A Comparative Study of High-Contrast Fluorescence Lifetime Probes for Imaging Amyloid in Tissue

Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant alpha-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to alpha-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to alpha-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and alpha-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for alpha-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.

Automated summary

This study compares the use of a novel fluorescent probe, PAP_1, with the traditional Thioflavin T as a fluorescence lifetime imaging probe for imaging alpha-synuclein aggregates in brain tissue. Despite lower brightness, PAP_1 has a much stronger affinity for alpha-synuclein aggregates, leading to a significant decrease in fluorescence lifetime and improved contrast between background tissue and aggregates.