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Try to isolate the problem to the extent possible.

Problem Possible Reason Possible Fix
Communication error between software and hardware Power cycle the affected hardware and restart the software
One hardware element not responding Connect to the hardware using manufacturer's software or serial terminal for Tiger controller
Beams only come out of the objective if both beams are turned on Fibers going to the wrong scanner Exchange the fibers
Stage scanning cameras not getting triggered Hardware/firmware out of date Make sure you have Rev F XY card or later, make sure micro-mirror card has jumper on positions 11/12 of SV6
Acquisition images look different than alignment (look like epi view) Camera trigger cables swapped Swap the camera trigger cables
Inconsistent communication with Tiger controller Windows comm port problems Update USB to comm port driver (NOT universal version), disable USB suspend in Windows per Micro-manager recommendations


The piezo objective movers are the most fragile and failure-prone component of the diSPIM. See the wiki page for care and troubleshooting instructions.


If you encounter bugs in the software it is best to contact the developers directly. For Micro-Manager see the wiki page for instructions.


Some users have reported vibrations, e.g. diffraction-size beads will appear as a diagonal smear. Assuming you have the system on a floated air table, this is probably due to a combination of the vibration of the camera fan and the “vibration-ability” of the piezo objective movers. The exact threshold of when vibrations become noticeable depends on the experiment specifics.

There are three approaches to reduce the apparent vibration:

  1. Reduce camera vibration: Try swapping cameras, even with another one that is nominally identical, to see if the problem is reduced; there can be significant variability even within the same brand/type of camera. Some cameras can be water-cooled in which case the fan can be turned off, e.g. Hamamatsu Flash4. Some cameras can turn their fans off, e.g. Andor Zyla (only recommended for bursts of acquisition, e.g. if you acquire for a few seconds every minute). PCO.edge cameras seemed to have the worst vibration initially, but in mid-2015 their internal design was modified to correct this and now all three major camera brands seem roughly comparable.
  2. Mechanically decouple the camera and piezo: You can mount the camera to the air table instead of the the microscope. As of mid-2016 ASI offers a universal air table mount for this purpose, and several groups have rigged this up themselves. There is some evidence that the CDZ-R block used on RAMM-mounted diSPIM reduces the vibration compared with the CDZ-1000 used when the diSPIM is mounted on other inverted microscopes.
  3. Reduce susceptibility of piezo to vibrate: If you have an 2014 or older ASI piezo objective mover you can update to the version introduced early 2015 which is significantly stiffer and hence less prone to vibration. At the same time the mounting scheme was changed which makes alignment significantly easier and offers other minor benefits. The only downside to updating the piezo is that the travel range will be 150 um instead of 300 um. ASI performs such updates at cost, contact them for details. If you are only doing stage scanning the piezos can be eliminated entirely. As of late 2021 ASI offers a bulkier and more expensive piezo objective mover (“s-POM”) which is significantly less susceptible to vibration than the standard “f-POM”.

Out of Bushing Travel

This process only applies to the 2015 and later piezo mounting scheme.

If you are unable to cofocus your objectives because you need to screw the bushings in farther than they will go, disassemble a few pieces and put them back together again. You may only need to do this on the side you are having troubles with, but it may help to do it on both sides. On one side, usually the left, there is a spacer block between the piezo and the male dovetail piece. How that spacer gets bolted onto the piezo can shift things around by a mm or two. You want to assemble them so that the bushing is mounted far from the sample so it will need to be extended more to reach cofocus.

Notice: Before disassembling either objective mount assembly, turn off the controller.

To disassemble the side without the OBLPA (objective lateral positioner assembly), remove off the left objective/piezo/dovetail after loosening the set screw which secures the dovetail. Remove the objective from the bushing. Remove the 4 flathead screws which attach the male dovetail piece to the spacer block and take off the dovetail piece. That will expose two bolts that hold the piezo to the spacer block. Loosen those bolts slightly and then push the piezo so that its lowest edge will be as close to the XY stage and far from the centerline of the microscope as possible when remounted. Retighten those bolts, then reattach the flathead screws and objective and put the dovetail back on.

The procedure for the side with the OBLPA is similar but a bit easier. The dovetail is integrated into the OBLPA so it's just a matter of loosening the two bolts that connect it to the piezo, pushing them relative to each other so that its lowest edge will be as close to the XY stage and far from the centerline of the microscope as possible when remounted. Retighten the bolts and reassemble.

Troubles with scanner

There are two occasional problems people encounter with the scanner, one hardware-related and the second software-related.

If the light sheet scan is asymmetric or the piezo/slice correspondence is good over part of the scan but not the remainder there may be a damaged MEMS mirror.

If the beam/sheet seems to disappear or is wildly offset while using the Micro-Manager plugin then it could be the below software bug.

Damaged MEMS mirror

It seems that occasionally a bit of dust shorts out one of the corners of the MEMS mirror, which limits its travel in part of its normal range. It is often manifest by an asymmetry. This failure mode seems to be stochastic and requires the MEMS mirror to be replaced by ASI.

A good test is to observe the output position optically as it is moved around the center of its travel. If one side moves more than the other then it is almost certainly a damaged MEMS. This can be done by e.g. using the sheet in the Micro-Manager plugin if it's along the sheet axis. In external mode you can move move in equal voltage steps and see whether the resulting optical response is very different on the two sides of center. Observe optically nearly conjugate to the sample/C-mount, either at the sample with a microscope, with the scanner off the microscope and a witness target cut to C-mount size and dropped into the scanner, or else with the scanner connected to a tube lens and then point the beam at a wall many focal lengths away to simulate the far field. At either of these places the MEMS deflection should give a linear displacement but if one region is strongly attenuated then the MEMS mirror is damaged.

Plugin confusion

Go to the Navigation panel. The “scanner sheet” axis should be at the following places depending on the state: - beam disabled: at 4 degrees (to deflect the beam completely) - beam is enabled but not the sheet: at 0 degrees (if you haven't explicitly changed it) - beam and sheet both enabled: changing

In the “plugin confused” situation, the middle of these cases will show 4 degrees instead of ~0 degrees. Normally you can fix by hitting the “go to zero” button in the plugin and then moving on.