Efficient removal of naturally-occurring autofluorescence from human nervous system tissue using high intensity white light

Authors

  • MR Sapio
  • DM King
  • SR Shah
  • D Maric
  • T Talbot
  • A Ghetti
  • MJ Iadarola
  • AJ Mannes

Abstract

Autofluorescence in human tissue prevents optimal RNA fluorescence imaging and accurate quantification of signal. One pervasive source of autofluorescence is lipofuscin, an age-associated agglomeration of oxidized lipoprotein, which emits light strongly at 400-650 nm, interfering with signal detection in critical wavelengths for multiplex microscopic imaging. This study aimed to improve the signal-to-noise ratio of multiplex fluorescent in situ hybridization (FISH) experiments in human nervous system tissue. We first explored chemical methods to reduce lipofuscin, as these methods are rapid and inexpensive to implement. While chemical methods were successful, we were unable to achieve reduction of autofluorescence without impact to assay signal, which led us to focus primarily on light-based approaches using phosphor-coated white light-emitting diodes (LEDs). Formalin-fixed paraffin embedded human dorsal root ganglion was used to establish the photobleaching protocol, with most of the benefits observed by 24 hours, and a maximum effect by 72 hours. After photobleaching, multiplex FISH was completed to assess improvement in signal-to-noise. This resulted in an approximately 5-fold decrease in autofluorescent signal in several wavelengths compared to untreated controls, while having negligible impact on assay sensitivity or tissue quality. Similar photobleaching methods have been used with lower light units/mm2 and/or different spectral peaks, though notably the existing literature is unclear about which wavelengths and/or intensities are safe to use for RNA assays. Our usage of phosphor-coated blue LEDs is aligned with the maximal absorbance wavelength of lipofuscin (~400-420 nm), and provides a simple method for removing autofluorescence, enabling multiplex imaging in highly autofluorescent human tissues.

Scientific Focus Area: Neuroscience

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