One of the most important tests to verify the potential of a light microscope is to use a diatom slide of Amphipleura pellucida (Kützing) mounted in a high refractive index medium, such as Zrax (n=1.70+) (Figure 1). This
type of diatom lives in freshwater lakes and rivers. From the Victorian era to the
present day, separation of the lines (striae) and dots (punctae) of this diatom
has always been a challenge for the microscopist. The striae of this diatom have
a separation of 250 nm (40 to 42 lines in 10 µm; 50 to 52 dots in 10 µm) which is
near the limit of optical resolution of a modest light microscope.
click image to enlarge (188K)
To achieve the separation of the striae and dots you must have
accurate control over every single element of the microscope, including, first
of all, the objective and condenser of the microscope. You can use a high power
objective such as the 63X or 100X with a numerical aperture of at least 1.25,
and possibly of the fluorite or apochromatic type with high correction for chromatic
and spherical aberration (Figure 2).
click image to enlarge (194K)
The substage condenser must have the
characteristics of N.A. ≥ 1.25. A very
good condenser of this type is the old Zeiss or LOMO aplanatic condenser with numerical
aperture of 1.4 whose aperture diaphragm is on rack and pinion, and rotatable for
obtaining oblique illumination from any azimuth (Figure 3).
click image to enlarge (126K)