Preparation

1. The surface of coverslips is hydrophobic which means, liquid droplets do not spread out easily and beads tend to aggregate at the edges. For PSF measurements you want individually spread out beads.
2. Bath the coverslips for 10 minutes in 100% ethanol.
3. Use forceps to remove the coverslip. Shake off excess liquid and run through bunsen burner flame.
4. This makes the surface slightly hydrophilic which means, the droplets and beads spread out easier.
5. Ideally, use ZEISS coverslips with a defined thickness of 170 μm. However, coverslips and mounting media for bead measurements must be identical to the ones used for the sample, the image of which shall be deconvolved. Beads should have a diameter below the resolution limit of the objective, e.g. 0.175 µm. Smaller diameters are better, but smaller beads are dimmer and can therefore be difficult to locate on the cover slip.
6. Tetraspeck beads from Thermo Fisher Scientific have the advantage of covering four colors which are frequently used in imaging research, but some batches can show rapid loss of fluorescence. Single color beads are typically brighter.
4. Break up agglomerates by sonicating stock suspension in a waterbath for 20 minutes. Stocks suspensions are way too dense, so dilute 1:100 with 70% ethanol.
5. Create further dilutions of 1:1.000 and 1:10.000 by adding 100 µl to 900µl 70% ethanol. Mix well using a Vortex mixer.
6. Put one 5 µl drop for each dilution on a cover slip using a 20 µl Eppendorf pipette
7. Let dry. This should take less than 5 minutes. You can speed it up by putting the cover slip on a warm surface.
8. Put 10-20 µl mounting medium on the coverslip. For aqueous mounting media seal edges of coverslip with valap (1:1:1 mixture of vaseline, lanolin, paraffin), nailpolish or paraffin.

1. In the next step, you acquire an image.

Imaging

1. Locate the beads on the microscope (e.g. use a 20 x lens first, then move to 63x oil). Start at the 1:100 spot which should have tons of beads and be easy to find.
2. When found, move to a sparser spot and try to find an area with a couple of single beads in the FOV. This can be tricky, but usually you will be able to find good areas if you keep looking around.
3. Acquire a Z-stack of a suitable area, observing the following rules.
4. No saturation. This can be tricky with LSM’s. Use 12-bit mode at least.
5. Only fill the dynamic range in the histogram to about 80%.
6. Make sure to focus up and down when setting up the exposure times to measure the exposure suitable for the bright bead center to avoid the risk of saturation.
4. Set up the z-stack as follows: in the Z-stack tool, click on the Optimal button.
   
5. This sets the distance according to Nyquist. For bead measurments further reduce the slice distance to about half of what Optimal suggests.
5. Also, define the top and bottom of the stack in such a way that the airy disc of the beads cannot be distinguished any longer.
6. When ready, save and name the image properly.

1. Look at the result in OrthoView: Do you see spherical aberrations? Are the beads symmetrical? Are there enough individual beads in the stack? Is the background low enough?

Processing in ZEN

1. Use the PSF wizard (Processing/Deconvolution) to extract the PSF from the bead-z-stack. For more information, see Creating a PSF - With Wizard and Without.

1. The PSF wizard guides you step by step through the necessary procedure.
2. The result of the wizard is a PSF file which you use in deconvolution for images acquired under the same conditions.