I’ve been getting a lot of questions in my inbox about darkfield microscopy for compound microscopes–one of the more advanced forms of optical microscopy. Being a more specialized use for a microscope, there isn’t a whole lot of information out there written in easy to digest terms to explain its benefits, and in what applications it’s needed for.
So, what is darkfield microscopy exactly? Darkfield microscopy is an illumination method used in a microscope that essentially bends the light coming from the light source in a way to where the sample is lit up from the sides (called oblique lighting), and has a dark background it is contrasted against. For some samples when viewed in brightfield, the bright background (created by the light travelling directly through the sample) doesn’t provide enough contrast to really see the sample–particularly with live blood cells and spirochetes (used in Lyme disease detection). The extra contrast allows you to view samples that normally must be dyed or stained to view without doing so. Stains and dyes kill the sample off, so viewing them without killing them can only be achieved by using darkfield (or phase contrast, however that’s a discussion for another day).
Microscope 101: What is Darkfield Microscopy?
How does darkfield microscopy work then? We briefly touched on the light being bent in a manner which allows illumination to the sample without going straight up and through it, but how is it bent? Typically, the condenser on the microscope needs to be changed out. Most Abbe condensers (the brightfield ones) only focus the light in a straight direction through the sample and into the objective, so without heavy modification, it won’t work for darkfield.
A separate condenser is typically sold through the microscope manufacturer or brand name if you want to do darkfield microscopy, however your condenser needs to be swappable. If you have a fixed brightfield condenser on your microscope, you won’t be able to add a darkfield to it, and would have to get a whole new microscope.
Also note that there are multiple kinds of darkfield condensers–primarily, dry condensers and oil condensers. Dry condensers are meant for use with low numerical aperture rating objectives (NA rating); usually, this means objectives ranging from 2x to about 40x. The condensers have a NA rating range on them that tells you what objective NA ratings it can pair with. For higher magnifications, such as a 60x or 100x (oil immersion) objective, you will need to use an oil condenser. They have higher NA ratings and require the condenser to have immersion oil dropped onto the lens, and moved up to contact the slide. The oil serves to prevent the light from scattering as much as it does through air, keeping it in the area and able to enter the small aperture of the higher magnification objectives (exactly the same as oil immersion techniques used with 100x objectives, just on the condenser and moved up instead of on the slide and the objective lens moved down).
Once you have the correct darkfield condenser for the magnification levels you’re interested in viewing, you want to remove the Abbe condenser on your microscope (the method differs by brand and model, so check your user’s manual or ask their technical support department if you need help determining how), and insert the darkfield condenser. Typically, you will need to center the condenser (unless it’s precentered from the factory for your unit) using the mechanism on your microscope’s condenser mount, or the condenser if it has an adjustment to it.
Once centered, the condenser has 2 reflective surfaces that serve to bend the light. The first is a conical pyramid, pointed down towards the light. This serves to reflect the light coming from the light source out and down to the second surface, which is an angled ring inside the condenser, which points the light up and to the sample at an angle, and around a black coin shaped surface (which serves to block the light out from contacting the sample directly, which would create a light background). Here’s a diagram provided by Rice University to demonstrate the basic properties of a darkfield condenser, for the visual learners:
If you’re using a specific brand of microscope that you need a condenser for, I suggest contacting them to see what options are available. If you have an AmScope brand microscope, as many of the products I use and suggest are, you can get the appropriate condensers depending on the optical quality of your microscope and objectives from here:
DK-DRY100 & DK-OIL100 (for basic achromatic units, like the T490)
DK-DRY120 & DK-DRY120 (for the T120)
DK-DRY200-INF & DK-OIL200-INF (for infinity microscopes like the T690)
Hope this helps out! Please feel free to let me know if you have any questions that I can answer or help clarify for you!