OPERATIONZEISS Illumination and contrast methods in transmitted light Axiolab 584 430037-7444-001 05/20194.2.2 Configuring transmitted light darkfield microscopy using the KÖHLER method(1) General principleDue to their transparency, unstained biological specimens, such as bacteria or living cell cultures, areoften barely or not at all visible in transmitted light brightfield microscopy. This is radically changed whensuch specimens are observed in transmitted light darkfield microscopy. In principle, the specimen isexposed to light from an illumination aperture which is larger than that of the objective used.In darkfield microscopy, only the diffracted and scattered light components which are important forimaging reach the objective, while the direct unchanged light bundles are routed past the objective. Thisis one of the reasons why even fine structures that are sometimes below the resolving power of the lightmicroscope can be resolved and appear very bright on a dark background.(2) InstrumentationAll Axiolab 5 microscopes, except stands for reflected light, are suitable for darkfield applications.Condenser with darkfield stop in position D e.g.:− Condenser 0.9/1.25 H with modulator disk BF, DF, Ph 1, Ph 2, Ph 3− Condenser, achrom.-aplan. 0.9 H D Ph DIC− Darkfield condenser with dry darkfield (465505-0000-000 applicable aperture from 0.6 – 0.75)− Ultra condenser (465500-0000-000 applicable aperture from 0.75 – 1)(3) Configuring transmitted light darkfieldmicroscopy• Adjust the brightness using the KÖHLERmethod as for transmitted light brightfieldmicroscopy. Instead of the 10x objective,however, swivel in the objective with thehighest aperture which does not exceed thelimit aperture for the darkfield with thecondenser used.• Position the turret/modulator disk of thecondenser at D and swivel in the condenserfront lens (if existing).• Remove the eyepiece from the tube (or replaceit with an auxiliary microscope) and check thecentering of the darkfield diaphragm in the exitpupil of the objective. If the central darkfieldstop D in the universal condenser is partlyoutside of or de-centered to the exit pupil ofthe objective, and if the exit pupil is nothomogeneously dark, the darkfield stop mustbe re-centered.• To center the darkfield stop (not possible with all condensers), use two Allen wrenches (AF 1.5)(Fig. 4-5/1) to turn the two centering screws (Fig. 4-5/2 and 3) until the exit pupil of the objectiveappears homogeneously dark. After centering, remove both Allen wrenches (AF 1.5) from thecondenser.Fig. 4-5 Centering the darkfield stop oncondenser, achromatic-aplanatic 0.9H D Ph DIC
