TITLE: Sample preparation for single molecule localisation microscopy and imaging.
AUTHORS: gurvir.virdi

[DESCRIPTION]
This protocol describes the sample preparation to perform SMLM on midbrain dopaminergic neurons using phalloidin and a DNA aptamer in a combination of STORM and DNA-PAINT. 
It also describes the experimental set up on the microscopy with the image acquisition details.

[STEPS]
SECTION: Fixation
1. For single molecule localisation microscopy (SMLM), neurons were grown on glass coated ibidi chambers.
SECTION: Fixation
2. Once neurons reached the desired age, they were washed once in PBS
SECTION: Fixation
3. They are then fixed for 15 min  in 4% paraformaldehyde + 0.1% glutaraldehyde (both from Electron Microscopy Services) in PBS at room temperature.

SECTION: Fixation
4. The neurons were then reduced in 0.1% sodium borohydride (Sigma) in PBS for 7 min at room temperature.

SECTION: Labelling cells
5. 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 10 min  at room temperature

SECTION: Labelling cells
6. 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 120 min  at room temperature

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


SECTION: Labelling cells
8. After incubation, cells were washed 1x in PBS
SECTION: Labelling cells
9. They are then incubated with phalloidin-647 (1:400) (Thermo Fisher Scientific) made up in the blocking solution for 60 min at room temperature.

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

SECTION: Labelling aggregates in cell lysate
11. Cells were lysed mechanically in PBS before being centrifuged at 3600 x g, 5 min, 4 °C .

SECTION: Labelling aggregates in cell lysate
12. The supernatant was collected and the protein concentration was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific).
SECTION: Labelling aggregates in cell lysate
13. 22 x 40 mm, 1 mm thick glass slides, were cleaned with an argon plasma for 60 min  before 22x22 gaskets (Bio-Rad) were affixed to the surface to create a well.

SECTION: Labelling aggregates in cell lysate
14. Cell lysate was diluted 1 in 10 with filtered PBS (0.02 μm) and added to the glass slides
SECTION: Labelling aggregates in cell lysate
15. 100 nM DNA-aptamer was added to the glass slide and incubated with lysate for 10 min .

 

SECTION: Labelling aggregates in cell lysate
16. Sample was then washed off with filtered PBS three times before imaging
SECTION: Microscopy imaging
17. 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. 
SECTION: Microscopy imaging
18. To ensure efficient stochastic blinking for STORM (AF647-tagged phalloidin), the samples were incubated with a blinking induction buffer (B cubed, ONI).
SECTION: Microscopy imaging
19. 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. 

 

SECTION: Microscopy imaging
20. The laser illumination angle was set to 51o for all imaging leading to total internal reflection fluorescence (TIRF).
SECTION: Microscopy imaging
21. 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. 
SECTION: Microscopy imaging
22. 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).