Fluorescence Filter Combinations
Blue Excitation Filter Sets
Included in the Nikon blue excitation fluorescence filter portfolio are six carefully balanced combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide band of the green, yellow, red, and near-infrared spectral regions. These filter combinations cover an excitation wavelength range between 420 and 495 nanometers with bandpass width profiles of 20, 30, 40, and 70 nanometers. Five of the combinations employ the same dichromatic mirror, while the sixth set has a mirror with a lower wavelength cut-on to increase the acquired signal. Bandpass barrier filters for the Nikon blue excitation filter combinations all have a spectral width of 40 nanometers. One of the filters (B-3A) is designed to be employed with tungsten-halogen illumination.
Performance of the blue 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 intestine and stained with a combination of three fluorophores. Mucus of the goblet cells in the thin section was labeled with Alexa Fluor 350 (ultraviolet excitation and blue emission) wheat germ agglutinin. The filamentous actin inhabiting the brush border were stained with Alexa Fluor 568 (red emission) phalloidin, while DNA in the nuclei was counterstained with SYTOX Green (the green emission prevalent in Figure 1) to round out the labeling regime.
Sets having bandpass emission filters in the Nikon blue fluorescence excitation series, the B-1E, B-2E, and B-2E/C, produce images at different levels of contrast with a deep green color on a jet-black background (Figures 1(a), 1(b), and 1(c)), and are ideal for use in multi-color fluorescence imaging with other excitation filter combinations. The bandpass emission combinations are designed to eliminate, or at least significantly reduce (depending upon spectral characteristics), fluorescence from yellow, red, and near-infrared fluorophores in specimens labeled with multiple probes. Generally, filter combinations utilizing bandpass emission components produce higher signal-to-noise ratios than those employing longpass filters, and the images exhibit visually darker backgrounds, although with less overall brightness.
All of the three filter combinations having bandpass emission filters utilize the same dichromatic mirror (505-nanometer cut-on) and similar 40-nanometer bandwidth emission filters. The B-1E set is equipped with a narrow-bandpass excitation filter centered on 480 nanometers, while the B-2E set differs only in having a wide excitation band centered at 470 nanometers. The emission passband of the B-2E/C combination is shifted by 5 nanometers to shorter wavelengths in order to optimize detection of popular fluorophores (FITC and others). The medium bandwidth of the excitation filter in the B-2E/C set is designed to exclude crossover detection of yellow and orange fluorochromes utilized in specific multiple labeling or counterstaining techniques.
Interactive Java Tutorial
Blue 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 blue (420-500 nanometers) region.
The B-1A filter combination (Figure 1(d)) incorporates a narrow-bandpass excitation filter (20-nanometers) that is intended to minimize photobleaching as well as specimen autofluorescence when excited by the shorter wavelengths in the blue spectral region. The dichromatic mirror in the B-1A filter set has a cut-on wavelength of 505 nanometers, as is used in most of the blue excitation sets. The longpass barrier filter passes emission above the 520-nanometer cut-on, allowing detection of a wide range of fluorochromes emitting in the green, yellow, orange, and red wavelength regions. Containing similar components, but with a wider 40-nanometer excitation passband, the B-2A filter set (Figure 1(e)) is considered the standard Nikon blue-excitation set. The wide excitation band, in the center of the blue spectral region, is combined with a 5-nanometer lower dichromatic mirror cut-on wavelength (500 nanometers) than the other blue-excitation filter blocks. This combination provides a brighter emission signal than is observed with the B-1A set for many popular fluorochromes.
The B-3A filter set (Figure 1(f)) combines the same dichromatic mirror and emission filter as the B-1A set, but with a very wide 70-nanometer excitation passband (420-490 nanometers) to maximize the energy available to fluorochromes absorbing at essentially any wavelength in the blue spectral region. The set is designed to be utilized primarily with tungsten-halogen illumination, although it may also be useful with arc-discharge lamps when employing probes having a very weak emission signal. The dichromatic mirror cut-on wavelength of 505 nanometers, coupled with a longpass emission filter (520-nanometer cut-on) enables detection of a large range of fluorochromes emitting green, yellow, orange, and red wavelengths. Specifications for the dichromatic mirrors and filters from the various Nikon blue filter combinations are listed in Table 1.
Nikon Blue Excitation Filter Combination Specifications
|
 |
||||||||||||||||||||||||||||||||||||
 |
 |
Table 1
B-1A - The B-1A filter combination is designed with a narrow excitation passband in order to reduce autofluorescence and photobleaching. The longpass barrier (emission) filter is capable of transmitting signals from green, yellow, orange, and red fluorophores that have significant absorption in the upper blue wavelength region corresponding to the excitation bandpass window.
B-2A - The B-2A filter combination is the standard filter set in the Nikon blue excitation group. Its design incorporates a wide excitation bandpass in order to provide an expanded absorption window for fluorophores compared to the B-1A combination, and the set also has lower dichromatic mirror and barrier filter cut-on wavelengths, producing enhanced image brightness when compared to the B-1A filter set.
B-3A - The B-3A filter combination utilizes the same dichromatic mirror and longpass emission filter as the B-1A set, but employs a very wide excitation band that makes it suitable for tungsten-halogen illumination. This combination enables the transmission of a significant amount of the signal from green, yellow, orange, and red fluorophores that have absorption bands in the blue wavelength region.
B-1E - The B-1E filter combination is one of three sets in the blue fluorescence excitation series that utilize bandpass, as opposed to longpass, barrier filters, and which differ primarily in the width of the excitation band. Each of the bandpass barrier filter sets provides performance suitable for imaging specimens labeled with fluorescein isothiocyanate (FITC) (green emission), while blocking yellow, orange, and red emission. The B-1E combination uses a narrow-bandpass excitation filter in conjunction with the bandpass emission (barrier) filter.
B-2E - The B-2E filter combination employs a medium excitation bandpass, extending 20 nanometers lower in wavelength than the excitation band of the B-1E set. The dichromatic mirror and bandpass barrier filter specifications are the same in the B-1E and B-2E filter combinations.
B-2E/C - The B-2E/C filter combination employs a medium-width excitation band in conjunction with a slightly lowered emission bandpass wavelength region compared to the B-1E and B-2E sets. The performance is optimized for detection of a number of popular fluorophores used in multiple-labeling experiments, while providing improved exclusion of yellow to red wavelengths.
A wide array of fluorophores has been developed for investigations using excitation wavelengths spanning the blue (420-500 nanometers) wavelength region. Catalogued in Table 2 are some of the most popular dyes and fluorescent probes that can be visualized with the Nikon blue 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-violet and green.
Fluorochromes with Blue Excitation Spectral Profiles
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
Table 2
Although the six filter combinations described above adequately serve in a majority of the fluorescence investigations with blue excitation wavelengths, several additional special filter sets are available from the aftermarket manufacturers. Some of these combinations incorporate blue-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 lamps and lasers, which occur in the appropriate spectral region. If no strong excitation line for the fluorochrome of interest is available, a wider excitation filter bandpass 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 are 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 (488-nanometer excitation), can be configured in two variations, each having a single excitation filter and dual emission filters, while utilizing different dichromatic mirror complements. 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. An example is the polar fluorescein derivative, BCECF, a commonly used indicator of intracellular pH, due to its pH-dependent spectral shifts. 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.
