Comparison of Results with Brightfield, Oblique and COL Illumination

My normal practice has been to align the components of the external fiber-optic illumination system using a 9X objective and establish good Koehler illumination using the aplanatic condenser with its aperture diaphragm left fully open. The aperture diaphragm at the light-guide end is used and the darkfield stops are also inserted at that location. I did the same with the prototype condenser and then opened the diaphragm fully at the light-guide end, and subsequently adjusted the diaphragm of the condenser for Koehler illumination. The 40X objective was then swung in on the turret and the field diaphragm imaged just outside the field of view of the eyepiece. I found that I had to raise the condenser from the height setting with the 10X objective in order to be able to fully fill the rear focal plane of the 40X objective with an image of the light source. The field diaphragm was then poorly imaged, as seen when the substage mirror was tilted slightly. I found that the stria pattern on Pleurosigma angulatum was not detectable until the illumination NA set with the aperture diaphragm almost matched that of the objective. This was the aperture setting also used for oblique and circular oblique lighting. The stria pattern in brightfield had very low contrast making detection and focusing difficult. The CoolPix^® was zoomed to record about half the field size and the resulting images were cropped to show the same field as the darkfield image in Figure 13. In order to attempt to match the eyepiece image quality, this image and the other images of the diatom have not had their contrast enhanced digitally. The cross hatch pattern from the stria is shown for brightfield in Figure 15.  The contrast is far inferior to the darkfield image in Figure 13. Oblique illumination from rotating the wheel to decenter the stop for the 40X objective gave much improved contrast as shown in Figure 16. The resolution is now directional, with only one set of parallel lines visible. There is now a camera lens artifact visible in this image as well as in the COL image. A concentric ring pattern is evident in these images and believed to result from residual tool marks in the mold subsequently replicated on the surface of one of the molded aspheric lens elements in the CoolPix^®. This artifact will not be present when using digital microscopy systems from the major manufacturers of microscopes. The contrast of the COL image in Figure 17 is far superior to the brightfield image in Figure 15. The stop for the COL images is the same stop used for the 4X objective, which is somewhat smaller than the stop for the 40X objective as seen in Figure 6.

click image to enlarge (95K) Figure 15

click image to enlarge (193K) Figure 16

click image to enlarge (67K) Figure 17