Fluorescence Filter Combinations
Green Excitation Filter Sets
The Nikon green excitation fluorescence filter combination series includes six carefully balanced sets that incorporate either single bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission within a narrow or wide band of the yellow, orange, red, and near-infrared spectral regions. These filter combinations encompass an excitation wavelength range between 510 and 560 nanometers with passband width profiles of 10, 25, 30, and 50 nanometers (including narrow, medium, and wide excitation bands). Three of the combinations employ the same dichromatic mirror (565 nanometers), while the other three have mirrors with higher wavelength cut-on values (570 or 575 nanometers). Two of the six Nikon green excitation filter sets incorporate bandpass barrier filters having bandwidths of 60 and 75 nanometers.
Performance of the green filter sets can be judged by comparing images from the same viewfield captured with each of the individual filter combinations, as illustrated in Figure 1. The specimen is a thin cryostat tissue section (16 micrometers) obtained from mouse kidney and stained with a combination of three fluorophores. Elements of the glomeruli and convoluted tubules in the thin section were labeled with Alexa Fluor 488 (green emission) wheat germ agglutinin. The filamentous actin inhabiting the glomeruli and the brush border were stained with Alexa Fluor 568 phalloidin (red emission), while DNA in the nuclei was counterstained with DAPI (blue emission) to round out the labeling regime. Note the variation in emission color and intensity exhibited by this specimen using the cadre of green excitation filter combinations.
Two of the Nikon green excitation filter sets incorporate bandpass emission (barrier) filters intended to reduce or eliminate interference from fluorophores emitting in the red or near-infrared spectral regions in specimens labeled with multiple probes. The G-2E/C set combines a narrow excitation band (25 nanometers) with a 60-nanometer emission bandpass. The Cy3 combination (a member of the Nikon HYQ filter group) incorporates similar components, but with wider-bandpass excitation and emission filters (30 and 75 nanometers, respectively), and with a dichromatic mirror cut-on wavelength of 570 nanometers compared to a 565-nanometer value for the G-2E/C set. Both of the sets utilizing bandpass emission filters are suitable for use with tetramethylrhodamine isothiocyanate (TRITC) and Cy3, having bandpass ranges of 590-650 nanometers for the G-2E/C set and 573-648 nanometers for the Cy3 combination. Images produced using the Cy3 set have a more yellow visual appearance, reflecting the extension of the emission bandpass to lower wavelengths (see Figures 1(a) and 1(d)), and the images are somewhat brighter due to the greater excitation and emission energy resulting from the wider bandpass values.
The G-1A and G-1B filter combinations have very similar optical components, and can be considered essentially interchangeable. Their primary application is with mercury arc-discharge lamps for selection of the 546-nanometer green emission line. Both are equipped with narrow 10-nanometer passband excitation filters (541-551 nanometers) to minimize autofluorescence, but differ somewhat in the cut-on wavelengths of their dichromatic mirrors and barrier (emission) filters. The G-1A set has a dichromatic mirror cut-on wavelength of 575 nanometers, coupled with a 580-nanometer (cut-on) emission filter. The G-1B filter set employs a lower wavelength dichromatic mirror (565 nanometers) in combination with a higher wavelength emission filter (590 nanometers). The increased wavelength separation between the mirror and emission filter wavelength values for the G-1B set compared to the G-1A can result in significant image differences with some fluorochromes. In addition, the higher emission filter cut-on excludes yellow wavelengths and results in a more reddish image appearance with the G-1B filter combination.
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Green Excitation Fluorescence Filters
Explore how the variations in the excitation and emission filter spectral profiles affect signal levels, overall filter performance, and image contrast in combinations designed for excitation of fluorophores in the green (510-560 nanometers) spectral region.
Considered the standard green-excitation filter set, the G-2A combination incorporates an excitation filter with a wide 50-nanometer passband (510-560 nanometers) that covers most of the green spectral region (Figure 1(c)). The longpass barrier filter passes emission from a large range of fluorochromes that emit at wavelengths above the 590-nanometer cut-on (orange through near-infrared). The dichromatic mirror employed in the G-2A set has a cut-on wavelength of 565 nanometers. A similar filter set, the G-2B combination utilizes the same excitation filter (50-nanometer bandpass) as the G-2A, but has a 10-nanometer longer wavelength dichromatic mirror cut-on (575 nanometers), coupled with a longpass barrier filter that is shifted toward the red spectral region by 20 nanometers, to 610 nanometers. Images produced with the G-2B set have darker backgrounds than those from the G-2A (Figure 1(f)), and also appear more reddish due to the increase in emission cut-on wavelength, which excludes more yellow-orange emission.
Nikon Green Excitation Filter Combination Specifications
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Table 1
G-1A - The G-1A filter combination for green wavelength excitation is designed with a narrow excitation bandpass in order to reduce autofluorescence and minimize specimen radiation exposure. The longpass barrier (emission) filter is capable of transmitting signals from yellow, orange, and red-emitting fluorophores that have significant absorption in the central green wavelength region.
G-1B - The G-1B filter combination has a similar component profile to the G-1A set, incorporating the narrow excitation bandpass to reduce autofluorescence and specimen damage, but with different dichromatic mirror and emission filter cut-on wavelengths. The shift of the emission filter cut-on to longer wavelengths produces images that appear redder due to the increased blocking of yellow emission.
G-2A - The G-2A filter combination is configured as the standard green filter block, with a wide excitation passband for application to a large number of fluorophores activated by green wavelengths. The longpass emission filter permits detection of all fluorescence wavelengths longer than the yellow spectral region.
G-2B - The G-2B filter combination is designed with the same excitation characteristics as the G-2A filter block. However, with higher dichromatic mirror and emission filter cut-on wavelengths, the G-2B set produces images having darker backgrounds and a color shift toward the red.
G-2E/C - The G-2E/C filter combination is designed for optimal performance with a number of popular fluorescent probes that are used in multiple labeling experiments. A narrow excitation bandpass provides selective excitation, while the bandpass emission filter reduces interference from fluorophores emitting in the red and near-infrared.
Cy3 HYQ - The Cy3 green-excitation filter combination is similar in configuration to the G-2E/C set. However, with a wider excitation passband and broadening of the emission bandpass to encompass lower wavelengths, the images produced by the Cy3 filter block are somewhat brighter and reflect the detection of more yellow spectral region emission.
TRITC HYQ - The TRITC HYQ filter combination for green-wavelength excitation is designed for improved performance with fluorochromes such as TRITC and DiI. Compared to some green-excitation sets typically used in this application, a broadened excitation passband and red-shifted dichromatic mirror cut-on contribute to higher signal level for these fluorophores, and allow improved detection of variants of DsRed and the far-red-fluorescing HcRed proteins.
A wide array of fluorophores has been developed for investigations using excitation wavelengths spanning the green (510-560 nanometers) wavelength region. Catalogued in Table 2 are many of the popular dyes and fluorescent probes that can be visualized with the Nikon green excitation filter combinations. The localized environment significantly influences fluorophore absorption and emission spectra maximum (peak) wavelengths, so the values presented in Table 2 may vary with experimental conditions. This list is intended to serve only as a guide for filter and fluorophore selection and should not be considered a comprehensive or exhaustive compilation. Many of the fluorescent probes included in Table 2 are proprietary and have been developed to minimize photobleaching while ensuring a maximum overlap between the fluorochrome absorption and emission spectra and common fluorescence filter combinations. Note that due to broad absorption and emission bands, several of the fluorescent probes listed in Table 2 are also suitable for use with filter combinations in other excitation wavelength regions, including blue and yellow.
Fluorochromes with Green Excitation Spectral Profiles
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Table 2
Although the six filter combinations described above adequately serve in a majority of the investigations with green excitation wavelengths, several additional specialized filter sets are available from the aftermarket manufacturers. Some of these combinations incorporate green-band excitation with non-standard dichromatic mirrors and barrier filters, which may be chosen to match particular detector characteristics. In other variations, a narrow excitation bandpass may be designed to selectively isolate specific emission lines of sources such as mercury arc-discharge lamps, which occur in the appropriate spectral region. If no strong excitation line for the fluorochrome of interest is available, a wider excitation filter passband may be required in order to collect sufficient signal, and a similar approach is necessary when broadband low-intensity sources such as tungsten-halogen lamps must be used.
Other specialized filter sets intended for ratiometric analysis of probes that exhibit environment-sensitive fluorescence emission include two emission filters with distinct bandpass regions. Sets for ion-sensitive probes, such as SNARF, can be configured in several variations, each having a single excitation filter and dual emission filters, while utilizing different dichromatic mirror complements. This fluorochrome can be excited at 515, 534, or 546 nanometers in the green spectral region, and applicable filter sets may utilize different excitation filters. As configured for simultaneous ratiometric imaging of both emission wavelengths with an emission-splitting system, two dichromatic mirrors are included in the filter set. Only one dichromatic mirror is utilized for sequential ratiometric imaging with an emission filter wheel. In addition, filter combinations are tailored for specific fluorophores whose excitation profile is ion dependent. Using a pH-sensitive probe, such as SNAFL (see Figure 2), filter cubes equipped with two excitation filters and a single emission filter allow rapid ratiometric analysis of the fluorescence intensities measured at two different excitation wavelengths, which can be calibrated as an indicator of pH at the fluorescent probe location.
Contributing Authors
Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
John D. Griffin, Nathan S. Claxton, Matthew J. Parry-Hill, Thomas J. Fellers, Kimberly M. Vogt, Ian D. Johnson, Shannon H. Neaves, Omar Alvarado, Lionel Parsons, Jr., Michael A. Sodders, Richard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
