Imaging the Diatom Test Plate with the 4X Objective

Figure 8 shows the condenser in the lowered position with the top lens removed for use with the 4X Zeiss objective. (I now use a Zeiss 4X 160 mm tube length objective instead of the LOMO 4X objective because the Zeiss lens is corrected for use with a compensating Zeiss Kpl eyepiece found best for use with the higher power LOMO objectives.)  Figure 9 shows the diatoms imaged in brightfield with the 4X objective with the field diaphragm adjusted so its image falls just within the 18 mm diameter intermediate image field size of the stop in the 10X Zeiss Kpl high eyepoint eyepiece. Figure 10 shows the same field imaged in darkfield after selecting and centering the stop in the wheel and opening the aperture diaphragm in the condenser. This low magnification is important for surveying the field before switching to a higher power objective.

click image to enlarge (191K) Figure 8

click image to enlarge (114K) Figure 9

click image to enlarge (135K) Figure 10

Imaging Pleurosigma angulatum with Darkfield Illumination

The diatom test slide has the diatom Pleurosigma angulatum which serves as a very good resolution test target for the 40X 0.65 NA objective because the stria spacing of about 0.52 micrometers matches the theoretical resolution of a 0.65 NA objective, when used with a matching illumination NA of just under 0.65. In his response to a letter by Robert B. McLaughlin,⁴ Dr. Walter McCrone republished a very high resolution optical photomicrograph of Pleurosigma angulatum taken in the early 20^th Century by Spitta. This image is shown in Figure 11. It was evidently taken with darkfield illumination, probably with blue light and perhaps with even shorter wavelength ultraviolet. The definition of “just resolved” means that the periodicity of the structure will be detectable but this fine structure will not be faithfully resolved. Figure 12 shows Pleurosigma angulatum recorded with the LOMO 40X objective and darkfield illumination from the prototype condenser. The condenser height had to be raised from the brightfield setting in order for the high NA rays to illuminate the specimen for darkfield. This same problem exists with the Olympus Abbe condenser used with my darkfield inserts and will be discussed in a subsequent paragraph. The CoolPix^® lens zoom control was set so the diagonals of the recorded field covered the field seen with the 10X 18 mm FN eyepiece and the image was subsequently cropped in Adobe PhotoShop^®. The periodicity is recorded along with lines that initially were suspicious of being alias lines from the camera sensor resolution being close to the optical resolution. The CoolPix^® lens was zoomed to cover about half the field size of the eyepiece for the cropped portion of the field shown in Figure 13. The lines are still present and therefore not from digital camera aliasing because they are also evident on a close examination through the eyepiece. Tony Havics of pH2, LLC previously tested my modified Biolam. Tony found that darkfield, with the stop at the fiber-optic light guide end with the aplanatic condenser and the same 40X objective, was capable of resolving the first three sets of lines of the HSE/NPL Test Slide, as shown in Figure 14. Resolving the three sets of lines is a requirement for counting asbestos fibers using phase contrast microscopy.

Figure 11

click image to enlarge (163K) Figure 12

click image to enlarge (215K) Figure 13

click image to enlarge (164K) Figure 14