Fig. 2: Background suppression and high-sensitivity microscopy in human brain tissue.

a, Background suppression is achieved with the autofluorescence quencher Sudan Black (SB). b, Box plots showing the IQR and 5th–95th percentile bounds of autofluorescence (AF) intensity before (median of 4,400) and after treatment with 0.1% SB (median of 333). N = 180 images per sample. c, Before quenching, the fluorescence from Alexa Fluor 568 labelled small aggregates is masked by the background autofluorescence. After quenching, small aggregates can be easily visualized (both images excited at 561 nm, 26 W cm⁻²). d, A high NA objective collects a larger amount of light from the sample. e, The modelled signal-to-noise ratio for imaging punctate objects in post-quenched tissue background at 100× magnification across a range of NAs of objectives. Only at high NA (>1) large aggregates and oligomers become detectable. f, Images of p-syn stained PD tissue with 40× magnification, NA = 0.75 (top) and 100×, NA = 1.49 (bottom). Close-ups show that the same small aggregate is clearly visible at high magnification and high NA. g, Images of p-syn stained PD tissue with no background suppression and low NA (left), background suppression implemented and low NA (middle) and background suppression implemented and high NA (right). Several example puncta are shown in the closeups (oligomers) after background suppression is implemented and a high NA objective is used.