Stereomicroscopy Fluorescence

The application of stereomicroscopes for green fluorescence protein (GFP) observation is now so prevalent that stereo fluorescence illuminators are more frequently referred to as GFP Illuminators, even though they can be utilized for many other applications in both the life sciences and industry.

This interactive tutorial explores internal optical pathways of the Nikon SMZ1500 stereomicroscope equipped with an epi-illumination intermediate tube and lamphouse. The tutorial initializes with an Endow GFP fluorescence filter combination inserted into the light path and the Beam Intensity slider, which controls the intensity of the light passing through the microscope, set to the midpoint. Use the Filter Cube slider to toggle between three filter sets:

Table 1
Filter SetExcitation
Wavelength Range
Dichromatic
Mirror
Emission
Wavelength Range
Blue GFP/DAPI379-401 nm420 nm (DCLP)435-485 nm
Endow GFP Long PassHQ 450-490 nmQ 495 (LP)500 nm (LP)
TRITC (DsRed)HQ 530-560 nmQ 570 (LP)590-650 nm

Large specimens, such as larvae, nematodes, zebrafish, oocytes, and mature insects can be easily selected and manipulated when they are labeled with GFP and illuminated by fluorescence techniques with a stereomicroscope. The fluorescence illumination reveals which organisms are producing the fluorescent protein and the stereoscopic vision coupled to a large field of view and ample working distance enables observers to conduct experiments with forceps, pipettes, or micromanipulators. Other, more conventional, specimens are also easily observed and recorded using stereomicroscopes with fluorescence illumination.

The illuminator for epi-fluorescence on a stereomicroscope functions in a manner that is similar to those employed on more complex compound microscopes. Typically, the fluorescence illuminator consists of a xenon or mercury arc lamp contained in an external lamphouse that is attached to the microscope via an intermediate tube (or vertical illuminator) positioned between the microscope zoom body and observation tubes. This type of illumination is currently restricted to applications employing common main objective (CMO) stereomicroscopes, because it is not possible using commercially available parts to adapt a Greenough or converging-type stereomicroscope for fluorescence illumination.

Contributing Authors

John C. Long 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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Stereomicroscopy Fluorescence

Introduction