Fine-tuning of the fluorescence microscope excitation spectrum for imaging dual or multiply labeled specimens can be readily accomplished with a split-filter excitation balancer, which contains tandem shortpass and longpass interference filters that are translated across the illumination aperture to adjust the arc-discharge lamp wavelength distribution profile. This interactive tutorial explores how the Nikon Eclipse i-Series excitation balancer system affects the fluorescence emission intensity of multiply labeled specimens when employed in conjunction with dual and triple excitation band filter combinations.
The tutorial initializes with the image of a randomly selected dual or triple fluorescent labeled specimen appearing in the Specimen Image window. Adjacent to this window is a spectral plot entitled Excitation Passband Spectral Profiles, which displays the absorption spectra for the fluorophores utilized to stain the specimen along with the approximate transmission percentage (white line) of the excitation balancer filter combination at the illumination aperture (see the excitation balancer drawing) in the visible spectral region. The fluorescent probes and their targets are identified for each specimen in the lower left-hand portion of the tutorial window next to a colored bar that coincides with the color of the excitation spectral profile in the excitation passband graph. Individual spectral profiles on the graph can be toggled on or off using the appropriate fluorophore checkbox. New specimens can be loaded into the tutorial using the Choose A Specimen pull-down menu.
In order to operate the tutorial, use the Aperture Position slider to shift the microscope aperture between the longpass and shortpass regions of the excitation balancer (illustrated in the Excitation Balancer drawing in the lower right-hand portion of the tutorial). As the slider is translated to the far left, the aperture moves into the shortpass region of the balancer and the excitation passband changes to resemble the profile of a shortpass filter. The specimen image also changes to reflect the loss of fluorescence intensity from probes having emission spectra in the longer wavelength region. Shifting the slider to the far right moves the aperture into the longpass region and produces a corresponding change in the excitation passband graph and specimen image. In between these extremes is a Mixed Region where the excitation passband resembles a hybrid of the two cutoff filters (as does the specimen image).
The Nikon Eclipse i-Series fluorescence illuminator excitation balancer system enables the microscopist to fine-tune the excitation wavelength passband at the illumination aperture for maximum efficiency when using optical blocks with dual and triple excitation filter combinations. This feature is useful to adjust individual fluorophore intensities in specimens containing several probes to, for example, reduce the fluorescence emission from one probe while simultaneously increasing the intensity of another in order to optimize observation and the recording of digital images. Specimens stained with two or more fluorophores often exhibit unequal fluorescence emission intensity levels due to a variety of factors, including unequal quantum yields, overstaining or understaining, inadequate membrane permeability, poor blocking, fixer-masked antibody binding sites, low inherent antigen concentrations, and simple fluorophore and secondary antibody mixing errors. Excitation balancers can help alleviate fluorescence signal imbalance in these specimens.
The Nikon excitation balancer consists of a rectangular optical glass window containing two thin-film interference coatings that act as either a longpass or shortpass filter (or a mixture of both), depending upon the position of the window in the optical train, as illustrated in the tutorial and in Figure 1. The rectangular filter is mounted in a slider that is positioned adjacent to the microscope illumination aperture diaphragm, which is contained in a removable housing that inserts into the microscope epi-fluorescence illuminator (termed the Digital Imaging Head in Nikon Eclipse i-Series microscopes) preceding the field diaphragm in the optical pathway. The amount of light passing through the excitation balancer and the numerical aperture of the excitation illumination can be controlled by adjusting the size of the aperture diaphragm. When observing overstained specimens, excessive fluorophore emission can often be reduced using the excitation balancer in combination with neutral density filters and by constricting the aperture diaphragm size.
Illustrated in Figure 1 are the spectral profiles of the longpass (yellow curve) and shortpass (purple curve) excitation balancer filters, along with the normalized absorption spectra of three fluorophores (DAPI, FITC, and Texas Red) matched to the characteristics of the filter combination. In practice, the Nikon excitation balancer filter combination slider is translated across the illumination aperture by pulling or pushing the frame handle, thus exposing the excitation light to varying mixtures of the wedge-shaped cutoff filters. When the slider is positioned so that the illumination aperture coincides with the shortpass portion of the excitation balancer (area under the purple curve), only fluorophores with absorption spectral profiles in the region below 520 nanometers will be excited to a significant degree by illumination from the arc-discharge lamp. As the slider is moved to the opposite end of the excitation balancer window, the longpass thin-film filter portion (area under the yellow curve) is illuminated by the aperture to excite fluorescent probes having wavelengths greater than 430 nanometers. Note that fluorophores, such as FITC and Alexa Fluor 488, having excitation bands in the blue spectral region (450-500 nanometers) are excited with a high degree of efficiency regardless of the excitation balancer position.
When the microscope illumination aperture is positioned in "mixed" regions that contain portions of both the longpass and shortpass filters, the excitation efficiency varies for fluorophores having absorption bands in the ultraviolet and yellow-green regions of the visible light spectrum. As the portion of the slider in front of the illumination aperture is translated from the region containing exclusively the shortpass interference filter into the region containing equal proportions of both filters, the absorption efficiency of ultraviolet-absorbing fluorophores drops continuously from approximately 100 percent to 50 percent. Simultaneously, the absorption efficiency of green-absorbing fluorophores increases steadily from zero to 50 percent, while that of blue-absorbing fluorophores remains constant. The opposite effect occurs when the slider is motion is continued into the regions containing larger proportions of the longpass filter. In this case, the absorption efficiency of ultraviolet-absorbing fluorophores drops from 50 percent to zero as the efficiency of green-absorbing fluorophores increases from 50 percent to 100 percent. Again, the absorption efficiency of fluorophores excited in the blue region remains constant.
A variety of common fluorophores, including the Alexa Fluors, cyanine dyes (the CyX series), BODIPYs, fluorescent proteins, MitoTrackers, SYTOX nucleic acid stains and many traditional synthetic and natural probes can be readily resolved using the excitation balancer in combination with either dual or triple excitation filter sets. For example, the series of images presented in Figure 2 illustrate a monolayer culture of albino Swiss mouse embryo cells stained with Alexa Fluor 405 (tubulin), SYTOX Green (nuclei), and Texas Red (the filamentous actin network) and imaged with a DAPI-FITC-Texas Red triple filter combination using the excitation balancer. In Figure 2(a), the excitation balancer slider is positioned with the shortpass thin-film filter region in front of the illumination aperture to preferentially excite the violet and blue fluorophores (Alexa Fluor 405 and SYTOX Green). Translating the slider into the central region of the excitation balancer (containing equal proportions of both filters) produces an image containing contributions from all three fluorophores (Figure 2(b)). Finally, moving the slider into the region containing the longpass filter (Figure 2(c)) eliminates the violet fluorophore to yield an image produced by the fluorophores (SYTOX Green and Texas Red) excited in the blue and yellow-green regions of the visible light spectrum. Replacing the DAPI-FITC-Texas Red triple excitation band filter combination with one designed for DAPI, FITC, and TRITC will produce similar results, but optimized for fluorophores having absorption profiles shifted from yellow-green to green (TRITC versus Texas Red) wavelengths.
The excitation balancer is also ideal for separating fluorescence emission in specimens containing two fluorescent probes using the popular dual bandpass excitation filter combinations, such as DAPI-FITC, FITC-TRITC, and FITC-Texas Red. Illustrated in Figure 3 are several examples. Figure 3(a) presents a thin tissue section of mouse small intestine stained with the nuclear probe Hoechst 33258 (blue fluorescence) and Alexa Fluor 488 conjugated to phalloidin (green fluorescence) imaged through a dual filter DAPI-FITC combination with the shortpass portion of the excitation balancer in front of the illumination aperture to excite both fluorophores. Translating the excitation balancer into the longpass region (Figure 3(b)) eliminates the ultraviolet-absorbing nuclear dye (Hoechst 33258). Employed in this manner, the excitation balancer is very useful for controlling fluorescence emission in specimens that have overstained nuclei.
Coupled to blue and green dual excitation filter combinations (FITC-TRITC and FITC-Texas Red), the excitation balancer can be used to control emission intensity levels from specimens labeled with fluorophores absorbing in the green through orange wavelength region. Illustrated in Figure 3(c) is the image of an adherent monolayer culture of Indian Muntjac deer skin fibroblast cells stained with Alexa Fluor 555 (orange fluorescence) conjugated to phalloidin, and Alexa Fluor 488 conjugated to goat anti-mouse antibodies targeting anti-peroxisomal membrane protein (PMP-70) primary antibodies. The image was captured using a FITC-TRITC dual band excitation filter set in combination with the excitation balancer positioned so that the longpass thin-film filter region coincides with the illumination aperture. Translating the excitation balancer to the shortpass region (Figure 3(d)) reduces (or eliminates) the fluorescence emission from the green-absorbing fluorophore (Alexa Fluor 555). Similarly, the FITC-Texas Red filter produces comparable effects (Figures 3(e) and 3(f)). The specimen in the latter two frames was a rat thoracic aorta cell culture labeled with Alexa Fluor 594 conjugated to phalloidin (actin) and Cy2 conjugated to secondary antibodies targeting mouse anti-alpha-tubulin primary antibodies.
Matthew J. Parry-Hill, John D. Griffin, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.