Midinfrared absorbance spectra obtained from spatially inhomogeneous and finite samples often contain scattering effects characterized by derivative-like bands with shifted peak positions. Such features may be interpreted and accurately modeled by Fano theory when the imaginary part of the complex dielectric function is small and Lorentzian in nature—as is the case for many biological media. Furthermore, by fitting Fano line shapes to isolated absorbance bands, recovery of the peak position and pure absorption strength can be obtained with high accuracy. Additionally, for small and optically soft spherical scatterers, recovery of one or the other of constant refractive index or radius (given approximate knowledge of the other) is possible.

To discover the mechanisms underlying the progression of diabetic retinopathy (DR), a more comprehensive understanding of the biomolecular processes in individual retinal cells subjected to hyperglycemia is required. Despite extensive studies, the changes in the biochemistry of retinal layers during the development of DR are not well known. In this study, we aimed to determine a more detailed understanding of the natural history of DR in Akita/+ (type 1 diabetes model) male mice with different duration of diabetes. Employing label-free spatially resolved Fourier transform infrared (FT-IR) chemical imaging engaged with multivariate analysis enabled us to identify temporal-dependent reproducible biomarkers of the individual retinal layers from mice with 6 weeks,12 weeks, 6 months, and 10 months of age. We report, for the first time, the nature of the biochemical alterations over time in the biochemistry of distinctive retinal layers namely photoreceptor retinal layer (PRL), inner nuclear layer (INL), and plexiform layers (OPL, IPL). Moreover, we present the molecular factors associated with the changes in the protein structure and cellular lipids of retinal layers induced by different duration of diabetes. Our paradigm provides a new conceptual framework for a better understanding of the temporal cellular changes underlying the progression of DR.