Microscope condensers and resolution kohler illumination

[u0362565]

Hi all,

I have a couple of questions about the process of kohler illumination (optimised condenser alignment) in brightfield microscopy and how this affects the final resolution attained.

Resolution concerns the minimum detectable distance between two objects and the closer to structures are, then a greater degree of diffraction occurs. Ultimately this means that higher resolution information is encoded by light rays that are at a more obtuse angle if the optical axis is zero degrees, hence the wider the acceptance angle of the lens (NA) the higher the resolution.

The light leaving the specimen can be seen as a mirror image of the light cone emanating from the condenser. If the condenser is closed down too far then the angles over which the objective lens can receive light is reduced and thus the full NA of the lens is not realized. Is that an acceptable way of thinking about this?

I'm slightly confused though by the opposite case, if the condenser aperture is opened too far. The acceptance angle range of the lens is fixed, so why does a wider light cone mean that the NA of the lens is not realized? Does closing the aperture force light into the lens, an over simplification i know..

The first image on this page gives a nice illustration of how I'm visualising the light cones emanating from the condenser and specimen.
http://www.olympusmicro.com/primer/anatomy/condensers.html

My second question concerns the field diaphragm. Why do you need a physical barrier to prevent some light from reaching the condenser? The crucial step is controlling the angles of light allowed to pass to the objective lens so why not let the condenser do all the work?

Thanks for your help!

Matt

 

[Andy Resnick]

There are a few issues here to discuss. First, a clean definition of Kohler, and critical, illumination: critical illumination images the source onto the sample plane, while Kohler illumination images the (spatial) Fourier transform of the source onto the sample plane. Both illumination conditions maximize the illumination efficiency, both provide independent control of the field and aperture diaphragms, and in both cases aberrations in the illumination optics do not reduce the optical performance of the objective lens.

Because the condenser NA is independent of the objective NA, two main effects occur, corresponding to NA_cond << NA_obj and NA_cond > NA_obj. Overall, resolution is given by

When NA_cond > NA_obj, image contrast is often lowered due to the 'mixing' of unscattered high NA rays and light from low NA rays scattered by then sample into higher NA. By slightly stopping down the condenser, image contrast is often increased, as is the depth of focus.

When the NA_cond << NA_obj, the sample is essentially illuminated by a plane wave, which can be used to emphasize diffraction effects. For example, if you are looking at a grating, stopping the condenser all the way down will provide improved images of the diffracted orders.

As for *why* the condenser should be stopped down at all, the answer has to do with aberrations- lens aberrations increase as the diameter of the stop increases- stopping down *any* lens will reduce aberrations (except for distortion).

Does this help?

 

[u0362565]

Hi Andy,

Yes thanks you made some good points there. Ultimately when I'm describing use of the condenser i'll make it clear that the achievable resolution is equal to the Na of the objective + NA condenser and NA condenser is changed by adjusting the condenser aperture. Would you say that the field diaphragm is purely used to ensure that the source is focussed at the front focal plane of the condenser rather than at the specimen plane?

Thanks again,

Matt

 

[Andy Resnick]

The field diaphragm defines the illuminated region of the sample. I only stop down the condenser field diaphragm for two reasons- during initial alignment, to ensure the condenser and objective are focused to the same sample plane, and if I am concerned about sample bleaching (fluorescence imaging)- I don't want to illuminate portions of the sample I am not looking at.

With Kohler illumination, focusing the source requires the ability to view pupil planes, either with a Bertrand lens or some other method. This is not always easy to do, which is why many lamps have a diffuser plate.