It sounds like minor collimation errors? These would move the image at different magnification settings, I think.

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artmar09/pj-collimation.html

The Nikon Eclipse is generally not a parfocal microscope, meaning each change of magnification requires refocusing and re-centering.

Hi David,

The graph you've made is awesome, but we'd need some additional information to troubleshoot further:

The Nikon Eclipse covers a broad range of microscopes, so knowing the exact model would help.

The axes "Y" and "X" can be confusing in microscopy due to the potential difference between the on-screen image, the eyepieces and the stage movement. Could you clarify how the image X and Y corresponds to the "real-world" XY? e.g. when you rotate from one objective to another, I would consider this the X direction (i.e. the objective is moving left/right). When you move the sample on the stage towards and away from you, I would consider this the Y direction. Does this match the movement in your image?

Thanks for the replies. 

Romuald, I will try the experiment you suggested and let you know what happens.

James, the exact model is Nikon Eclipse Ti-E.  By x- and y-position, I am referring to the position of the object I'm imaging (a cell) within the image itself.  So x is the horizontal direction and y is the vertical direction in the image, in pixels.

I am attaching some of the actual images so that it's easier to see.  The images differ only by the objective used.  I didn't move the stage or do any refocusing.  The arrow in the image indicates the cell whose position is graphed in my original post.  I positioned the cell at the center for the 10x image, but as you can see its position drifts to the right as I change to other objectives.

In case it matters, the 10x, 20x, and 40x objectives are from the Plan Apo Lambda product line.  The 60x image was acquired using the 40x objective along with the 1.5x setting on the microscope.

Thanks David.

I might be chasing a red herring here, but this is what comes to mind...

Does the XY position in the image correspond with the XY axis in the real world?

i.e. when you change to the next objective, does this correspond to X in the image or when you move left/right with the stage, does this correspond with X in the image?

I'm trying to establish if it's the attachment of the nosepiece onto the focus motor. I've seen cases (on the TiE) where the nosepiece (holding the objectives) is not fully secured into the focus drive (not blaming Nikon here - I probably built the microscope myself without fully checking it!), causing the objectives to droop toward the user slightly.

From what I recall, this resulted in parcentricity issues in the (real-world) Y axis. Depending on how the camera is mounted of course, this could show as either X or Y movement.

This is also trivial to check if you are able to remove the stage. The nosepiece is held on with two 4mm hex screws and has a tendency to droop forward, especially on the PFS model. It needs to be held / supported whilst securing the hex screws otherwise there is a risk it will not be sitting flat.