Visualizing the invisible: inner plexiform layer stratification with conventional spectral-domain optical coherence tomography

Abstract

Background: The inner plexiform layer (IPL) of the retina plays a key role in visual processing, consisting of five stratified sub-bands (S1-S5) that segregate ON and OFF visual pathways. Until now, resolving these IPL sub-layers was only possible with experimental high-resolution (HR-OCT) or visible-light OCT (VIS-OCT), which remain inaccessible for clinical use. This study provides the first demonstration that IPL stratification can be visualized using commercially available spectral-domain OCT (SD-OCT) with optimized imaging and grayscale inversion.

Methods: This retrospective, cross-sectional image analysis study included three healthy individuals who underwent macular OCT imaging. Two subjects were imaged with SD-OCT devices (Nidek RS3000 Advance and Zeiss Cirrus 6000), while one subject was imaged with a swept-source OCT (SS-OCT) device (Topcon Triton DRI). High-density B-scans (1024 A-scans per B-scan) with 120 repetitions for noise reduction were analyzed in both standard and inverted grayscale display modes. The impact of scan size (12 mm, 6 mm, and 3 mm) on IPL visualization was also evaluated.

Results: In conventional grayscale, IPL stratification was indistinct. However, inverted grayscale revealed five IPL sub-bands in all cases, particularly in the parafoveal region where the IPL is thicker. Hyperreflective dots near IPL-1, likely representing the superficial capillary plexus, were also identified. The 3-mm scan protocol provided superior sub-layer differentiation compared to 12-mm scans. However, SS-OCT images did not allow for the distinction of the five IPL strata.

Conclusions: This study challenges the belief that IPL stratification cannot be identified with conventional SD-OCT. By refining imaging parameters and using grayscale inversion, this approach enhances retinal circuit analysis with standard technology. While SD-OCT enables detailed IPL visualization under specific conditions, SS-OCT does not appear to be well-suited for this purpose. These findings redefine SD-OCT's diagnostic capabilities, opening avenues for research in ophthalmology and neurodegenerative disease monitoring. Further studies should establish best practices and expand clinical applications for this novel methodology.

Keywords: Inner plexiform layer; Optical coherence tomography; Retinal imaging; Spectral-domain.

Fig. 1

Horizontal 12-mm macular OCT B-scan illustrating the inner plexiform layer (IPL) in standard and inverted grayscale modes. (A) Standard grayscale OCT image acquired using a horizontal 12-mm scan with the Nidek RS3000 Advance device. The scan parameters included ultra-fine resolution, active eye tracking, 1024 A-scans per B-scan, and 120 repetitions of B-scans for digital image enhancement (denoising). In this standard grayscale mode, the differentiation of the IPL into its five distinct sub-bands (IPL 1– IPL 5) is not evident, as the hyper-reflective and hypo-reflective layers within the IPL remain indistinct. Magnified views of the parafoveal region (blue and green boxes) highlight the absence of a clear stratification. (B) Inverted grayscale OCT image of the same horizontal scan. By reversing the grayscale polarity, hyper-reflective regions appear black, and hypo-reflective regions appear white, enhancing the visualization of the IPL’s sub-bands. Partial delineation of hyper-reflective lines corresponding to the IPL strata becomes discernible, as indicated in the magnified parafoveal regions (blue and green boxes). These findings illustrate that inverted grayscale enhances contrast within the IPL, facilitating the identification of its multilaminar architecture

Fig. 2

Horizontal macular OCT B-scan acquired with the Zeiss Cirrus 6000 device illustrating the inner plexiform layer (IPL) in inverted grayscale mode using 12-mm, 6-mm, and 3-mm scan lengths. (A) 12-mm scan with a highlighted area shown at higher magnification in (B). (C) 6-mm scan with a highlighted area shown in (D). (E) 3-mm scan with a highlighted area shown in (F). In all scans, the differentiation of the IPL into its five distinct sub-bands (IPL 1– IPL 5) is visible, with smaller scan lengths providing more evident stratification

Fig. 3

Macular OCT imaging using horizontal and vertical 3-mm scans to visualize the inner plexiform layer (IPL) in high detail. (A) Infrared fundus image illustrating the location of the vertical scan (red arrow indicating the scan direction). (B) Horizontal 3-mm macular B-scan obtained using the Nidek RS3000 Advance device. The scan parameters included ultra-fine resolution, active eye tracking, 1024 A-scans per B-scan, and 120 repetitions of B-scans for digital image enhancement (denoising). The orange dashed rectangle highlights the parafoveal area, shown in greater detail in panel C. (C) Magnified view of the parafoveal area (orange dashed rectangle in panel B), allowing the identification of five distinct sub-bands (IPL-1 to IPL-5) that represent the IPL’s stratification. (D) Detailed annotation of the IPL sub-bands (IPL-1 to IPL-5) within the magnified region (orange solid rectangle in panel C). Adjacent retinal layers, including the ganglion cell layer (GCL) and inner nuclear layer (INL), are labeled to provide anatomical context. (E) Infrared fundus image illustrating the location of the horizontal scan (red arrow indicating the scan direction). (F) Horizontal 3-mm macular B-scan using the same parameters as the vertical scan. The orange dashed rectangle highlights the parafoveal area, shown in greater detail in panel G. (G) Magnified view of the parafoveal area (orange dashed rectangle in panel F), allowing the identification of five distinct sub-bands (IPL-1 to IPL-5) that represent the IPL’s stratification. (H) Detailed annotation of the IPL sub-bands (IPL-1 to IPL-5) within the magnified region (orange solid rectangle in panel G). Adjacent retinal layers, including the GCL and INL, are labeled to provide anatomical context

Fig. 4

Horizontal macular OCT B-scan acquired using a 6-mm scan length in inverted grayscale mode. (A) Inverted grayscale OCT image acquired using a 6-mm scan length with the Topcon Triton DRI device. (B) Magnified view of the region outlined by the orange rectangle in (A). Orange arrows indicate the IPL region where differentiation of the five strata (IPL 1–IPL 5) is not distinguishable

Fig. 5

Retinal Cellular Organization in the Parafoveal Area Demonstrated by SD-OCT and Schematic Overlay. The B-scan spectral-domain optical coherence tomography (SD-OCT) image of the parafoveal region, displayed in an inverted grayscale format (top panel), highlights a specific area delineated by an orange rectangle. This region is magnified in the bottom panel, where a detailed schematic overlay illustrates the retinal cellular organization across distinct layers. The highlighted region includes the ganglion cell layer (GCL), the inner plexiform layer (IPL), and the inner nuclear layer (INL). The schematic overlay in the bottom panel visualizes the ON and OFF systems of the IPL, with retinal neurons color-coded by cell type for clarity. The GCL is composed of ganglion cell bodies (dark blue), which form synapses within strata 1 and 2 of the IPL (ON system) and strata 4 and 5 (OFF system). The IPL also contains processes of amacrine cells (red), bipolar cells (green), and Müller cells (pink), which contribute to maintaining synaptic architecture. Adjacent to the image, a grayscale depiction of retinal layers and boundaries corresponds directly to the SD-OCT reflectance data, providing anatomical and functional context. The INL accommodates the cell bodies of amacrine cells, bipolar cells, Müller cells, and other retinal structures, integral to the retinal microarchitecture