images jumping around a bit

Jim, this issue can be related to one of three primary issues.

1) Ultrasonic Motors in Lens – You will want to confirm the ultrasonic image stabilizers are in the off position when shooting with microscope objectives attached. The reason is because the stabilizer is designed to trigger when the camera is being exposed to light. Ever time this happens, there is an internal mechanism that braces internal optics. The result is a stable lens when a photo is being captured which generates an overall sharper image less prone to lens blur. This does not work when stacking because the mechanism stabilizing the glass will always be in a slightly different position between frames and over a period of time. In the case of stacking, you do not want anything touching or altering the lease between shots and because the system is supported by a rugged tripod, the need for stabilization is not necessary or recommended.

2) Not enough pictures – Serene Stacker is phenomenally good at aligning features in images, even if the there is a slight wiggle between frames. Therefore, the simple solution is to capture a greater number of images. Allow for 50-75% of overlap. For example, at 50x, i generally take between 200-300 images to complete a stack. The patience it requires is far more efficient than a do-over.

3) High Precision Mode – there isa mode in the stackshot controller called “High Precision Mode.” This will help fix this issue without having to allow for greater overlap. The difference is that in normal mode the rail position is accurate to roughly 0.005 mm uncertainty, while in high precision mode the accuracy is increased to roughly 0.001 mm uncertainty. Obviously high precision mode is better for fine work, such as through a high power microscope objective where the nominal step size may be only 0.010 mm or less. However, high precision mode has the drawbacks that 1) it causes the stepper motor to produce a nearly continuous high-pitched whining sound that can be annoying to people around it, and 2) the motor drive currents are larger so the motor will become warm. Often a good strategy is to set the high precision threshold to something like 0.020 mm so that large steps are done in normal mode and the rail stays cool and quiet except when it is actually moving, while small steps are done in high precision mode where the increased accuracy is required.