MicrobeHunter.com Microscopy Forum

Hi-

I am very new to the field of microscopy and have recently learned about the existence of darkfield techniques. However, I don't understand everything that is involved. On a basic level, darkfield imaging can be achieved with a "normal" microscope using a homemade mask filter with a circular opaque region in the center that blocks light from traveling in a straight line from the light source to the eyepiece, while still allowing oblique illumination of the specimen. This gives an image of the specimen against a dark background. Am I correct so far? This seems pretty straightforward.

The thing that has me confused is that there also exist special darkfield condensers, darkfield objectives, and darkfield microscopes. I don't understand what these are doing. Are these just different ways of achieving the same thing? Can someone please explain, at a basic level, what these things do, how they differ from each other (and from the simple filter mask), and the pros and cons of each?

This video shows an amazing shot (to me) of pond life under darkfield illumination. The guy who made it says that he used an American Optical 110 with a filter mask like I described above. Is that really all there is to it?



Thank you!

I'm certainly no expert, but the two typical ways of doing darkfield, patch stop or darkfield condenser are just two different ways of doing much the same thing. While the result is generally comparable, they get there in different ways which can affect efficiency and performance. I have two styles as designed by the same manufacturer, a patch stop and a cardioid darkfield condenser, and they both produce the same effect; however, the darkfield condenser seems to transmit more of the light and appears to work better with high power objectives than the patch stop does. That said, both seem to work fine.

At lower magnifications the simple darkfield stops work well. They will almost surely work up to 20x (200x) and often to 40x (400x). Above this it gets progressively more challenging. A special darkfield condenser is needed to precisely control the illumination and at highest powers the condenser will need oil immersion to the slide and above the slide.

One also doesn't want the objective to have a wider view (higher numerical aperture) than the condenser, so some sort of iris or stop within the objective lens is common for darkfield at 100x (1000x with 10x objectives).

Good news is that many things - including protists - don't need to be magnified 500 to 1000x.

Thank you both. The fog is already starting to lift a little bit!

Can a darkfield condenser typically be adjusted to also act as a brightfield condenser, or are they usually dedicated to only doing darkfield work?

Thanks...

-Matt

There are some which can operate in various "modes" such as the Heine condenser, but I believe that sort of capability is quite unusual. The example I have and the other darkfield condensers I have seen are expressly for darkfield and don't adapt to other methods; though the patch stops, being filters for a typical condenser, do.

Here are brief instructions for the two ‘dedicated’ Leitz darkfield condensers:
http://microscope.database.free.fr/Acce ... oscopy.pdf

http://www.science-info.net/docs/leitz/ ... denser.pdf

MichaelG.

Thanks MichaelG. The cross section diagrams in those PDFs really help me understand what is going on in the condenser.

If one puts a DF stop under or in a condenser, they are turning that condenser into a DF condenser. The stop will cause the condenser to pass a cone of light, rather than a beam of light. The walls of the cone will have a width, much like a funnel made of light, with the o.d. of the light funnel being represented by the N.A. of the condenser and the i.d. of the light funnel being represented by the N.A. at which the edge of the stop sits. Let's say for the sake of argument that the condenser is an oiled 1.25 N.A. abbe being used dry. It's functional N.A. is about .90 and the stop blocks everything below .75N.A. So as a DF condenser, it now has an N.A. range of .75 to .90. In order for a DF condition to be obtained with that condenser, an objective must have an N.A. below .75. and to achieve really dark DF it would need to be somewhat less, maybe .65. The upper N.A. of the condenser will increase to 1.25 if oiled but the minimum N.A. will stay the same. In order to accomodate objectives of higher N.A. and therefore resolution, a wider stop would have to be fitted or move the existing one closer to the objective. Both solutions require a fairly high degree of precision. As well, the condenser will still provide a quality of illumination consistent with it's class or category. If it is a 2 lens abbe condenser, the quality of the cone of light will be adversely affected by it's innate defects and it will be chsracterized by whatever aberrations and distortions in DF that it has in BF.

DF condensers on the other hand are designed to reduce those aberrations and distortions to from a very low level to non-existant. Cardioid types have virtually no ca.
The reason for this is that in DF, chromatic abberation is more apparent, so can become a huge defect in the imaging. Additionally, DF condensers usually work at very high N.A.s with the illumination cone typically sitting somewhere around 1.2 to 1.4 N.A. or only slightly lower. It is virtually impossible to duplicate that quality of performance installing stops in a refracting condenser of limited colour correction and high spherical aberration and field curvature.
That's why DF condensers exist.