May 09, 2024

Public workspacePreparation and imaging of lipid bilayer-coated silica microspheres

Nature Methods
DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzjy2rlx1/v1
  • Ezra Bruggeman1
  • 1University of Cambridge
Ezra Bruggeman
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzjy2rlx1/v1
External link: https://doi.org/10.1101/2023.02.07.527479
Protocol Citation: Ezra Bruggeman 2024. Preparation and imaging of lipid bilayer-coated silica microspheres. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjy2rlx1/v1
Manuscript citation:
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
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 99426
Keywords: ASAPCRN, imaging of lipid bilayer, lipid bilayer, instant molecular orientation microscopy for the life science, lipid vesicle, microscopy, lipid extrusion, scale glass bead, instant molecular orientation, coated micrometer, 3d localization of dipole, tip probe sonicator
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000509
Abstract
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:


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).

Materials
Reagents:
  • DPPC in chloroform (850355C, Avanti Polar Lipids, sold by Merck). Note: solid at -20 °C.
  • Cholesterol (C8667-5G, Sigma-Aldrich). Note: will need to be diluted in chloroform, after which it is liquid at -20 °C.
  • Chloroform (366927, Sigma-Aldrich). Note: chloroform is toxic (SDS).
  • Tris buffer: 100 mM NaCl, 10 mM Tris base, pH 7.4
  • Tris-Ca2+ buffer: 100 mM NaCl, 3 mM CaCl2, 10 mM Tris base, pH 7.4
  • 5 μm diameter silicon dioxide (SiO2, i.e. silica) microspheres (44054-5ml-F, Sigma-Aldrich)
  • 0.01% poly-L-lysine solution (P4707, Sigma-Aldrich).

Equipment:

Other:
  • 0.02 μm syringe filters (6809-1102, Whatman) with compatible syringes.
  • Glass viles with PTFE (Teflon) cap liners (14-955-327, Thermo Fisher Scientific, or 600460, Avanti Polar Lipids, sold by Merck). If you don't have vials with PTFE caps, you can use PTFE sealing tape (Z104388-1PAK, Merck) before screwing a regular cap on a vial.
  • Parafilm (P7543-1EA, Sigma-Aldrich)
  • Cover glass.
  • Frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad).
Troubleshooting
Preparation of reagents
Prepare Tris buffer: 100 mM NaCl, 10 mM Tris base, pH 7.4
Prepare Tris-Ca2+ buffer: 100 mM NaCl, 3 mM CaCl2, 10 mM Tris base, pH 7.4
Filter 50 ml of Tris buffer and Tris-Ca2+ buffer using a 0.02 μm syringe filter (6809-1102, Whatman).
Preparation of DPPC + 40% cholesterol lipid vesicles
1h 22m
Dissolve DPPC (850355C, Avanti Polar Lipids) in chloroform (366927, Sigma-Aldrich) to a concentration of Concentration25 mg/mL in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need Amount23 µL per sample.

Dissolve cholesterol (C8667-5G, Sigma-Aldrich) in chloroform (366927, Sigma-Aldrich) to a concentration of Concentration10 mg/mL in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need Amount20 µL per sample.

Prepare a DPPC + 40% cholesterol mixture by combining Amount23 µL Concentration25 mg/mL DPPC with Amount20 µL Concentration10 mg/mL cholesterol in a new glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific).
Evaporate the solvent DurationOvernight under vacuum.

Re-hydrate the lipids/cholesterol mixture using Amount1 mL of Tris-Ca2+ buffer (Concentration100 millimolar (mM) NaCl, Concentration3 millimolar (mM) CaCl2, Concentration10 millimolar (mM) Tris base, Ph7.4 ).

Duration00:40:00 Vortex for Duration00:00:30 .

40m 30s
Sonicate the solution using a tip sonicator for Duration00:40:00 (cycles of 45 seconds on and 15 seconds off, 60% amplitude) until the solution runs clear.

40m
Centrifuge the solution for Duration00:01:30 at Centrifigation14000 rcf to remove residu from the sonicator probe. Keep the supernatant.

1m 30s
Coating silica microspheres with the DPPC/40% cholesterol mixture
35m
Dilute the glass beads (5 µm diameter, 44054-5ML-F, Sigma-Aldrich) to approximately Concentration2.8 mg/mL .

Clean the beads by centrifuging and replacing the supernatant with Tris-Ca2+ buffer.
Heat the glass beads and the lipid vesicle solutions to Temperature65 °C using a heated water bath.

Once heated to Temperature65 °C , mix the glass bead and lipid vesicle solution together in a 1:1 ratio. Leave the mixture at Temperature65 °C and wait for Duration00:30:00 .

30m
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.
Gradually replace the buffer by Tris (100 mM NaCl, 10 mM Tris base, pH 7.4):
Centrifuge for Duration00:05:00 at Centrifigation0.3 rcf .

5m
Gently replace two thirds of the supernatant with Tris.
Repeat the last two steps (14.1 and 14.2) a total of 6 times.
Store the lipid-coated glass beads at Temperature4 °C and use as soon as possible, or within a week of preparation.

Imaging
50m
Argon plasma clean cover glass (VWR collection, 631-0124) for Duration00:30:00 in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).

30m
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.
Pipet Amount70 µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for Duration00:15:00 . The PLL will coat the surface of the cover glass.

15m
Use a pipet to remove the excess PLL from the well and immediately replace it with Amount70 µL of filtered PBS.

Use a pipet to remove the excess filtered PBS from the well and immediately replace with Amount70 µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

Use a pipet to remove the excess PBS from the well and immediately replace with Amount50 µL of the lipid bilayer-coated beads. Wait for Duration00:05:00 .

5m
Use a pipet to gently remove the excess PBS from the well and immediately replace with Amount50 µL of a dye of choice, e.g. Concentration1 nanomolar (nM) Nile red for PAINT of the lipid membrane.

Image the sample the same day.