Protocol for Tetraspeck Calibration Slide Preparation used for generating Biplane PSF

Updated: 11/21/19

Note – I now add the tetraspeck beads onto my sample. That way the calibration is performed in the same environment, stage, chamber holder, and coverslip as the sample. Therefore, PSF generated in the calibration will resemble the one registered in the actual imaging. For details on how to add the beads follow this link: Day-of-experiment protocol, including fluidics

Adapted from Bruker Vutara

  1. Deposit 100 μl of poly-L-lysine solution (Sigma-Cat# P8920-100 ml) to the center of an 18 mm square size #1.5 (0.17 um thickness) (see note A). While depositing, smear the drop to extend poly-L-lysine coverage.
  2. Leave poly-lysine on the coverglass for 10 minutes, aspirate with a pipette, leaving only a thin layer of liquid, and let air dry (should take ~20 minutes). Once dry, an outline is visible on the coverglass. When it’s about to be dry, you can start the next step.
  3. Aliquot 1 μl of 0.1 um Tetraspeck beads (Molecular Probes #T7279) into a 0.6 mL centrifuge tube, spin down and sonicate for 10 minutes at room temperature (RT).
  4. Dilute the beads to 1:500 in MilliQ or high purity water. Vortex beads and sonicate again for 10 minutes at RT. Vortex beads again after sonication.
  5. Add 15-40 μl (see note B) of 1:500 bead sample onto the center of the 18 mm cover glass. Make sure that the beads are added to the same area as the poly-L-lysine. Let stand for 10 minutes and aspirate with a pipette. You can leave a thin layer, and let air-dry.
  6. Add a small drop of the appropriate objective oil (see note C) using the Immersol mini-brush onto the 18mm square cover glass.
  7. Place coverglass on the slide and seal with nail polish (See note D).


Note A – Coverglass. No. 1.5 square coverslips from Carl Zeiss (Cat# 474030-9000-000) are high performance and precision with coverslip thickness 0.17 mm +/- 0.005 mm and hence are recommended to ensure an optimal PSF. You can also use 22 mm square size #1.5.

Note B – The volume that gives the optimal density will vary depending on the batch of Tetraspeck beads, and the coverage of the poly-L-lysine, and will have to be empirically determined.

Note C – Use Immersol W 2010 Zeiss for water objective, and 2-3 μl of Fluoromount G (Electron microscopy services Cat#17984-25) if using oil objective. Use Olympus silicone oil for Olympus silicone objective.

Note D – Fluoromount G acts also as a sealant so let air dry for at least half an hour before using.

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