Careful alignment of the phase contrast microscope is essential in order to produce the maximum contrast effect without introducing artifacts that degrade specimen appearance or lead to erroneous interpretation. This interactive tutorial examines variations in how specimens appear through the eyepieces (at different magnifications) when the condenser annulus is shifted into and out of alignment with the phase plate in the objective.
The tutorial initializes with a randomly selected phase contrast specimen appearing in the Microscope Image window on the right-hand side of the tutorial. Adjacent to this window is a view of the objective rear focal plane (entitled Objective Rear Focal Plane; on the left) illustrating the proximity of objective phase plate and condenser annulus. By default, the objective phase plate and condenser annulus light cone are out of alignment and must be repositioned using the X-Translation and Y-Translation sliders. Translating these sliders enables the operator to adjust the position of the condenser annulus light cone with respect to the fixed objective phase plate. When the two annuli are centered, the maximum possible specimen contrast is obtained.
As an alternative to translating the sliders, the mouse cursor can be used to drag the condenser annulus image within the window so that it can be positioned anywhere within the periphery of the objective phase plate. New specimens can be loaded into the tutorial by using the Choose A Specimen pull-down menu, and the objective magnification can be changed using the Select Objective menu. Each time a new specimen or objective is chosen, the tutorial automatically reloads a random misalignment of the condenser annulus and objective phase ring.
While viewing the fixed phase ring in the objective using a Bertrand lens or a phase telescope, the operator can then perform adjustments to the condenser annulus position to align the two for phase contrast observation (see Figure 1). Presented in Figure 1 are images of the objective rear focal plane in misalignment (Figure 1(a) and 1(c)) and after the condenser annulus and phase ring have been properly aligned (Figure 1(e)). Also illustrated in this figure are the corresponding images that appear when viewing through the eyepieces (Figure 1(b), 1(d), and 1(f)), which demonstrate how the specimen appears when the microscope is misaligned (Figure 1 (b) and 1(d)) and carefully aligned (Figure 1(f)) according to the procedure outlined below.
Before attempting to align a microscope for phase contrast observation, examine the instrument carefully to ensure that all objectives contain phase plates and are firmly seated in the nosepiece. The objectives should also be sequentially ordered in their arrangement on the nosepiece, from lower to higher magnification, in order to minimize changeover frequency between one condenser annulus and another. Often the 10x and 20x objectives share a common condenser annulus, as do the 40x and 60x objectives. Highly corrected objectives, such as apochromats designed for oil immersion, often have similar numerical apertures and utilize the same condenser annulus over a wider range of magnification (40x to 100x). Low power phase contrast (4x and 5x) usually requires a swing-lens condenser and a specialized condenser annulus.
The condenser annular plates should also be sequentially arranged, starting with the annulus designed for the lower magnification objectives and proceeding up to the one designed for the highest magnification. In general, the entire magnification range can be covered with three or four individual annuli. If a universal condenser is employed, position the lowest magnification annulus (for example, PhL or Ph1) to the right of the brightfield slot, and the other plates in sequential order in the neighboring slots. Often, the specimen must be examined in brightfield either before or after phase contrast observation, so this arrangement will provide an easy work flow.
The following steps are recommended for the alignment of a phase contrast microscope.
- Place a brightly stained specimen on the stage and rotate the 10x phase contrast objective into the optical pathway in brightfield illumination mode. Focus the specimen, and close the field diaphragm until it enters the edges of the viewfield. Using the condenser height adjustment knob, position the substage condenser so the individual leaves of the field diaphragm are in sharp focus, and use the main condenser centering adjustment knobs to ensure the field diaphragm is centered in the field of view. Carefully review the microscope configuration to ensure that Köhler illumination has been achieved, and the specimen is in sharp focus.
- Remove the stained specimen and place a phase specimen on the microscope stage. In cases where the target specimen has only minimal optical path differences (and may be difficult to visualize), align the microscope with a specimen known to produce high contrast in phase contrast mode. Rotate the condenser turret until the appropriate annulus is positioned in the optical pathway (Ph1 or the equivalent for the 10x objective). Check to ensure that the condenser annulus color code or inscription matches that of the objective. Examine the position of the condenser aperture diaphragm lever and move it to the widest iris position (condensers designed for phase contrast may do this automatically).
- If the microscope is equipped with a Bertrand lens, use the thumbwheel control to swing the lens into place. Alternatively, remove one of the microscope eyepieces and insert a phase telescope into the observation tube.
- While peering through the eyepieces or phase telescope, adjust the focus of the Bertrand lens or telescope focus eye tube until the phase plate in the objective is in sharp focus, and the overlap between the bright image of the condenser annulus and dark neutral density material in the phase plate is apparent. In many cases, the microscope will initially be out of alignment and the annulus image will not accurately overlay the neutral density material in the phase plate (as illustrated in Figure 1(a) and Figure 1(c)).
- Locate the condenser annulus centering pins (or screws) and adjust the position of the annulus with a pair of screwdrivers or the appropriate knobs until it is coincident with the objective phase plate (Figure 1(e)). Note: do not attempt to adjust the position of the condenser annulus with the main condenser centering knobs (usually located on the condenser mounting bracket attached to the microscope). This effort will probably not achieve the intended condenser annulus alignment and will definitely compromise Köhler illumination conditions that should have been previously established.
- When the condenser annulus is properly centered, the ring of light passing through the condenser will be attenuated by the neutral density material applied to the objective phase plate, reducing the intensity of the annulus image. Therefore, if the condenser annulus is improperly centered, a bright crescent edge will appear adjacent to the neutral density material in the objective phase plate (Figures 1(a) and 1(c)). If the image of the annulus does not fit within the dark circle of the phase plate, then either the condenser is out of focus (and Köhler illumination is not established), or the phase telescope (or Bertrand lens) is not focused on the objective rear focal plane. In some cases, even when the phase telescope (or Bertrand lens) and substage condenser are properly focused, the condenser annulus image is blurry and appears out of focus. This may be caused by low frequency diffraction from the specimen. If this occurs, remove the specimen from the stage and proceed with alignment of the microscope.
- If the condenser annulus image is significantly different in size from that of the objective phase plate, check to see if the wrong annulus plate has been installed in the condenser (the most probable cause). Another possibility, if high numerical aperture oil immersion objectives are being employed, is that the condenser front lens element is designed to be immersed. In this case, it may be difficult (or impossible) to superimpose the condenser annulus on the phase plate without a drop of oil between the condenser lens and the microscope slide (and/or between the objective and the coverslip).
- When the condenser annulus and objective phase plate are in proper alignment, the image illustrated in Figure 1(e) should appear in the phase telescope or eyepiece with a Bertrand lens in place. At this point, the microscope is properly configured for observation of the specimen with phase contrast illumination.
- Replace the phase telescope with the eyepiece, or rotate the Bertrand lens out of the optical pathway, and examine the specimen. The background should appear a neutral gray in color (depending upon the neutral density of the objective phase plate) with the specimen visible in high contrast.
Once the microscope has been aligned for phase contrast, it will generally hold its centration for a considerable number of objective/annuli changes, but should be checked periodically to ensure proper alignment. If the microscope starts to slip out of alignment, the images appearing in the eyepieces (or on a computer monitor) will appear increasingly more like those observed with brightfield illumination.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew Parry-Hill, Cynthia D. Kelly, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.