Hi,
When running the auto calibration on my microscope, flatfield correction with the 4x lens does not work as expected, a pink circle remains in the corners of the image:
I was able to get rid of the pink shadow playing with the illumination and removing the lens from the condenser. But shouldn’t the software also be able to do that with flatfield correction?
Also I am planning to use the openflexure microscope for a project where I need to use a lens with an even lower magnification. Got these 1x and 2x lenses from Aliexpress with 45mm parfocal distance: https://www.aliexpress.com/item/1005005885257912.html?spm=a2g0o.order_list.order_list_main.23.56cb18021e0qZ6
In theory a lens with these specifications should work, right?
The image with the 2x lens looks like this:
So basically I can get a sharp image with this lens, but most of the image is black. Also the object needs to be very close to the lens, much closer than the 23.5mm working distance that were advertised in the product description. What could be the issue here?
The calibration can only go so far, if the illumination is too far from being uniform then it will not work.
You are running up against a hardware issue in the illumination. The microscope is designed to work from 20x to 100x, and is optimised for the higher magnification.
In the illumination, a 100x lens needs a very high illumination angle, but over a small area. You see that small illumination area in your images when you use a low magnification lens. A 2x lens needs a large area of illumination, but does not need steep angles. Replacing the condenser lens with one that has a longer focal length would achieve that. Removing the lens altogether gives a large area, but over most of the area the light is heading outwards and will not go into the lens. A wide source, like a small screen backlight, placed readonably close abovevthe sample would give a broad area illumination with enough illumination angle.
The working distance issue might be that you are using an infinity corrected objective with a standard optics module, or the other way round?
You may also find that the motion is not appropriate for wat you want. At 20x the images are good, but the motion starts to feel slow because the step size is small to work at higher magnification. At 1x to 4x the translation speed will be very limiting.
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Thanks for your reply, William. Yes, now that you say it it is quite obvious that the light source has to be bigger but with a narrow lens for the low magnifying lenses. Tested it on my computer monitor, nice and even image. Was not able to resolve the issue with the low working distance tough, I guess it is a problem of the objectives themselves. If anybody knows where to find reliable reasonably priced 1x and 2x objectives please let me know.
Yeah true, the motion speed is rather slow, this is why I made faster gearing. For my upcoming project I don’t need the moving stage at all and actually a simpler microscopy imaging setup would be sufficient. However, as the Openflexure microscopes are fun and easy to adapt, I thought I’d use those 
Talking about adoption, I will use reflected light microscopy and I made a led ring that holds 9x 5mm LEDs around the objective of the upright microscope:
upright_LED_mount.stl (200.6 KB)
Do you think it would be worth considering an epi- illumination lens setup to achieve a more uniform illumination? I.e. the epifluorescence optics with just the beam splitter and without the color filters, and would that even work?
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The reflectance optics module works for normal reflectance as well as for fluorescence. As you say, just use a neutral beamsplitter and no additional illumination filter.
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