An Alternative to Dye-Based Approaches to Remove Background Autofluorescence From Primate Brain Tissue
AffiliationUniv Arizona, Evelyn F McKnight Brain Inst
Univ Arizona, ARL Div Neural Syst Memory & Aging
Univ Arizona, Dept Psychol Neurol & Neurosci
KeywordsSudan Black B
MetadataShow full item record
PublisherFRONTIERS MEDIA SA
CitationPyon WS, Gray DT and Barnes CA (2019) An Alternative to Dye-Based Approaches to Remove Background Autofluorescence From Primate Brain Tissue. Front. Neuroanat. 13:73. doi: 10.3389/fnana.2019.00073
JournalFRONTIERS IN NEUROANATOMY
RightsCopyright © 2019 Pyon, Gray and Barnes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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AbstractBrain tissue contains autofluorescing elements that potentially impede accurate identification of neurons when visualized with fluorescent microscopy. Age-related accumulation of molecules with autofluorescent properties, such as lipofuscin, can possess spectral profiles that invade the typical emission range of fluorophores commonly utilized in fluorescent microscopy. The traditional method for accounting for this native fluorescence is to apply lipophilic dyes that are able to sequester these unwanted signals. While effective, such dyes can present a range of problems including the obstruction of fluorescent probe emissions. The present study utilizes aged primate midbrain tissue stained for tyrosine hydroxylase and calbindin to investigate an image processing approach for removing autofluorescence utilizing spectral imaging and linear unmixing. This technique is then compared against the traditional, dye-based autofluorescence sequestration method using Sudan Black B (SBB). Spectral imaging and linear unmixing yielded significantly higher cell numbers than SBB treatment. This finding suggests that computational approaches for removing autofluorescence in neural tissue are both viable and preferential to dye-based approaches for estimation of cell body numbers.
NoteOpen access journal
VersionFinal published version
SponsorsNIA [R01 AG050548, P51 RR000169, F31 AG055263]; McKnight Brain Research Foundation