Epi-Fluorescence Imaging Help

Hi All,

We have created a custom epi-fluorescence microscope using the delta stage main body and a custom made filter cube wheel. The distance between the picamera and the achromatic lens is exactly the same and the objective has been moved further away. We believe there is stray light bouncing around between the dichroic lens, the emission lens and the camera. When imaging with the filter lenses there is a green blob/circle appearing in the middle of the image. However, when there are no filter lenses between the camera and objective no such blob appears. Any advice on how to fix this problem would be greatly appreciated!

Here are some image examples of the problem.

The above image has filter lenses in place and a white LED located above the sample.

The above image has a blue dichroic, a green emission filter and a blue light source that hits the 45degree dichroic.

We believe we have fixed this error by changing the flat-field correction settings in the openflexure software.

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Hi, Could you share the process to build your custom microscope?

All being well @robarch may be able to give details from a user. The parts for a fluorescence (or reflection) microscope are in the main STLs of the microscope, but there are no instructions there yet. The Delta stage shares the same design, with instructions for assembling the reflection opitcs here.

Note: You must use the parts from the Microscope full STL download, not from the Delta stage instructions. They are optically the same, but the Delta stage camera is rotated by 45 degrees and the reflection illumination module axis is rotated by 60 degrees (I think) from the microscope version. The STLs also have slightly different names in the two versions, but they should be easy to find.