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

Ricardo Luz Leitão Guerra; Luiz Roisman; Jay S Duker; Giuseppe Querques; Luiz Filipe Adami Lucatto; Emmerson Badaró; Gabriel Castilho S Barbosa; Eduardo Amorim Novais

doi:10.1186/s40942-025-00692-3

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

Int J Retina Vitreous. 2025 Jun 15;11(1):65. doi: 10.1186/s40942-025-00692-3.

Authors

Ricardo Luz Leitão Guerra^{1

2}, Luiz Roisman^{2

3}, Jay S Duker^{4

5}, Giuseppe Querques^{6

7}, Luiz Filipe Adami Lucatto^{2

3}, Emmerson Badaró^{2

3}, Gabriel Castilho S Barbosa^{8

9

10}, Eduardo Amorim Novais^{2

3}

Affiliations

¹ Retina Department, Leitão Guerra - Oftalmologia, Salvador, Brazil.
² Orbit Ophthalmo Learning, Salvador, Brazil.
³ Department of Ophthalmology, Federal University of São Paulo, São Paulo, Brazil.
⁴ New England Eye Center, Tufts Medical Center, Boston, MA, USA.
⁵ Tufts University School of Medicine, Boston, MA, USA.
⁶ School of Medicine, Vita-Salute San Raffaele University, Milan, Italy.
⁷ Division of head and neck, Medical Retina & Imaging Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
⁸ Orbit Ophthalmo Learning, Salvador, Brazil. gabrielcastilho.oftalmo@gmail.com.
⁹ Department of Ophthalmology, University of São Paulo, São Paulo, Brazil. gabrielcastilho.oftalmo@gmail.com.
¹⁰ Cole Eye Institute, Cleveland Clinic, Cleveland, OH, USA. gabrielcastilho.oftalmo@gmail.com.

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.