Sample preparation for single molecule localisation microscopy and imaging.

For single molecule localisation microscopy (SMLM), neurons were grown on glass coated ibidi chambers.

Once neurons reached the desired age, they were washed once in PBS

They are then fixed for 0h 15m 0s in 4% paraformaldehyde + 0.1% glutaraldehyde (both from Electron Microscopy Services) in PBS at room temperature.

The neurons were then reduced in 0.1% sodium borohydride (Sigma) in PBS for 0h 7m 0s at room temperature.

To label cells with the DNA-aptamer and phalloidin, after fixation and PBS washes, cells were permeabilised with 0.25% triton X-100 in PBS for 0h 10m 0s at room temperature

The cells were then blocked in blocking solution (0.1% triton X-100, 10% normal goat serum (Abcam), 10% salmon sperm DNA (Thermo Fisher Scientific)) in PBS for 2h 0m 0s at room temperature

The samples were then incubated with 100 nM of the DNA-aptamer made up in the blocking solution at 4°C overnight.

After incubation, cells were washed 1x in PBS

They are then incubated with phalloidin-647 (1:400) (Thermo Fisher Scientific) made up in the blocking solution for 1h 0m 0s at room temperature.

Cells were then washed 1x in PBS and either imaged, or incubated with DAPI (1:10000) in PBS for 0h 10m 0s at room temperature followed by 2x PBS washes before imaging.

Cells were lysed mechanically in PBS before being centrifuged at 3600x g,4°C .

The supernatant was collected and the protein concentration was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific).

22 x 40 mm, 1 mm thick glass slides, were cleaned with an argon plasma for 1h 0m 0s before 22x22 gaskets (Bio-Rad) were affixed to the surface to create a well.

Cell lysate was diluted 1 in 10 with filtered PBS (0.02 μm) and added to the glass slides

100 nM DNA-aptamer was added to the glass slide and incubated with lysate for 0h 10m 0s .

Sample was then washed off with filtered PBS three times before imaging

SMLM was performed on a Nanoimager super-resolution microscope (Oxford Nanoimaging Ltd) equipped with an Olympus 1.4 NA 100x oil immersion super apochromatic objective.

To ensure efficient stochastic blinking for STORM (AF647-tagged phalloidin), the samples were incubated with a blinking induction buffer (B cubed, ONI).

Separately to this, AD-PAINT was also employed which relies on the addition of an imaging oligonucleotide strand

to the buffer. 1 nM of the imaging strand was added to the B cubed buffer before imaging.

The laser illumination angle was set to 51^o for all imaging leading to total internal reflection fluorescence (TIRF).

AF647-tagged phalloidin was first imaged for 4000-8000 frames using the 640 nm laser (80% power). After this, 4000-5000 frames at 30% power for the 561 nm laser was used to image and super-resolve the aptamer. Both were recorded at a frame-rate of 50 ms.

For imaging aggregates in neuronal lysate using AD-PAINT, 2 nM of the imaging strand was added. Images were acquired on Oxford Nanoimager at 20 frames s^-1,for 8000 frames (20% 635 nm laser power, TIRF).