Biological Cells Photography Question

[Moriama]

Greetings folks.
I am a bit of an odd bird here as the best thing i know about photography is how to press that small bottom on my cell. fingers crossed, that's going to be changed.
I am a scientist and now we are inspecting a project to see have gone so terribly wrong. the attached picture is from one of our tests in which we pull a living cell inside a small tube and we measures the pulled length. from the picture we can the tip of the pipette is more clear than the root of the tube. we are concluding this means that there is an "in depth" distance between tip and root of the pipette. a diagram is provided in figure 2. the camera which have captured the image belonged to an inverted microscope its placed precisely to be perpendicular to the test plane and focus on that plane, the subject cell is "fairly" in the center of the lens of the camera.
my question is this: could this decrease in picture quality be a result of any factor beside the in depth distance? say some lens feature or any thing like that?
Many thanks

 

[Buckster]

While it's remotely possible that it's an aberration of the lens itself, I strongly suspect it's what you surmise; That the pipette is not exactly perpendicular to the camera and lens. At macro/micro scales and distances like this, depth of field is EXTREMELY thin, and the slightest variation will cause the blur you see.

 

[weepete]

Hi Moriama,

If your diagram is correct it looks to me like your pipette wasnt perpindicular to your camera lens and you need to move it from the actual position into the ideal position to get the whole pipette in the focal plane.

Or it may be possible to use a method called "focus stacking" where the lens (or I assume mircroscope in this case) is refocused between shots so "slices" of focus are taken to cover the image. The in focus areas of each image are then combined in post processing to form one in focus image.

 

[Moriama]

Bless you