Set the Stage Limits

When first setting up your microscope you should set the limit magnets on the motorized stages to prevent crashes that can break coverslips or worse. This is especially important when using the 24 x 50 mm coverslip chamber, where there is not much lateral travel room and the coverslip glass is easily broken. Although the TG-1000 firmware supports software-defined limits, setting hardware limits using the provided magnets is the most foolproof way to prevent crashes.¹⁾ The limit magnets move with the stage body; when the magnet passes the Hall effect sensors affixed in the stage body the firmware detects a limit condition and stops the stage.

There are several methods of seeing whether the stage is at a limit. One method is to continually query the limit condition over serial, e.g. using the RS <axis>- command; the controller will reply with or for upper or lower limit respectively. Some programs, such as Advanced Serial Port Monitor, allow sending such a command repeatedly for easy monitoring. Another method is to watch the LED of the corresponding drive card on the controller front panel; the LED will flash rapidly when one of its axes is at a limit. Third, a feature is planned for the new version of ASI Tiger Console where an audible beep can be heard when the axis is at a limit.

The lower limit of the upper Z stage (the LS-50 that raises and lowers the SPIM assembly) is the most important limit to set, and also the most difficult. For the typical Nikon 40x SPIM objectives, the objectives nominally sit approximately 250 um above the cover slip, so the limit must be set within 0.25 mm accuracy to allow the needed range while preventing a crash. To set the lower limit of the upper stage:

1. Prepare a coverslip with a feature on the upper surface (e.g. make a small dot with a permanent marker) and place it in the sample holder.
2. Use the lower objective to focus on the top surface of the coverslip (e.g. focus on the dot). If you cannot focus on the top coverslip surface, the limit magnet is probably set too conservatively and you can move it upwards.
3. Gradually lower the SPIM assembly (e.g. using the joystick or knob set appropriately in the Micro-manger diSPIM plugin Navigation tab) until reaching the coverslip, watching carefully for the moment that the objectives touch the coverslip by noting the change in focus in the bottom camera.
4. While monitoring the limit condition (see Section ), move the appropriate limit magnet (the top one on the LS-50, very easily accessible) until the limit condition is reached. There is a range of a few mm over which the limit condition is reported; adjust so that the magnet is on top end of that range, i.e. where the controller reports a limit condition but moving it slightly higher takes it out of limit condition.
5. Check that the hardware limit stops the motion appropriately by moving a mm or so up and then back down the same amount (e.g. by issuing the command followed by or dialing back and forth with a control knob). The stage should stop itself slightly short of the prior position, with the objectives nearly touching the bottom of the sample holder (get the position by issuing the command before and after the move).

It is also important to set the limits of the XY stage to prevent the objectives from crashing into the sides of the sample holder, which will perturb objective alignment. To set the limits of the XY stage:

1. Start with the SPIM assembly lowered down to its limit position as set in the prior sequence. When the XY stage is moved too far the objectives will crash into the sides of the sample chamber.
2. Move the XY stage (e.g. with the joystick) until the objectives are just about to crash (or barely crashing) into the side of the sample chamber. Query the appropriate limit status of the appropriate axis (e.g. or ) and move the limit magnet until the limit condition is barely reached. You may need to turn the limit magnets around so the dots are facing each other. Repeat for all 4 limits.
3. For all of the four directions, check that the hardware limits stop the motion before the objectives crash into the sample holder using the joystick with the fast speed setting.

This step prevents the lower Z stage from damaging itself, which can happen in the lower LS-50 motorized stage is lowered so much that the attached objective mount crashes the cube that is attached to the LS-50 .

This step does not apply to SPIM systems mounted on non-ASI inverted microscopes. For ASI inverted microscopes this limit magnet is generally set during factory assembly.

To set the upper limit²⁾ of the lower Z stage:

1. Gradually lower the lower objective (e.g. using the joystick wheel) until there is a few millimeters of space between the cube mounted to the side of the lower stage and the lower objective holder.
2. While continuously monitoring the limit condition (e.g. ), move the appropriate limit magnet (the top one on the LS-50) until the limit condition is reached. Adjust so that the magnet is on the top end of the range. If needed, the magnet can be flipped around so that the set screw doesn’t attach in the region where the magnet track is expanded.
3. Check that the limit stops the motion appropriately by moving a mm or so up and then back down the same amount (e.g. by issuing the command followed by ). The objective should stop itself slightly short of the prior position, well before a crash could occur.

This step is less critical because the lower Z stage (the LS-50 that raises and lowers the inverted objective) is unlikely to be moved around much, but it is still recommended to set its hardware limit to prevent crashing the objective into the bottom of the sample chamber. The limit can be set anywhere between the focus point of the lower objective in the sample and where it touches the sample chamber bottom, typically 1 to 3 mm depending on the working distance of the lower objective.

This step does not apply to SPIM systems mounted on non-ASI inverted microscopes.

To set the lower limit³⁾ of the lower Z stage:

1. Raise the SPIM assembly up a few mm from the coverslip in the sample holder so the subsequent steps won’t break the coverslip.
2. Gradually raise the lower objective (e.g. using the joystick wheel) until the sample holder begins to move upwards, being pushed by the lower objective. Watch the sample holder from the side to see its vertical movement. Lower the objective until the sample holder is stationary again.
3. While continuously monitoring the limit condition (e.g. ), move the appropriate limit magnet (the bottom one on the LS-50) until the limit condition is reached. Adjust so that the magnet is on the bottom end of the range. If needed, the magnet can be flipped around so that the set screw doesn’t attach in the region where the magnet track is expanded.
4. Check that the limit stops the motion appropriately by moving a mm or so up and then back down the same amount (e.g. by issuing the command followed by ). The objective should stop itself slightly short of the prior position, but still very close to the bottom of the sample holder.