Efficient removal of naturally occurring lipofuscin-derived autofluorescence in human nervous tissue using high intensity white light

Authors

• MM Jezek
• L Wang
• S Baek
• HE Arda

Abstract

Autofluorescence in human tissue impedes optimal multiplex fluorescence imaging and accurate signal quantification. One pervasive source of autofluorescence is lipofuscin, an age-associated agglomeration of oxidized lipoprotein, which absorbs and emits maximally between 400 and 600 nm which are critical wavelengths for multiplex imaging, preventing full utilization of the technique. When lipofuscin is present alongside target signal, signal detection is occluded, especially for low-expressed targets.

Previous experiments done on formalin-fixed paraffin embedded human dorsal root ganglion (DRG) tissue (healthy controls, AnaBios Corp.) showed that prolonged exposure to high intensity white light prior to multiplex staining effectively photobleaches the autofluorescence, substantially reducing lipofuscin intensity. Because the samples were paraffin embedded and remained at 2˚C for the duration of the exposure, the tissue RNA was minimally impacted. This was assessed by measuring fluorescence intensity in DRG using four Opal dyes in a 4-plex TSA-amplified in situ hybridization protocol. Subsequently, we tested the rigor of the technique on brain tissue from patients with Alzheimer’s Disease. Because lipofuscin accumulates in direct association with age, samples taken from older individuals tend to be greatly affected. Additionally, Alzheimer’s pathogenesis causes especially high levels of autofluorescent plaques, often being so severe as to limit the accuracy of data interpretation.

Our preliminary data shows effective photobleaching of the Alzheimer’s tissue, thereby validating this technique for both healthy and pathologic human samples. The technique can be used on multiple slides simultaneously which enhances its utility for comparative experiments evaluating multiple disease states, brain regions and treatment conditions.

Scientific Focus Area: Neuroscience

This page was last updated on Monday, September 25, 2023