OPERATIONCarl Zeiss Illumination and Contrasting Method Axio Scope.A174 M60-2-0007 e 05/084.1.3 Adjusting the Transmitted Light/Phase-Contrast(1) General principle of operationThe phase-contrast method is ideal for examining thin uncolored samples, e.g. culture cells. Generally thehuman eye cannot detect phase differences (variations in refractive index or thickness) within thedifferent cell components.The phase-contrast method converts with the aid of optical modulators, such as "annular phasediaphragm and phase ring", the small phase differences into intensity and color differences which arevisible to the human eye. Important for the generation of such images is the interference of differentbeams in the intermediate image.With the aid of the optically defined ring channel "annular phase diaphragm and phase ring", the high-intensity direct light portions are damped and provided with a constant phase shift. The indirect lightportions, however, which are diffracted by different cell particles, bypass this optical channel and theirphase is affected by the difference in the sample’s refractive index and thickness.In the intermediate image plane, the partial beams thus differently affected achieve interference andstrengthen or weaken each other – depending on their phase. As a result, these interferences createimage contents with intensity differences visible to the human eye.(2) Instrumentation− Phase-contrast objectives with the phase rings Ph 1, Ph 2 or Ph 3 for various medium numeralapertures which can also be used in the bright-field.− Condenser with revolver / modulator disk which contains centerable ring diaphragms Ph 1, Ph 2and Ph 3 for various medium numeral apertures.− The phase annular diaphragm used on the condenser must be consistent with the labeling on theobjective which is used, e.g. Ph 1.(3) Adjusting the transmitted light/phase-contrast• Put the phase-contrast objective, e.g. labeled Ph 1, into the beam path.• Switch on the annular phase diaphragm on the condenser’s revolver disk which has the same label asthe phase-contrast objective, e.g. 1.• In order to check the centering and the overlap of the lighter annular diaphragm (in the condenser)with the darker phase ring (in the objective), remove an eyepiece from the tube and replace it with anauxiliary microscope. Use the adjusting fixture on the auxiliary microscope to focus the annulardiaphragm and the phase ring in the objective exit pupil.
