May 08, 2024

Public workspaceImaging single SYTOX Orange molecules on a PLL-coated cover glass V.1

Nature Methods
DOI
dx.doi.org/10.17504/protocols.io.x54v9pbbmg3e/v1
  • 1University of Cambridge
Ezra Bruggeman
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x54v9pbbmg3e/v1
Protocol Citation: Ezra Bruggeman 2024. Imaging single SYTOX Orange molecules on a PLL-coated cover glass. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pbbmg3e/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: 93146
Keywords: ASAPCRN, Single-molecule, Single molecules, SYTOX Orange, Microscopy, Imaging, Fluorescence, Fluorescence microscopy, POLCAM
Funders Acknowledgements:
Aligning Science Across Parkinson's
Abstract
This is a protocol for the preparation of a microscopy sample of single SYTOX Orange molecules on a PLL-coated cover glass. This protocol was used to generate the data shown in Figure 1a, 1b and 1c of the following publication:

Protocol
Protocol
1h 30m
1h 30m
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
In the meantime:
  • Filter phosphate-buffered saline (PBS) using a 0.02 μm syringe filter (6809-1102, Whatman).
  • Dilute SYTOX Orange (S11368, Invitrogen) in filtered PBS to a concentration of Concentration1 nanomolar (nM) .

Note
Always use a new aliquot of SYTOX Orange to prepare the 1 nM dilution, as dye doesn't store well at low concentrations.

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.

Note
Always use a freshly thawed aliquot of PLL. You can aliquot the PLL and store it in a -20 °C or -80 °C freezer.

15m
Use a pipet to remove the excess PLL from the well and immediately replace it with Amount70 µL of filtered PBS.
Note
It is important to always have liquid on top of the PLL-coated glass and not let it dry out.


Use a pipet to remove the excess filtered PBS from the well and immediately replace withAmount70 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 Concentration1 nanomolar (nM) SYTOX Orange (S11368, Invitrogen). The SYTOX Orange molecules will stick to the surface of the PLL-coated cover glass.

Image the sample straight away and make sure it doesn't dry out during imaging.