. 6b, 6c. One minute later, the tear film gap was imaged again at the same position. Figure 6d shows that the scattering media has been diluted because of the existence of tear exchange between anterior and post lens tear film. This experiment demonstrates that ultrahigh resolution OCT can be applied in the research of CL fitting and ocular comfort.

Fig. 6

Tracking tear exchange underneath the lens. The post-CL gap (yellow arrow) was imaged using our newly developed ultrahigh resolution OCT before and after the instillation of OCT-scattering media (Soothe XP, Bausch & Lomb, Rochester, New York). The light scattering from the 10 × 10 pixels within the gap was quantified and normalized by the background noise. The intensity of the scattering was used to quantify the mixing/exchanging of the tears with the scattering media. The increase of scattering was evident in OCT images after instillation of the media. The inserted graph showed calculated intensity of the scattering in the post-lens tear film, which demonstrated possible tear exchange.

3.2.

Imaging the Posterior Segment of the Human Eye

For retinal imaging, an ocular lens was added in front of the eye in the sample arm. Figure 7 shows an ultrahigh resolution OCT image in gray scale of the human retina in the region of the macula. Three structures (vitreous, retina, and choroid) are presented in this image. In the retina, a total of nine structural layers can be identified by the brightness of the pixels, as shown in Fig. 7a. A narrow white line at the bottom of the outer nuclear layer is the external limiting membrane, which is usually not recognized by commercially available OCT systems. The cloudy structure at the bottom of the image is the choroid, where cross-sections of vascular structures are visible. Based on this picture, we can segment these layers in order to measure the thicknesses of each [Fig. 7b]. In Fig. 8, an optic nerve head was imaged. In the en face view of optic nerve head, the artery, vein, cup, and optic disk are clearly visualized. In the horizontal cross-sectional OCT image, the cup and disk are identified, and the cup/disk ratio is calculated to be 0.73.

Fig. 7

Human macular image acquired by SD-OCT. Retinal sublayers are visualized clearly (a) and can be segmented using the semiautomatic method (b). These sublayers are the RNFL, ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer limiting membrane (OLM), inner segment layer/outer segment layer junction (IS/OS), outer segment layer/retinal pigment epithelium junction (OS/RPE), and RPE+choriocapillaris.

Fig. 8

ONH acquired by fundus photography and SD-OCT. The ONH in the fundus photo (a), matched the en face view obtained with OCT (b). The cup and disk can be seen in the horizontal cross-sectional OCT image (c) and the same image was marked to show the cup/disk ratio (d), which is 0.73. The rim of the ONH was also marked in the image.