Microscope Alignment for Khler 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
Khler 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 Khler 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
Khler in the late 1800s, and is still widely (almost exclusively) employed for modern

microscopes over 100 years later. Khler'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.

Contributing Authors
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.