Epi-Fluorescence Illumination

Until very recently, fluorescence illumination was an option available only on research-level compound microscopes equipped with specialized high-numerical aperture objectives. The need for this technique in stereomicroscopy has escalated with the introduction of genetically encoded and biologically specific fluorescent proteins such as GFP (Green Fluorescent Protein).

The application of stereomicroscopes for 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 the electronics manufacturing industry. 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. 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; see Figures 1 and 2) 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.

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Stereomicroscopy Fluorescence

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Explore the light paths in Nikon's SMZ1500 stereomicroscope equipped for fluorescence illumination using an intermediate tube and external lamphouse.

Light generated by the arc lamp is directed through an adjustable collector lens to an excitation filter housed in a combination filter block (as illustrated in Figures 2 and 3), which allows only light having a specific wavelength range (or bandpass) to pass through. Filtered light is then deflected through the microscope imaging path to the lower portion of the microscope (zoom body and objective in a stereomicroscope) and onto the specimen by a dichromatic mirror that is also tuned to selectively filter, reflect, and/or transmit a specific set of wavelength regions. The term dichromatic (or dichroic) refers to the filter or mirror's ability to differentiate between two color ranges by reflecting colors below the specified wavelength limit while transmitting those wavelengths above the limit.

The focused excitation light passes through the microscope zoom body and objective, where it forms an inverted cone of illumination that bathes the specimen, exciting any fluorophores that are present and have absorption bands corresponding to the excitation wavelength bandpass range. Secondary fluorescence emission (usually of longer wavelengths than the excitation light) emitted from the specimen is captured by the stereomicroscope's common main objective and directed back through the zoom body and to a barrier filter that blocks excitation wavelengths and only allows a selected region of emission wavelengths to pass. The microscope body tube in Figures 1 and 2 is constructed so that longer wavelength fluorescence emission light passing back through the left and right zoom optical channels is focused independently before reaching the epi-fluorescence illuminator. Light from the left channel passes directly through a barrier filter before being directed into the observation tubes or to the camera port. In contrast, light from the right channel first travels back through the dichromatic mirror and then to the barrier filter and eyepieces. This light does not have a pathway to the camera port, and can only be utilized for observation of specimens.

Construction details of the fluorescence filter combination block are presented in Figures 2 and 3. Each block contains a single excitation filter, two barrier filters and a dichromatic mirror. Light generated by a mercury arc lamp enters the block through the excitation filter and is reflected by the surface of the dichromatic mirror, as discussed above and illustrated in Figure 3. Secondary fluorescence emission passes through the barrier filters. The excitation filter, dichromatic mirror, and barrier filter for the left channel are glued into place, but the barrier filter for the right channel is mounted in a small frame that can be removed from the block by loosening a set of retaining screws. Removing the barrier filter frame permits access to the dichromatic mirror positioned inside the filter block. When gluing replacement filters into the block, be careful not to spread glue onto the filter surface and do not hold the filters or dichromatic mirror with ungloved hands to avoid contaminating the surfaces with oily fingerprints.

The fluorescence vertical illuminator can accommodate three filter blocks and a dia-filter dummy block (devoid of filters) that enables normal brightfield observation. Filter blocks are mounted on a sliding rack and can be inserted into the optical path by means of a lever that is utilized to control the rack position. Each block has an accompanying identification plate that can be inserted into a slot on the illuminator exterior housing in sequential order to enable the operator to easily select the proper block for fluorescence observation.

Currently, Nikon offers a modest lineup of filter combinations, which are listed in Table 1. These filters cover a wide range of fluorescence excitation and emission conditions, and should be useful for many of the commonly utilized fluorescent probes in biological research. The filter combinations are also suitable for industrial applications such as examination of integrated circuit wafers for contamination by fluorescent photo resist polymers. Fluorescent probes with excitation wavelengths ranging between 380 and 510 nanometers can be utilized by choosing the appropriate excitation/emission filter combination (see Table 1). The filter combinations are also quite useful for studies employing various green fluorescent protein mutants, including both the cyan and blue versions.

In living cell cultures having fluorescent protein labels, the signal intensity can be significantly improved when filter combinations are critically matched to the excitation and emission profile of the fluorophores. For example, in the case of DS-Red signals, visual and image sensor detection of red fluorescence can be dramatically improved by shifting the red signal to more orange emission. In addition, filter combinations designed for plant specimens having intense autofluorescence background emission from chlorophyll often benefit from careful and precise selection of the filter specifications with respect to the corresponding signal emission. Microscope engineers take many of these design criteria into consideration when they are optimizing wavelength bandpass regions for stereomicroscope filter combinations.

Stereomicroscope Fluorescence Filter Combinations
Filter Set Excitation
Wavelength Range
Dichroic Mirror Emission
Wavelength Range
Blue GFP/DAPI 379-401 nm 420 nm (DCLP) 435-485 nm
Cyan (EGFP) GFP 426-446 nm 455 (DCLP) 460-500 nm
Endow GFP Bandpass HQ 450-490 nm Q 495 (LP) 500-550 nm
Endow GFP Long Pass HQ 450-490 nm Q 495 (LP) 500 nm (LP)
TRITC (DsRed) HQ 530-560 nm Q 570 (LP) 590-650 nm
Yellow GFP Bandpass HQ 490-510 nm Q 515 (LP) 520-550 nm
Table 1

As with other sensitive interference filters, the combination block filters will deteriorate with time upon exposure to high-intensity light and ultraviolet wavelengths. Characteristics such as bandpass frequencies and transmission values will also be affected if the filters are exposed to conditions of high humidity. In order to increase the useful lifetime of these filters, they should be stored in a desiccator or a hermetically sealed container containing a drying agent. Also, reduce the amount of time that light is being passed through the filters by keeping the illumination shutter closed when specimens are not being directly observed or imaged with a digital or film camera system. The filters should only be cleaned with dry air from an ear balloon, soft camelhair brush, or oil-free pressurized gas cylinder. Never attempt to wipe the surface of the filters with lens tissue to avoid introducing scratches or abrasions into the soft interference coating.

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Focusing and Alignment of Arc Lamps

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Gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.

A wide range of specimens can be imaged under fluorescence illumination with a stereomicroscope such as the Nikon SMZ1500. With objective magnifications of 0.5x through 1.6x and a zoom span of 15x, the microscope is capable of providing a total magnification breadth of 4x to 540x, taking the stereomicroscope observation range into that of a classical compound microscope. This wide latitude of magnification enables the microscopist to image both large living specimens and minute details present on fluorochrome-labeled thin sections that are mounted on microscope slides. An example of fluorescence observation in the high magnification range is illustrated in Figure 4 for a triple-labeled thick section of mouse kidney tissue. The specimen is labeled with DAPI, Alexa Fluor 488 WGA, and Alexa Fluor 568 (the Alexa Fluor probes and specimen were obtained from Molecular Probes) and was imaged using three filter combinations listed in Table 1: Blue GFP/DAPI, Endow GFP Bandpass, and TRITC DsRed. This image clearly illustrates the high-magnification capabilities of fluorescence stereomicroscopy to observe specimens originally prepared for compound microscopes.

Other stereomicroscope manufacturers offer alternative illumination strategies for fluorescence excitation and observation. The most popular configuration, presented in Figure 5, involves an external pathway for fluorescence excitation that does not utilize the microscope imaging optical system. Illumination from the arc lamphouse is first passed through an excitation filter, and then channeled through a tube positioned at the rear of the main microscope body. The lower portion of the tube is equipped with a lens system that directs excitation wavelengths onto the specimen. This configuration ensures that light is guided directly onto the specimen at all magnification zoom positions, providing an equally intense fluorescence illumination at all magnifications with a uniformly dark background.

Secondary fluorescence emission from a labeled specimen is captured by the common main objective (Figure 5) and passed through the zoom channels and into a set of barrier filters positioned in the microscope head. From there, the light is directed into the eyepieces for direct observation or into a camera tube for digital imaging or photomicrography. The major benefit of this configuration is the lack of a requirement for dichromatic mirrors, and an independence from pre-configured filter combination blocks, which allows the investigator somewhat wider latitude in filter selection. However, the configuration can also lead to errors by inexperienced operators who attempt to observe specimens with incorrect filter combinations.

The intense ultraviolet radiation generated by mercury arc lamps and utilized as an excitation source in fluorescence microscopy can cause damage to the retina of the observer's eye. To avoid this situation, many microscope manufacturers include a protection device on the microscope body that filters ultraviolet light bathing the specimen on the microscope stage. Other safety precautions include ultraviolet barrier filters in the observation path and stray-light protection surrounding the lamphouse. In addition, dummy filter carriers are often inserted into unoccupied filter positions in sliders and rotating frames designed for fluorescence interference filters.

Fluorescence stereomicroscopes are usually equipped with a light stop positioned somewhere between the mercury lamphouse and the vertical illuminator to block damaging ultraviolet radiation from the lamp when specimens are not being observed or imaged. This stop should always be inserted into the light path when observations are not being conducted.

Reflexes, or hot spots can occur in the lower portion of the viewfield when observing specimens using higher magnification apochromatic objectives (1.6x to 2.0x) in fluorescence stereomicroscopy. This artifact is usually only visible at the lower zoom ratios and often disappears when the zoom factor is increased. In most cases, reflex problems do not occur with lower magnification objectives (0.5x and 1.0x), regardless of the optical correction factor, and are usually absent from high magnification objectives of lower correction (achromats and plan achromats).

There exists a broad range of applications for fluorescence investigations utilizing stereomicroscopes, as presented in Table 2. The number of specimens that benefit from this observation mode is considerable and covers a wide spectrum of disciplines including those ranging from the biological arena to industrial manufacturers.

Applications for Fluorescence Stereomicroscopy
Field Application
Biology Gene Expression, Cell Sorting, Dissection, Developmental Processes, Eye and Muscle Investigations
Botany Plant Cells, Tissue Autofluorescence, Soil Samples, and Parasites
Pharmacology Capillary Flow, Drugs, and ELI Observation
Hydrology Water Quality, Cell Structures, and Filter Membrane Analysis
Agronomy Seed Observation, Genetic Expression, and Transgenetics
Electronics Solder Paste, Epoxy Resin Analysis, Monitor Coatings, Polymer Castings for Integrated Circuits
Semiconductors Photo Resist Contamination, Foreign Particles, Process Errors
Polymers Foreign Particles, Voids, Bead Analysis, Non-Polymerized Areas
Metalworking Cracks and Surface Defects, Contamination, Weld Control, Fracture Analysis
Materials Cracks, Welds, Carbon Bonding, Fractures, Orientation Studies
Papermaking Fiber Coatings and Inclusions
Forensics Textile Fibers, Body Fluids, Fingerprints, Bank Notes, Forgeries
Table 2

Fluorescence observation in stereomicroscopy offers the unique property of three-dimensional observation when compared to the view presented by a classical compound microscope. In addition, the much larger working distances and depth of field afforded by the stereo technique enable a wider panoramic field of view and more intense fluorescence. These features are of tremendous benefit to investigators who are attempting to manipulate large biological specimens and materials technicians that are carrying out preparation work, such as embedding, electronics inspection, or cutting. As more clearly defined filter combinations become available for specialized applications, the use of fluorescence in stereomicroscopy should continue to climb.


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Contributing Authors

William Chambers - Industrial Division, Nikon Instruments Inc., Melville, New York 11747.

Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.