User manual

Manipulator selection

Select the current manipulator with numbers (1, 2).

Ranges of axes

To measure the range (minimum and maximum) of the axes of the unit and microscope, type M, then move the unit axes and microscope Z to all limit positions (min and max for all axes). This can be done for example by moving to two opposite corners. The same must be done for all units and for the XY stage. For the XY stage, note that the calibration process uses the camera and therefore it must be checked that not only the positions are reachable, but that the image is also acceptable (in particular, there must be light). Type M again to end the measurement process.

Calibration

The coordinate systems of the XY stage and manipulators must be matched to the coordinate system of the camera and microscope. This operation is called calibration. The way it works is by matching photos of the pipette tip. Thus, it is very important that the image is clean and sharp at all positions reached by the calibration procedure.

  1. Mount a pipette on the manipulator. In principle, any object could be used, the main requirement being that it should be sharp and with a good contrast when seen under the microscope.

  2. Put water on the coverslip, as much as possible.

  3. Move the microscope as high as possible. If it is an immersion objective, move it up as much as possible while remaining immersed. If it is not an immersion objective, focus on the top of the water drop.

  4. Move the pipette manually with the tip in focus, in the center of field.

  5. Run the calibration (key: C). The program will first move the XY stage, then move each axis of the unit. If the unit is mounted on the stage, it will also make compensatory movements with the stage when the unit is moved beyond the field of view.

The calibration runs a maximum number of calibration_moves exponential moves, which consist in a sequence of movements of the axis, each movement being twice larger than the previous one, with a minimum distance equal to half stack_depth. The program also stops if the next movement would not be reachable by the axes, or if it would go beyond the minimum vertical position (Z). The minimum vertical position for the microscope or floor can be set with key F. It has not been set, then the program chooses 300 um below the current position. Initially, minimum and maximum ranges of axes are not set, which means that all positions are considered reachable.

Save the calibration with Ctrl+S.

Secondary calibration

When the pipette is changed, or if the pipette appears to be miscalibrated, for example after a large movement, then the pipette should be recalibrated. The idea is that the axis angles should be stable but there can be a shift (translation) in the coordinate systems. To recalibrate, move the pipette in focus and right click on the tip.