Troubleshooting

The golden rule of troubleshooting is to bisect the issue until the root cause is clear. Below are some hints.

In case of communication errors between software and hardware, first power cycle the affected hardware and restart the software. If that doesn't resolve things make sure you can communicate with the specific piece of hardware using manufacturer's software (or for Tiger controller, with a serial terminal).

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

If the beam/sheet seems to disappear or is wildly offset then it is likely the below “beam displaced” software bug.

If the light sheet scan range 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.

On rare occasion the beam disappears because it gets stuck at the edge of its range. This is a software glitch with a straightforward fix.

Go to the Navigation panel. Depending on the state of the beam/sheet controls the “scanner sheet” axis position should be:

1. beam disabled: at 4 degrees (to deflect the beam completely)
2. beam is enabled but sheet is disabled: at 0 degrees (if you haven't explicitly changed it)
3. beam and sheet both enabled: changing

In the “beam displaced” situation, when the beam is enabled but the sheet is disabled (middle case0 the “scanner sheet” position will be 4 degrees instead of ~0 degrees. It is resolved by hitting the “go to zero” button in the plugin which moves it back to the normal position, and things should proceed as expected from there.

If you find a way to reproduce this “beam displaced” situation please contact ASI. We have spent hours in vain trying to figure out what causes this occasional glitch.

The most common hardware failure of the scanner is when a bit of dust shorts out an actuator on one corner of the MEMS mirror, which is manifest by an asymmetry in scanner. This failure mode seems to be stochastic and can happen after months or years of use without any problem. It requires the MEMS mirror to be replaced by ASI. The damage can occur with either the “slice axis” or “sheet axis”.

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.

Observe the beam or sheet optically conjugate to the sample/C-mount. There are a few ways of doing this. One is at the sample with a microscope in epi view and a uniform sample. Another is with the scanner off the microscope and a witness target placed near the C-mount image plane (decent at the outside edge of the scanner, better at the lip at the bottom of the threads, and best 17.5mm inside the outside edge. Another is with the scanner connected to a tube lens and pointed at a wall several meters away. Then move the MEMS from the center position. This can be done either by moving the static position of the beam or else adjusting the amplitude of a continuous movement, either with serial commands or using the plugin. The displacement of the beam should be linear with commaded MEMS deflection, but if one region is strongly attenuated then the MEMS mirror is damaged.

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 Micro-Manager see the wiki page for reporting instructions.

If there is inconsistent communication with Tiger controller make sure you have the recommended Windows driver (NOT universal version).

Also make sure you disable USB suspend in Windows per Micro-manager recommendations.

If stage scanning is not getting triggered look at the instructions on the page about stage scanning.

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”.

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.

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.

If the beams only come out of the objective if both beams are turned on then likely fibers going to the wrong scanner and the fibers should be exchanged.

If acquisition images look like the epi view in live mode instead of the imaging view then the camera trigger cables need to be interchanged.