Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration

Abstract

PurposeWe investigated the autofluorescence (AF) signature of the microscopic features of retina with age-related macular degeneration (AMD) using 488 nm excitation.MethodsThe globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496–610 nm, 6 nm resolution). The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch’s Membrane, as well macroscopic features, were considered.ResultsOverall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch’s Membrane across all the tissue cross-sections (p = 0.02).ConclusionsThe AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation) compared with the control tissue. The magnitude of these differences is small (~4 nm) and highlights the potential challenges of detecting these subtle spectral differences in vivo.

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell

“…In this study, we opted to investigate the autofluorescence at two wavelengths (530 and 585 nm),because the characteristic pattern of fluorescence detected from the human lipofuscin granules [ 25 , 51 ] or in patients [ 52 ], exhibits a broad peak around 580–620 nm, and, therefore, the inclusion of 585 nm would provide a better correlation with the fate of human lipofuscin in vivo. Furthermore, a recent study established an autofluorescence peak of 568 nm in AMD eyes and 572 nm for control eyes, values much closer to a wavelength of 585 nm than to 530 nm [ 53 ]. Generally, there appeared to be some similarity in fluorescence increases across both wavelengths.…”

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding

“…dExPath removed the autofluorescence from lipofuscin found in senescent brain tissues (43)(44)(45)(46)(47)(48)(49)(50), improving immunohistochemistrymediated detection of intracellular structures. Although other methods exist for the masking or quenching of lipofuscin autofluorescence, such as with Sudan Black B (51), they have been associated with limitations, including interruption of antibody binding and reduction of on-target fluorescence (44,46,49,50).…”

“…Fluorescence microscopy of clinical tissues is often hindered by lipofuscin (43)(44)(45)(46)(47)(48)(49)(50), an autofluorescent (throughout the visible optical spectrum) material that is composed of aggregates of oxidized proteins, lipids, and metal cations and that accumulates in many cell and tissue types (51)(52)(53)(54). We imaged regions with lipofuscin in normal human cortex (ages 19 to 45 years old), in the preexpansion state (Fig.…”

Decrowding Expansion Pathology: Unmasking Previously Invisible Nanostructures and Cells in Intact Human Brain Pathology Specimens

“…We discovered that dExPath has the unexpected capability to remove the intense autofluorescence from lipofuscin aggregates found in senescent brain tissues [42][43][44][45][46][47][48][49] , improving the accuracy of IHC-mediated detection of intracellular structures. While other methods exist for the masking or quenching of lipofuscin autofluorescence, such as with Sudan Black B 50 , they have also been associated with limitations including interruption of antibody binding, and reduction of on-target fluorescence 43,45,48,49 , which are in stark contrast to the enhanced immunostaining results from dExPath.…”