TITLE: Preparation and imaging of lipid bilayer-coated silica microspheres
AUTHORS: Ezra Bruggeman

[DESCRIPTION]
This protocol describes the preparation and imaging of lipid bilayer-coated micrometer-scale glass beads. This protocol was used to generate the data presented in Figure 2d-g in the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

The described protocol is based on a previously published protocol that uses lipid extrusion instead of a tip probe sonicator to generate lipid vesicles:

Tingting Wu, Jin Lu and Matthew D. Lew. pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters. bioRxiv (2022). https://doi.org/10.1101/2021.12.30.474544

[GUIDELINES]
Chloroform is toxic (SDS).


[STEPS]
SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
4. Dissolve DPPC (850355C, Avanti Polar Lipids) in chloroform (366927, Sigma-Aldrich) to a concentration of 25 mg/mL in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need 23 µL per sample.

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
5. Dissolve cholesterol (C8667-5G, Sigma-Aldrich) in chloroform (366927, Sigma-Aldrich) to a concentration of 10 mg/mL  in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need 20 µL per sample.

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
7. Evaporate the solvent   under vacuum.

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
6. Prepare a DPPC + 40% cholesterol mixture by combining 23 µL 25 mg/mL DPPC with 20 µL 10 mg/mL cholesterol in a new glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). 
SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
8. Re-hydrate the lipids/cholesterol mixture using 1 mL of Tris-Ca2+ buffer (100 millimolar (mM) NaCl, 3 millimolar (mM) CaCl2, 10 millimolar (mM) Tris base, pH 7.4).

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
9. 40 min Vortex for 30 s.

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
10. Sonicate the solution using a tip sonicator for 40 min (cycles of 45 seconds on and 15 seconds off, 60% amplitude) until the solution runs clear. 

SECTION: Preparation of DPPC + 40% cholesterol lipid vesicles
11. Centrifuge the solution for 90 s at 14000 rcf to remove residu from the sonicator probe. Keep the supernatant.

SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
12. Dilute the glass beads (5 µm diameter, 44054-5ML-F, Sigma-Aldrich) to approximately 2.8 mg/mL.

SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
13. Clean the beads by centrifuging and replacing the supernatant with Tris-Ca2+ buffer.
SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
14. Heat the glass beads and the lipid vesicle solutions to 65 °C using a heated water bath.

SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
15. Once heated to 65 °C, mix the glass bead and lipid vesicle solution together in a 1:1 ratio. Leave the mixture at 65 °C and wait for 30 min.

SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
16. Turn the heating element of the water bath off, and let the glass bead/lipid vesicle mixture slowly cool down to room temperature inside the water bath. The lipid vesicles will attach to the glass beads, open and form a lipid-bilayer on the glass surface of the beads.
SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
17. Gradually replace the buffer by Tris (100 mM NaCl, 10 mM Tris base, pH 7.4):
SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
17.1. Centrifuge for 5 min at 0.3 rcf.

SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
17.2. Gently replace two thirds of the supernatant with Tris.
SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
17.3. Repeat the last two steps (14.1 and 14.2) a total of 6 times.
SECTION: Coating silica microspheres with the DPPC/40% cholesterol mixture
18. Store the lipid-coated glass beads at 4 °C and use as soon as possible, or within a week of preparation.

SECTION: Imaging
19. Argon plasma clean cover glass (VWR collection, 631-0124) for 30 min in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).

SECTION: Imaging
20. Create a sample well on the cleaned cover glass by sticking a frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad) on the cover glass.
SECTION: Imaging
21. Pipet 70 µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 15 min. The PLL will coat the surface of the cover glass.

SECTION: Imaging
22. Use a pipet to remove the excess PLL from the well and immediately replace it with 70 µL of filtered PBS.

SECTION: Imaging
23. Use a pipet to remove the excess filtered PBS from the well and immediately replace with 70 µL  filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

SECTION: Imaging
25. Use a pipet to gently remove the excess PBS from the well and immediately replace with 50 µL of a dye of choice, e.g. 1 nanomolar (nM) Nile red for PAINT of the lipid membrane.

SECTION: Imaging
24. Use a pipet to remove the excess PBS from the well and immediately replace with 50 µL of the lipid bilayer-coated beads. Wait for 5 min.

SECTION: Preparation of reagents
1. Prepare Tris buffer: 100 mM NaCl, 10 mM Tris base, pH 7.4
SECTION: Preparation of reagents
2. Prepare Tris-Ca2+ buffer: 100 mM NaCl, 3 mM CaCl2, 10 mM Tris base, pH 7.4
SECTION: Preparation of reagents
3. Filter 50 ml of Tris buffer and Tris-Ca2+ buffer using a 0.02 μm syringe filter (6809-1102, Whatman).
SECTION: Imaging
26. Image the sample the same day.