1.

Introduction

The human retina is composed of several tissue layers with varying optical properties, including scattering, absorption, and refractive index. While the retina primarily contains vertically aligned cells transparent to visible and near-IR light, the pigmented epithelial layer strongly scatters and absorbs light due to the presence of melanin. The posterior choroidal tissue, consisting primarily of vasculature, shows bloodlike wavelength-dependant absorption. The variation of the optical properties of the individual retinal layers produces the fundamental optical coherence tomography (OCT) image contrast. Recently, spectral or Fourier-domain optical coherence tomography (SD/FD-OCT) introduced a new principle with significantly improved sensitivity and scanning speed.^{1, 2, 3, 4} SD/FD-OCT can be realized either by a spectrometer-based^{5, 6, 7, 8} or a rapid tunable source^{9, 10, 11, 12, 13, 14, 15, 16, 17} design. The later is known as optical frequency domain imaging (OFDI) or swept source OCT (SS-OCT). The capability of imaging retinal volumes at higher speeds enables for a reduction in measurement time and motion artifacts, making this second-generation OCT technology more appealing for retinal studies.

The majority of current OCT instruments for retinal imaging applications use wavelengths in the $845\text{-}\mathrm{nm}$ region.^{5, 7, 8, 18, 19, 20, 21} The advantages of imaging the posterior segment at $845\mathrm{nm}$ include minimal optical power loss from the double pass through the vitreous humor and the possibility for ultra-high-resolution imaging using available wideband optical sources.^{22, 23, 24, 25} More recently, an alternative wavelength centered at $1050\mathrm{nm}$ was investigated for retinal imaging.^{26, 27, 28, 29, 30, 31, 32, 33, 34} The increasing interest in imaging at this wavelength is primarily due to the apparent improvement in depth penetration into the choroid compared to $845\mathrm{nm}$ . Other advantages include no visibility of the scanning beam to the subject and improved image quality in patients with cataracts.²⁹ Comparison studies of these two approaches were reported by Povožay ²⁶ and Unterhuber ²⁷ using time-domain systems and by Lee ²⁸ using SD-OCT at $845\mathrm{nm}$ and OFDI at $1060\mathrm{nm}$ . Clinical studies for these two wavelengths have also been reported.^{33, 34}

To increase our understanding of the optical properties and the OCT contrast mechanisms of retinal structures, it is important to quantitatively investigate the backscattered intensity distribution within the individual retinal segments. Hammer ³⁵ systematically studied the optical scattering of four posterior eye segments: the neural retina, the retinal pigment epithelium (RPE), the choroid, and the sclera. Scattering and absorption coefficients $({\mu }_s,{\mu }_a)$ were determined in vitro from bovine/porcine samples using a visible to $1064\text{-}\mathrm{nm}$ spectral range. The study was also followed by an A-line analysis on OCT data.³⁶ In this paper, we directly compare the optical scattering properties from retinal layers scanned in vivo at the two wavelengths. Two equivalent OFDI systems at 1060 and $845\mathrm{nm}$ were used to image the same human subject, generating two sets of volumetric image data. A comprehensive signal-processing algorithm was developed to facilitate a point-based quantitative comparison of the optical backscattered signal from the layered retinal structures.

2.

Material and Methods

3.

Results and Discussion

Figure 1 shows a single pointwise matched frame with the overlaid retinal layer segmentation volumes. The average intensity within each retinal layer volume is plotted in decibel units, as shown in Fig. 2, demonstrating the relative scattering intensities for the two wavelengths. The noise floor at both wavelengths was below $70\mathrm{dB}$ . The red horizontal line in Fig. 2 is the average intensity within the retinal volume delineated by the RNFL and the OS layer, providing relative contrast of the different retinal layers. The respective average scattering intensities and the standard deviations of each layer are listed in Table 1 .

Fig. 1

Typical pointwise matched frame (left: $1060\mathrm{nm}$ , right: $845\mathrm{nm}$ ). The cutout shows a $3\times$ enlarged view of the retinal layers; color-coded overlays show the areas in which the backscattered intensity was calculated for each layer.

Fig. 2

Measured mean backscattered intensity from each retinal layer volume. The horizontal line at $81\mathrm{dB}$ is the mean intraretinal backscattered intensity. RNFL, retinal never fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, photoreceptor inner segment/outer segment junction; U-RPE, upper retinal pigment epithelium; L-RPE, lower retinal pigment epithelium; CH, choroid.

Table 1

Mean backscattered intensity and variance for each retinal layer at 845 and 1060nm in decibel units. (dB).

The most striking differences between the contrast at 845 and $1060\mathrm{nm}$ are the $2.6\text{-}\mathrm{dB}$ smaller reflected intensity in the RNFL and the $5.6\text{-}\mathrm{dB}$ larger reflected intensity in the shallow choroidal tissue at $1060\mathrm{nm}$ as compared to $845\mathrm{nm}$ . Reflected intensities in the GCL, IPL, INL, OPL, ONL, and the boundary between the inner and outer segments of the photoreceptors are very similar. A major difference develops in the RPE. To obtain a more detailed attenuation profile in the RPE region, the layer corresponding to the tip of the RPE and the deeper portion was segmented in two, an upper RPE (U-RPE) and a lower RPE (L-RPE). At the L-RPE the reflected intensity at $1060\mathrm{nm}$ is $4.3\mathrm{dB}$ larger. This suggests that the thin melanin rich RPE tissue, with strong wavelength-dependent absorption, impacts the sensitivity enhancement observed in the choroidal tissue at $1060\text{-}\mathrm{nm}$ light the most. As demonstrated from the scattering and absorption measurements by Hammer,³⁵ the scattering coefficient ${\mu }_s$ is relatively constant for the RPE and only slightly decreases within the intraretinal layers from $780\text{to}1064\mathrm{nm}$ . Retinal tissue anisotropy further reduces the net contribution of ${\mu }_s$ to the total attenuation; however, the absorption of RPE is approximately four times greater at 780 than at $1064\mathrm{nm}$ . Based on the in vitro measurement by Hammer and assuming a $10\text{-}\mu \mathrm{m}$ RPE thickness, the calculated round-trip attenuation yields an intensity difference of $2.4\mathrm{dB}$ following the RPE attenuation. This calculation is less than our in vivo measurement finding of $4.3\mathrm{dB}$ . The backscattered intensity (Table 1 and Fig. 2) suggest that the increased attenuation of $845\text{-}\mathrm{nm}$ light likely begins at a shallower depth (IS/OS junction) before the RPE. From Table 1 we conclude also that $4.3\mathrm{dB}$ of the $5.6\text{-}\mathrm{dB}$ intensity difference for the two wavelengths at the shallow choroid can be attributed to the light path from the photoreceptor layer to and including the RPE.

An important aspect for 3-D volumetric image data of the human retina is segmentation of the different retinal layers for quantitative analysis, e.g., for the automated determination of RNFL thickness maps.^{37, 38} Better contrast between retinal layers improves the robustness of these segmentation algorithms. Table 2 shows the scattering intensity difference or relative contrast between individual retinal layers at 845 and $1060\mathrm{nm}$ . The $1060\text{-}\mathrm{nm}$ acquisition wavelength has a significantly better sensitivity in deeper layers, but from Table 2 we can conclude that the $845\text{-}\mathrm{nm}$ wavelength produces an overall better contrast at the inner-retinal layers, particularly between the RNFL and the GCL layers. At $845\mathrm{nm}$ , the relative contrast between RNFL and GCL is $8.24\mathrm{dB}$ , $2.2\mathrm{dB}$ more than for $1060\mathrm{nm}$ . Therefore segmentation algorithms for the RNFL and GCL might perform better for $845\mathrm{nm}$ wavelength data. The relative contrast between the GCL and IPL was only $0.33\mathrm{dB}$ , making the two layers difficult to discern in $1060\text{-}\mathrm{nm}$ wavelength data.

Table 2

The relative contrast or backscattered intensity difference between adjacent retinal layers for 845 and 1060nm in decibel.

4.

Conclusions

Finely matching the reflectivity of retinal layers from equivalent OFDI systems at two separate acquisition wavelengths enables a quantitative comparison of optical properties such as scattering and attenuation. The relative reflectivity at 845 and $1060\mathrm{nm}$ of nine intraretinal layers and the choroid were measured and compared. The results indicate a relatively weaker signal intensity in the RNFL $(-2.6\mathrm{dB})$ for the $1060\text{-}\mathrm{nm}$ images as compared to the $845\text{-}\mathrm{nm}$ images, but a significantly stronger signal return from the RPE $(+4.3\mathrm{dB})$ and choroid (at least $+5.6\mathrm{dB}$ ). OFDI imaging at the alternative $1060\text{-}\mathrm{nm}$ wavelength reduces the contrast between the RNFL and the GCL/IPL intraretinal layers, but significantly increases the signal at larger depths.

Better contrast between RNFL and GCL from $845\text{-}\mathrm{nm}$ OFDI images might improve the robustness of segmentation algorithms to obtain RNFL layer thickness. It might be difficult to segment between GCL and IPL for $1060\text{-}\mathrm{nm}$ OFDI retinal data. The quantitative reflected intensity comparison at the photoreceptor and RPE layers suggests the absorption from these tissue structures results in larger sensitivity degradation of $845\text{-}\mathrm{nm}$ light at the choroid.