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 α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to Thioflavin T, PAP_1 binds to α-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 α-synuclein aggregates resulting in a separation of >1.4 standard-deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between high fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.


Figure S1 .
Figure S1.A) Structure of amyloid binding fluorophore PAP_1.B) Surface-plasmon resonance binding affinity curves of PAP_1 and ThT binding to late-stage aggregates of α-Syn.B) These data have been previously published in reference.S1 C) Normalized absorption and fluorescence emission spectra of PAP_1 in PBS buffer and ethanol (EtOH).

Figure
Figure S2.Α) Recapitulation of the decay curve shown in Figure 1D and the corresponding weighted residuals versus time associated with the bi-exponential fit of Bii) PAP_1 on PLL and Bii) PAP_1 bound to αSyn fibrils.

Figure
Figure S3.A) Molecular structure of ThT.B) Intensity and color-coded τFl images of ThT stained αSyn fibrils.C) Comparison of fluorescence decay curves of PLL-control and αSyn bound ThT from single-point measurements with elevated integration time.τFl values and errors were determined from a mean and standard-deviation of ≥ four single-point measurements with the same bi-exponential tail-fitting model used for image analysis.

Figure S4 .
Figure S4.Schematic showing the imaging of αSyn fibrils artificially embedded in HFB brain tissue.A brain-tissue slide was coated with PLL upon which aSyn aggregates were immobilized and subsequently stained with either PAP_1, ThT or syn211-AF647.A glass coverslip was subsequently placed on top and the sample imaged.The relative thicknesses of the layers in this schematic are not to scale.

Figure S5 .
Figure S5.Images showing different regions of PAP_1 stained αSyn aggregates in HFB mouse brain tissue sample with A) fluorescence intensity contrast and B) τFl contrast.Full view intensity and τFl images are maintained at the same contrast (0 photons minimum, 640 photons maximum and 1.9 ns minimum, 4.6 ns maximum respectively).Magnified inset images have been set in contrast independently, values of which are shown in the respective calibration bars.Scale bars for the insets=2 μm.

Figure S6 .
Figure S6.Images showing different regions of ThT stained αSyn aggregates in a HFB mouse brain tissue sample with A) fluorescence intensity contrast and B) τFl contrast.Full view intensity and τFl images are maintained at the same contrast 240 photons minimum, 1740 photons maximum and 1.5 ns minimum, 2.6 ns maximum respectively).Magnified inset images have been set in contrast independently, values of which are shown in the respective calibration bars.Scale bars for the insets =2 μm.

Figure S7 .
Figure S7.Component images showing Ai) fluorescence intensity of PAP_1 stained and Aii) ThT stained αSyn aggregates and Bi) τFl of PAP_1 stained and Bii) ThT stained αSyn aggregates in HFB brain tissue.Dotted lines show spatial positions used to construct profile plots.Profile plots across Ci) PAP_1-stained and Cii) ThT-stained αSyn fibrils against the WT HFB brain tissue showing the spatial variation of intensity (blue) and τFl (orange).Both the intensity and τFl contrast of PAP_1 and ThT stained images have been set independently.

Figure S10 .
Figure S10.Images showing the of Ai) fluorescence intensity and Aii) τFl of PAP_1 stained sonicated αSyn fibrils in HFB.Bar graphs showing the mean and standard deviations of pixel values above and below a constant threshold in the Bi) fluorescence intensity and Bi) τFl domain of PAP_1 stained sonicated αSyn fibrils in HFB in order to illustrate the achieved contrast in each imaging mode upon binding to small aggregates in complex background.

Figure S11 .
Figure S11.Images showing the Ai) fluorescence intensity and Bi) τFl of sonicated fibrils of αSyn stained with Syn211-AF647 in HFB as well as the Aii) fluorescence intensity and Bii) τFl of Syn211-AF647 in HFB as a control.Bar graphs showing the mean and standard deviations of pixel values above and below a constant threshold in the Ci) fluorescence intensity and Di) τFl domain of sonicated fibrils of αSyn stained with Syn211-AF647 in HFB and Ci) fluorescence intensity and Di) τFl domain of the Syn211-AF647 in HFB control.

Figure S12 .
Figure S12.Images showing Ai) fluorescence intensity and Bi) τFl of PAP_1 stained αSyn aggregates and Aii) fluorescence intensity and Bii) τFl of ThT stained αSyn aggregates both imaged in vHFB mouse brain tissue sample.Histograms comparing the pixel values αSyn aggregates and vHFB stained with PAP_1 in the Ci) fluorescence intensity and Di) τFl domains and stained with ThT in the Cii) fluorescence intensity and Dii) τFl domains.Both the intensity and τFl contrast of PAP_1 and ThT stained images have been set independently.

Figure S13 .
Figure S13.Images showing different regions of PAP_1 stained αSyn aggregates in vHFB mouse brain tissue sample with A) fluorescence intensity contrast and B) τFl contrast.Full view intensity and τFl images are maintained at the same contrast (0 photons minimum, 2977 maximum and 2 ns minimum, 4 ns maximum respectively).Magnified inset images have been set in contrast independently, values of which are shown in the respective calibration bars.Scale bars for the insets = 2 μm.

Figure S14 .
Figure S14.Images showing different regions of ThT stained αSyn aggregates in vHFB mouse brain tissue sample with A) fluorescence intensity contrast and B) τFl contrast.Full view intensity and τFl images are maintained at the same contrast (0 photons minimum, 2977 maximum and 2 ns minimum, 4 ns maximum respectively).Magnified inset images have been set in contrast independently, values of which are shown in the respective calibration bars.Scale bars for the insets = 2 μm.

Figure S15 .
Figure S15.Component images showing Ai) fluorescence intensity of PAP_1 stained and Aii) ThT stained αSyn aggregates and Bi) τFl of PAP_1 stained and Bii) ThT stained αSyn aggregates in vHFB brain tissue.Dotted lines show spatial positions used to construct profile plots.C) Profile plots across a Ci) PAP_1 stained and Cii) ThT stained αSyn fibril against the vHFB brain tissue showing the spatial variation of intensity (blue) and τFl (orange).Both the intensity and τFl contrast of PAP_1 and ThT stained images have been set independently.