May 09, 2024
Imaging single AF647 molecules immobilised in PVA on a cover glass
- 1University of Cambridge
Protocol Citation: Ezra Bruggeman 2024. Imaging single AF647 molecules immobilised in PVA on a cover glass. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj5bplx9/v1
Manuscript citation:
POLCAM: Instant molecular orientation microscopy for the life sciences
Ezra Bruggeman, Oumeng Zhang, Lisa-Maria Needham, Markus Körbel, Sam Daly, Matthew Cheetham, Ruby Peters, Tingting Wu, Andrey S. Klymchenko, Simon J. Davis, Ewa K. Paluch, David Klenerman, Matthew D. Lew, Kevin O’Holleran, Steven F. Lee. bioRxiv 2023.02.07.527479, 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: January 09, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93171
Keywords: ASAPCRN, Single-molecule, Single molecules, Microscopy, Imaging, Fluorescence, Fluorescence microscopy, POLCAM
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000509
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
Protocol
Protocol
1h
Prepare a 1% poly(vinyl) alcohol (PVA) solution:
Filter 100 mL Milli-Q water using a syringe filter (0.02 μm syringe filter, Whatman, 6809-1102).
Dissolve 1 g of solid PVA into 100 mL 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 00:30:00 or until the PVA is completely dissolved.
30m
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.
Note
The filter tends to clog if you try to filter too large a volume of 1% PVA in one go. If this happens, filter in separate smaller batches, replacing the filter each time for a new one.
Argon plasma clean cover glass (VWR collection, 631-0124) for 00:30:00 in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
30m
Dilute Alexa Fluor 647 (AF647) to 500 picomolar (pM) in the filtered 1% PVA solution.
Place a clean cover glass into a spincoater (WS-650Mz-23NPPB, Laurell) and spin-cast 10 µL of the AF647 solution onto the cover glass for 00:00:45 at 3000 rpm .
Note
To avoid forgetting which side of the cover glass the sample was put on, you can use a marker to place a small dot in the corner of the cover glass before spin coating.
45s
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.