I am trying to setup our microscope for Köhler illumination with a 60x objective. However, the manual for our microscope states that the image of "field iris" cannot be observed in the sample plane with objectives having 40x or beyond magnification. How should I go about obtaining a Köhler illumination with a 60x objective then?
I tried a 10x objective to obtain the Köhler illumination but unfortunately, our stage is too high to focus 10x objective. [We plan to use the same stage height for imaging. ]
Hello Sankalp,
from the instructions it seems that the issue is that the field iris, even at its tightest setting, still has an aperture bigger than the microscope field of view. This means you will lose some image contrast, but in general, especially if you use a digital camera, you can still acquire respectable images.
The only problem in case you use it like that would be in correctly focusing the condenser lens, as you can not close the iris enough to see the aperture image and put it in the objective focal plane. You can work around the by closing the iris at its tightest setting, then move the condenser lens sideways (it usually has two screw actuators at 45 degrees) until you see the edge of the aperture, move the condenser focus until you have a sharp image of the edge, and move the condenser back sideways so that you don't see the edge anymore. It is not exactly a simple procedure, but it's better than not having a Kohler illumination.
Hello Sankalp,
from the instructions it seems that the issue is that the field iris, even at its tightest setting, still has an aperture bigger than the microscope field of view. This means you will lose some image contrast, but in general, especially if you use a digital camera, you can still acquire respectable images.
The only problem in case you use it like that would be in correctly focusing the condenser lens, as you can not close the iris enough to see the aperture image and put it in the objective focal plane. You can work around the by closing the iris at its tightest setting, then move the condenser lens sideways (it usually has two screw actuators at 45 degrees) until you see the edge of the aperture, move the condenser focus until you have a sharp image of the edge, and move the condenser back sideways so that you don't see the edge anymore. It is not exactly a simple procedure, but it's better than not having a Kohler illumination.
Thanks, Paolo and Peter for your valuable input. I thought of using a trick similar to what Paolo mentioned.
We only have 10x, 40x and 60x objectives.
I think using 40x objective and following steps similar to Paolo with this should give me fairly good results. Next, I will replace the 40x objective with 60x objective as suggested by Peter.
Seems like a good strategy, just make sure that the two objectives have the same parfocal distance (which means they focus on the same plane when you swap one for the other). If they were both sold togheter with the microscope, that's probably the case, but better safe than sorry.
Good luck with your experiments!
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