Darkfield microscopy allows a specimen to be viewed as a bright image on a dark background without chemical staining. Popular subjects for darkfield include environmental water samples containing aquatic organisms and live blood cell analysis.
Darkfield can be achieved using a simple darkfield stop (which blocks a small circle of light from the centre of the condenser) – these can be homemade and can often give reasonble results with 10x to 40x objectives. A more sophisticated solution for higher numerical aperture (NA) objectives is to use a cardioid condenser. A comprehensive technical explanation can be found here
In this test, we are using a standard iScope 1153PLi with an additional darkfield package which includes a 5W cardioid condenser and a 1.25 Oil Iris objective from Euromex. The Iris objective has an adjustable ring which is used to reduce the aperture of the objective. Microscopy images were taken using a Touptek 20MP E3CMOS camera (to be reviewed soon).
The first series of images are a preliminary sequence of a Kemp 8 Form test slide. Images are resized down to 2000 pixels wide but otherwise unmodified.
Once the condenser was in place, it was relatively straightforward to produce these images. It was possible to get a darkfield image with all of the objectives tested, although getting the camera settings was more challenging than for normal brightfield work – exposure time and gain had to be increased and best results were obtained when the darkfield correction was enabled in the Touptek software (to remove sensor background noise). Below are some images of some samples that were to hand.
The images above show excellent darkfield contrast but show some chromatic aberration (CA), charactistic of achromatic objectives. Some CA is not a problem for routine visual observation of samples but is more apparent during photography. Given that the cardioid condenser gave best results when placed as close as possible to the underside of microscope slide, it was not possible to place a standard green filter in an attempt to reduce CA. However, a very thin layer of green plastic (from a chocolate wrapper) provided a low tech solution which could be placed between the condenser and slide.
I think CA may have been reduced by use of the green filter but it is quite difficult to set the white balance correctly (compared to brightfield microscopy) as there is no white background to use as a reference. With the 100x oil iris objective, the iris can be adjusted to give a bright background as a reference but colour setting is still quite subjective. As with all photography, the colour of the final image can vary widely depending on how the camera is set but it is especially challenging with darkfield.
I photographed some sugar crystals. These were done very quickly – shots of 20-40 images with the iScope and stacked using cam software. Both the unmodified and cleaned/sharpened images are shown. The background gives a good contrast with the sugar crystal and I think I prefer darkfield to brightfield for this subject. Some CA is present but I think the levels are acceptable (no green filter used).
Next I had a look at some aquatic organisms on moss. Pretty tricky critters as they are quite fast moving. x100 total magnification seemed to work best for me.
I could centre the condenser by removing one eyepiece and lowering the substage until the circle of light could be seen through the eyepiece opening. The usual condenser centering screws could then be used to centre the circle of light to the best fit (while adjusting the height as necessary). The condenser substage is then raised as high as possible, so the condenser is almost touching the underside of the slide. When viewing a specimen, the condenser substage could then be lowered slightly to point of maxmium contrast.
Overall, the condenser works well in producing a bright image against a dark background.
A simple and inexpensive Darkfield stop from Euromex with a 20mm opaque disc can also be fitted to a standard iScope condenser. The slider itself is well made, fits the condenser well and slides into place with a satisfying precision click. Results are shown below. The contrast between subject and background is not as great as that seen with the cardioid condenser. It is simple solution for lower numerical aperature objectives.