Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Basic Microscopy

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 up to six unique filter sets (four cubes shown in tutorial) mounted in individual cubes.

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. Two other ports are shown, one located near the base at the front of the microscope, and the other with the image being projected to the right side, 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, the field diaphragm, and a series of filters before entering the condenser front aperture. After being focused by the condenser lens system, uniform 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.

Contributing Authors

Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.

Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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