If you have been making use of Knobby’s spatial calibration button, you can now move on to interacting with Knobby remotely.
The new Knobby scheduler panel looks a bit expanded. The speed (coarse/fine/super-fine), the mode (normal/rotate), and zero (XYZ/XYZA) buttons should be self explanatory. They do exactly the same as if you were to be using them on Knobby. The action you select on the panel will be reflected on Knobby’s screen as well.
The range, steps and frames entries provide a quick way to edit the scheduler’s table if you intend to do a simple z-stack. Range is the extent of the distance in z you want to span, steps is the the size of the step (in um), and frames is the number of frames you want to scan at each position.
Finally, there is new checkbox labeled “Mouse control”. When this is active and you are scanning you can easily move around the sample by clicking the cell you want to bring to the center of the screen, as shown by the video example above. Once you click on a cell, Knobby does the rest. We call this feature click-and-center. This works at all magnifications (even if you change it on the fly). At the same time, scrolling the mouse wheel allows you to focus up and down. The speed of the movement will be controlled by the selected speed. You may want to start at the slowest speed (Super-fine).
The latest Scanbox release switches its positioning mechanism to rely on the 3dconnection wireless SpaceMouse. A single controller provides a more intuitive, responsive and smoother control of microscope position. It is more intuitive because the panning controls align with the axes of the microscope, and a twist motion of the knob allows you to rotate the objective.
With the 3D mouse it is no longer necessary to specify an axis before movement — you just move the joystick in the desired direction and the axes will be selected. It also provides two buttons that allow switching between coarse/fine motion and normal/rotated modes. The interface is and faster through java-based listeners that take care of the motion without interfering with data acquisition and display.
Because the communication and control methods with the motor control box have changed substantially, the older ShuttleXpress wheel will no longer be supported in future releases. So make sure you have your 3D mouse before upgrading to the most recent version of Scanbox. It can be obtained here.
The movement of the microscope is achieved by four motors that control the position of the objective. The motors can be independent or coupled, depending on the operating mode selected in the Position panel in the Scanbox GUI.
There are three positioning modes: normal, rotated and pivot. To explain how these work, we need to define the coordinate system used first. So, imagine yourself standing behind the main vertical holding post of the microscope arm.
In the normal mode, moving along the positive x-axis will move the objective to the right; movement along the positive y-axis will move the objective away from you; moving along the positive z-axis will move the objective up. There is a fourth degree of freedom that comes from the ability to rotate the objective in the (x,z) plane. The angle between the objective and the negative z-axis is defined as a. In the normal mode all these motors are independent of each other. Manipulating each of the controls only changes the axis selected.
In the rotated mode, the x and z controls move the microscope along the x’ and z’ axes, which is obtained by rotating the (x,z) plane by a degrees. The z’ axis is along the line of sight of the objective and x’ is normal to it. In rotated mode, changing position of the x control will move the objective parallel to the x’ axis. In this mode the x and z motors are coupled so that the resulting movement is restricted to the plane normal to the objective. When changing the z control, movement will be along the z’ axis.
Finally, in the pivot move, movement of the x control will be coupled to that of z and the objective angle such that the focus point p remains under focus. To use this mode a quick calibration is required to measure the length between the focal point and the rotation axis of the objective. To do this set your objective to be in the vertical position and zero the position counters. Image some beads at a magnification of x1 and pick one near the center of the screen. Set the mode to normal, place your mouse cursor on top of the bead, and move the it to the left or right using the ShuttleExpress wheel. Now, switch to the a-axis and move the bead back to its original position by rotating the objective. When the bead is in its original position you can read out the movement in the x-axis (in um) and the a-axis (in deg). The ratio of these two numbers, in deg/um, defines the value of ‘pivotk’ in the sbconfig structure. In our system, we have sbconfig.pivotk = 5.9e-4 [deg/um].