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Table of Contents
Troubleshooting
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| Stage scanning cameras not getting triggered | Hardware/firmware out of date | Make sure you have Rev F XY card or later, make sure micro-mirror card has jumper on positions 11/12 of SV6 |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 |
Piezos
The piezo objective movers are the most fragile and failure-prone component of the diSPIM. See the wiki page for care and troubleshooting instructions.
Software
If you encounter bugs in the software it is best to contact the developers directly. For Micro-Manager see the wiki page for instructions.
Vibration
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:
- 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.
- 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.
- 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.
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.
