Imaging single AF647 molecules immobilised in PVA on a cover glass
Ezra Bruggeman
ASAPCRN
Single-molecule
Single molecules
Microscopy
Imaging
Fluorescence
Fluorescence microscopy
POLCAM
Abstract
This is a protocol for the preparation of a microscopy sample of single Alexa Fluor 647 molecules immobilised in PVA polymer on a cover glass. This protocol was used to generate the data shown in Figure 2i and 1j of 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
Steps
Protocol
Prepare a 1% poly(vinyl) alcohol (PVA) solution:
Filter 100mLMilli-Q water using a syringe filter (0.02 μm syringe filter, Whatman, 6809-1102).
Dissolve 1g of solid PVA into 100mL of filtered milli-Q water in a glass beaker.
Place the beaker on a stirring hot plate and heat the solution to 90°C under continuous stirring for 0h 30m 0s or until the PVA is completely dissolved.
Let the solution cool. Once cooled, filter it using a syringe filter (0.02 μm syringe filter, Whatman, 6809-1102). You can store the filtered 1% PVA solution at 4°C for later use.
Argon plasma clean cover glass (VWR collection, 631-0124) for 0h 30m 0s in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
Dilute Alexa Fluor 647 (AF647) to 500picomolar (pM) in the filtered 1% PVA solution.
Place a clean cover glass into a spincoater (WS-650Mz-23NPPB, Laurell) and spin-cast 10µL of the AF647 solution onto the cover glass for 0h 0m 45s at 3000rpm,0h 0m 0s.
Optional : Seal the sample by sticking it to a standard glass microscope slide (631-1550, VWR) using a double-sided frame-seal slide chamber sticker (9x9 mm, SLF0201, Bio-rad). Make sure the coated side of the cover glass is facing the microscope slide.
