Frequently Asked Questions

SPE and SPE2

Which way is up when I adjust the focus on the display?

When you adjust the focus using the mouse's track-wheel it isn't easy to tell whether the stage is moving up or down.

As you lower the Z Galvo stage insert, moving the sample away from the objective lens, the Z Position approaches zero or becomes negative. Likewise the Z Position value becomes greater as you move the sample towards the objective.

Can I use the SPEs to look at colocalisation in specimens?

Yes, but you may need to apply registration correction to your images if you need to colocalise features at very high resolution.

The SPEs have different dichroic mirrors for the laser pairs 405/532 and 488/635, which means that the motorised dichroic mirror block must move position when imaging green and red specimens (or blue and far red specimens). I suspected for a while that there might be a shift in registration caused by the movement of the dichroic mirror block but recent 20-minute stacks of 0.5 micron bead specimens using both the SPE and SPE2 have shown that although there is a small lateral shift between channels this is not linked to the dichroic mirror position because it also occurs between the green and far red channels and these use the same dichroic mirror. The lateral shift is in the order of 50 to 100 nm, which is below the resolution of the highest power lens on the machine. There is also a more pronounced axial shift of about 500 nm between the blue channel (e.g. DAPI, Hoechst) and the other channels. The mis-registrations are similar on both machines. The shifts represent imperfect corrections of chromatic shifts in the optics. Corrections can never be perfect but it may be possible to improve them on the SPEs. We are currently trying to find out whether this is possible.

The lateral shifts are less than the resolution of the highest power lens so it may not be necessary to correct them in all cases, however the axial shift between the blue and the other channels is more pronounced so you would need to correct that if you needed to register blue with everything else.

Correcting chromatic shift using Volocity

What steps should I take to avoid crashes and hangs - especially during time-lapse?

The SPE and SPE2 computers are not very powerful and aren't really designed for demanding applications like time-lapse. Nevertheless there are things you can do to optimise the disc so that errors are less likely.

For general acquisition sessions or time-lapses I would recommend that you do the following:

1. Quit LAS AF and reopen it before starting your session to clear the TEMP folder of old .tmp files. This is especially true if the machine has been on for a long time or if it has been used for overnight time-lapse.

For anyone wanting to do time-lapses I would make the following further recommendations:

1. Defragment the D drive before starting the time-lapse. Defragmenting the drive will make it easier for the software to write files to the TEMP folder. You will need to book time for this because it takes some time to defragment a drive.
2. After the time-lapse has finished save your data, quit LAS AF and defragment the D drive again to undo fragmentation caused during the time-lapse. Again, this may take some time.