Imaging single SYTOX Orange molecules on a PLL-coated cover glass
Ezra Bruggeman
ASAPCRN
Single-molecule
Single molecules
SYTOX Orange
Microscopy
Imaging
Fluorescence
Fluorescence microscopy
POLCAM
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:
- 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
Argon plasma clean cover glass (VWR collection, 631-0124) for 0h 30m 0s in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
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
1nanomolar (nM).
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 70µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 0h 15m 0s. The PLL will coat the surface of the cover glass.
Use a pipet to remove the excess PLL from the well and immediately replace it with 70µL of filtered PBS.
Use a pipet to remove the excess filtered PBS from the well and immediately replace with70µL 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 50µL of 1nanomolar (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.
