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Inverted Microscope Lightpaths

This interactive tutorial explores illumination pathways in the ZEISS Axio Observer research-level inverted tissue culture microscope. The microscope drawing presented in the tutorial window illustrates a cut-away diagram of the Axio Observer microscope equipped with lamphouses for both transmitted light (tungsten halogen; HAL lamp) and epi-fluorescence (mercury arc; HBO lamp) light sources. Also featured are the output ports, including the eyepieces, phototube, and front camera port. The fluorescence filter cube turret, housed underneath the nosepiece containing a set of objectives, is equipped with four unique filter sets mounted in individual cubes.

The tutorial initializes in Epi-Fluorescence illumination mode with the Light Beam Intensity slider set to a value of approximately 100 percent. An ultraviolet (excitation wavelength of 350 nanometers) interference filter set is positioned in the light path by default. To operate the tutorial, use the Light Beam Intensity slider to adjust the illumination intensity of either the mercury arc lamp (Epi-Fluorescence mode) or the tungsten halogen lamp (Transmission mode). Different filter sets can be selected using the Filter Cube slider and a magnified view of the objective turret can be obtained by clicking on the Zoom box. Under normal circumstances, the intensity of an arc lamp is not variable, however this option has been made available to allow visitors to adjust the intensity of light passing through the microscope optical train in order to compensate for differences in computer monitors used to view the tutorial.

Microscopes featuring an inverted-style frame are designed primarily for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. Epi-illuminators usually consist of a mercury, xenon, or external metal halide lamphouse (or laser system) stationed in or coupled to a port at the rear of the microscope frame. Fluorescence illumination from the arc lamp or laser passes through a collector lens and into a cube that contains a set of interference filters, including a dichroic mirror, barrier filter, and excitation filter. Light reflected from the dichroic mirror is restricted in wavelength by the excitation filter and enters the objective (now acting as a condenser) to bathe the specimen with a cone of illumination whose size and shape is determined by the objective numerical aperture. Secondary fluorescence, emitted by the specimen, returns through the objective, dichroic mirror and barrier filter before being routed through the microscope optical train to the eyes or a camera port. The microscope presented in the tutorial above contains a trinocular observation tube that is equipped with a port and extension tube for mounting a camera system. Another port, located near the base at the front of the microscope, can also serve as an attachment point for a camera system.

Transmitted illumination is provided by a tungsten halogen lamphouse that is positioned on the microscope pillar, above the stage. Light emerging from the lamphouse passes through a collector lens, a series of filters, and the field diaphragm before entering the condenser front aperture. After being focused by the condenser lens system, transmitted illumination is projected onto the specimen, which is placed on the stage. The light that is diffracted, refracted, and not absorbed by the specimen continues through the objective and into the microscope optical train where it can be directed to the eyepieces or to a camera system.