Quantitative analysis of lipofuscin in neurodegenerative diseases using serial sectioning two-photon microscopy and fluorescence lifetime imaging microscopy.

Lipofuscin, a cellular pigment that accumulates with age, serves as a significant marker of aging. Recently, studies have linked lipofuscin with neurodegenerative diseases, such as Alzheimer's disease (AD). Using an integrated serial sectioning optical coherence tomography (OCT) and two-photon microscopy (2PM) systems, we developed a method to examine the accumulation and distribution of lipofuscin in postmortem human brain samples. Lipofuscin was imaged with 2PM autofluorescence and quantitatively analyzed in specific structures revealed by OCT images. We involved samples from 15 people aged 60 to 90 years, including those with late-stage AD, chronic traumatized encephalopathy (CTE), and controls (NC). We developed a segmentation method for lipofuscin aggregates based on high-pass filtering and adaptive thresholding, achieving a Dice score of 61% using the integrated system at lower resolution when validated against high-resolution fluorescence lifetime imaging microscopy and phasor analysis. Quantitative metrics such as lipofuscin number density, area fraction, and radius were calculated, revealing distinct spatial distribution patterns across different brain regions and neurological conditions. AD cases exhibited a higher accumulation of lipofuscin in the gray matter sulcus regions compared with the controls, represented by the three metrics of density, area fraction, and size. The difference is particularly significant in number density. Furthermore, we discovered that lipofuscin forms layer structures in the cortical gray matter, which may be related to cell distribution in these regions. Further investigation of these areas revealed significant differences in CTE cases, especially in the infragranulary layer sulcus, compared with controls. In contrast to AD cases, the accumulation difference is significant in the sulcus of both the supergranular and infragranular layers compared with controls. These findings provide valuable information on the pathological role of lipofuscin in neurodegeneration.