Microscope Alignment for Köhler Illumination
Perhaps one of the most misunderstood and often neglected concepts in optical microscopy is proper configuration of the microscope with regards to illumination, which is a critical parameter that must be fulfilled in order to achieve optimum performance. The intensity and wavelength spectrum of light emitted by the illumination source is of significant importance, but even more essential is that light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm. This interactive tutorial reviews both the filament and condenser alignment procedures necessary to achieve Köhler illumination.
The tutorial initializes with a randomly selected specimen image appearing in the virtual microscope viewport and a variable amount of illumination passing through the optical train, which has an intensity level dependent upon the (randomized) initialization state of the lamp filament. Two windows are utilized by the tutorial, and they can be accessed (toggled) using the Filament Alignment and Condenser Alignment radio buttons. The virtual microscope is assumed to be using a 10x objective to image the specimen selected either randomly at initialization or by using the Choose A Specimen pull-down menu. The Reset button can be used to re-initialize the tutorial (choose a new specimen and lamp filament position) without reloading the browser.
In order to operate the tutorial, first select the Filament Alignment radio button to display the lamp filament in the microscope viewport. The Filament Control set of (three) sliders can be employed to adjust the lamp Intensity (ranging from zero to 12 volts), Focus (position of the lamp along the optical axis), and the Rotation axis of the lamp with regard to the lamphouse. In addition, the Filament Position sliders translate the filament laterally along the x and y axes of the virtual microscope.
Once the lamp filament has been centered, focused, and brought to an operating potential of approximately 9.0 volts, click on the Condenser Alignment radio button to view the specimen and condenser adjustment control sliders. If the lamp filament has been properly adjusted, the specimen should be evenly illuminated regardless of the fine focus state, condenser height, or field diaphragm opening size. To align the condenser, first focus the specimen using the Specimen Fine Focus slider, and then use the Condenser Height slider to bring the field diaphragm iris leaves into focus. Next, use the Condenser Lateral Adjustment sliders to translate the field diaphragm iris opening to the center of the viewport. Finally, use the Diaphragm Opening Size slider to open the field diaphragm to its maximum size. If the diaphragm opens off-center, use the x-translation and y-translation sliders to bring the opening into the center of the field. Alternatively, the mouse cursor can be placed in the small window (containing a set of crosshairs) and used to drag the image of the field diaphragm (appearing as a white circle) into the center. After the filament has been properly aligned and the virtual microscope adjusted for Köhler illumination, the Condenser Aperture slider can be utilized to simulate how varying the numerical aperture affects specimen contrast and resolution.
The currently accepted method of microscope illumination was first described by Dr. August Köhler in the late 1800s, and is still widely (almost exclusively) employed for modern microscopes over 100 years later. Köhler's technique requires a collector lens in or near the lamphouse that can be adjusted to focus an image of the lamp filament at the front focal plane of the condenser where the aperture diaphragm resides. If the lamp filament image is properly centered and completely fills the aperture, then illumination of the specimen plane is bright and even. In order to ensure that the filament image appears in the condenser focal plane, the height of the condenser itself must often be adjusted (a technique reviewed in the tutorial). This critical adjustment brings two sets of conjugate focal planes (referred to as the field set and the aperture set) into precise physical locations within the microscope optical train, and maximizes the performance of the instrument.
Matthew Parry-Hill, Robert T. Sutter, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.