Included in the Nikon blue-violet excitation fluorescence filter portfolio are four combinations that include either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide region of the cyan, green, and red wavelengths. These combinations cover an excitation wavelength range between 400-446 nanometers with bandpass width profiles of 10, 20, and 40 nanometers. Three of the combinations employ the same dichromatic mirror, while the fourth set has a mirror with a higher wavelength cut-on (5 nanometers) to conform with its other components.
Performance of the blue-violet filter sets can be judged by comparing images from the same viewfield captured with each of the individual filter combinations, as presented in Figure 1. The specimen is a culture of fibroblast Indian Muntjac deerskin cells that were immunofluorescently labeled with primary anti-oxphos complex V inhibitor protein monoclonal antibodies (mouse) followed by goat anti-mouse Fab fragments conjugated to Pacific Blue. The visible light absorption maximum of Pacific Blue is 410 nanometers and the emission maximum occurs at 455 nanometers. In addition, the specimen was simultaneously stained for F-actin with Alexa Fluor 568 (red) conjugated to phalloidin, and for DNA with SYTOX Green. In many cases, SYTOX Green stains a variety of cytoplasmic elements in addition to DNA, as is evident in several of the images. Note the presence of signal from both the red and green fluorophores in the longpass filter combinations (BV-1A, BV-2A, and BV-2B). The background fluorescence intensity observed in the longpass sets, which is absent in the single bandpass filter combination (CFP), varies directly as a function of excitation filter bandwidth.
Of the four filter blocks constituting the Nikon blue-violet excitation series, only the CFP set incorporates a bandpass emission filter. This combination has been designed for applications with cyan fluorescent proteins, particularly in dual and triple fluorophore techniques, and produces images with an intense cyan color on a jet-black background (Figure 1(a)). The bandpass emission filter in the CFP combination eliminates fluorescence from red, yellow, and green fluorophores in specimens labeled with multiple probes. This filter set typically excludes signal from yellow fluorescent protein, but not from green fluorescent protein. The BV-1A filter combination (Figure 1(b)) passes wavelengths from a wide range of fluorochromes that emit in the cyan-blue, green and red spectral regions (greater than 470 nanometers), and has a narrow 10-nanometer excitation band to minimize autofluorescence. The dichromatic mirror in the BV-1A filter set has a cut-on wavelength of 455 nanometers.
Commonly specified as the standard blue-violet filter set, the BV-2A combination is equipped with a 40-nanometer bandpass excitation filter that covers the entire blue-violet wavelength region. Coupled with a 455-nanometer cut-on dichromatic mirror and a longpass barrier filter (470-nanometer cut-on), the BV-2A produces the brightest images of any filter combination in the Nikon blue-violet group (Figure 1(c)). The BV-2B filter set is similar in configuration to the BV-2A, except that each component is red-shifted by 5 nanometers in center or cut-on wavelength specification. The result is that images acquired under the same conditions have darker backgrounds and improved overall contrast compared to those using the BV-2A filter block. Specifications for the dichromatic mirrors, excitation, and barrier filters from the various Nikon blue-violet filter combinations are listed in Table 1.
Table 1 - Nikon Blue-Violet Filter Combination Specifications
|Filter Set Description||Excitation Filter (nm)||Dichromatic Mirror (nm)||Barrier Filter (nm)||Remarks|
|BV-1A||435/10 (430-440)||455 (LP)||470 (LP)||Narrow Excitation Band Longpass Barrier Filter|
|BV-2A||420/40 (400-440)||455 (LP)||470 (LP)||Standard BV Cube Longpass Barrier Filter|
|BV-2B||425/40 (405-445)||460 (LP)||475 (LP)||Darker Background Longpass Barrier Filter|
|CFP||436/20 (426-446)||455 (LP)||480/40 (460-500)||Narrow Excitation Band Bandpass Barrier Filter|
- BV-1A - The BV-1A filter combination is designed as a filter block for blue-violet fluorescence excitation with a very narrow bandwidth to minimize specimen autofluorescence. The longpass emission filter allows detection of a wide range of fluorochrome wavelengths.
- BV-2A - The BV-2A combination is designed as the standard blue-violet fluorescence filter block. With a medium excitation filter bandpass and longpass emission filter, the images produced with this combination are the brightest of the Nikon blue-violet filter group.
- BV-2B - The BV-2B combination is designed as a filter block for blue-violet fluorescence that provides a darker background and higher contrast than the standard BV-2A filter combination.
- CFP (Cyan Fluorescent Protein) - The CFP filter block combines a narrow excitation bandwidth with a bandpass emission filter to limit autofluorescence as well as to exclude detection of red, yellow, and many green-emitting fluorochromes.
A wide array of fluorophores has been developed for investigations using excitation wavelengths spanning the blue-violet region. Catalogued in Table 2 are some of the most popular dyes and fluorescent probes that can be visualized with the Nikon blue-violet 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 violet and blue.
Table 2 - Fluorochromes with Blue-Violet Excitation Spectral Profiles
|Fluorochrome||Excitation Wavelength (Nanometers)||Emission Wavelength (Nanometers)||Recommended Filter Set(s)|
|Acriflavin||436||520||BV-1A, BV-2A BV-2B|
|AFA (Acriflavin Feulgen SITSA)||355-425||460||BV-2A, BV-2B|
|Alexa Fluor 405||401||421||BV-2A, BV-2B|
|Alexa Fluor 430||431||541||BV-1A, BV-2A BV-2B|
|APTS (Aminopyrenetrisulfonate)||424||505||BV-2A, BV-2B|
|Berberine Sulfate||430||550||BV-1A, BV-2A BV-2B|
|Beta-Lactamase||409||447 (520)||BV-2A, BV-2B|
|Bis-BTC (Bis-Benzothiazole Indicator)||405 (High Ion) 455 (Low Ion)||505 (High Ion) 529 (Low Ion)||BV-2A, BV-2B|
|Brilliant Sulphoflavin FF||430||520||BV-1A, BV-2A BV-2B|
|BTC (Benzothiazole Indicator)||401 (High Ion) 464 (Low Ion)||529 (High Ion) 533 (Low Ion)||BV-2A, BV-2B|
|BTC-5N (Benzothiazole Indicator)||417 (High Ion) 459 (Low Ion)||532 (High Ion) 517 (Low Ion)||BV-2A, BV-2B|
|Calcofluor White||440||500-520||BV-1A, BV-2A BV-2B|
|Cascade Yellow||402||545||BV-2A, BV-2B|
|CCF2 (GeneBLAzer)||402||520||BV-2A, BV-2B|
|CFP (Cyan Fluorescent Protein)||434||477||All|
|CFP/DsRedFP FRET||434||583||BV-1A, BV-2A BV-2B|
|CFP/YFP FRET||434||527||BV-1A, BV-2A BV-2B|
|Chromomycin A||436-460||470||BV-1A, BV-2A CFP|
|CTC (Chlorotetracycline)||400-450||602||BV-1A, BV-2A BV-2B|
|Diphenyl Brilliant Flavin 7GFF||430||520||BV-1A, BV-2A BV-2B|
|ECFP (Enhanced Cyan FP)||433, 453||475, 501||All|
|Euchrysin||430||540||BV-1A, BV-2A BV-2B|
|Flazo Orange||375-530||612||BV-1A, BV-2A BV-2B|
|FluoroJade||425||510||BV-1A, BV-2A BV-2B|
|Fura Red||436 (High Ca) 472 (Low Ca)||637 (High Ca) 657 (Low Ca)||BV-1A, BV-2A BV-2B|
|Genacryl Brilliant Yellow 10GF||430||485||All|
|Genacryl Yellow 5GF||430||475||All|
|Gloxalic Acid||405||460||BV-2A, BV-2B|
|HAT (Hydroxytryptamine)||370-415||520-540||BV-2A, BV-2B|
|HPTS (Hydroxypyrene Trisulfonic Acid)||403||513||BV-2A, BV-2B|
|Lucifer Yellow CH||425||528||BV-1A, BV-2A BV-2B|
|Lucifer Yellow VS||430||535||BV-1A, BV-2A BV-2B|
|LysoSensor Green DND-153, DND-189||442||505||BV-1A, BV-2A BV-2B|
|Maxilon Brilliant Flavin 10GFF||450||495||BV-2A, BV-2B|
|NBD (Chloronitrobenzofurazan)||450||530||BV-1A, BV-2A BV-2B|
|Nuclear Fast Red||289-530||580||BV-1A, BV-2A BV-2B|
|Olivomycin||430||545||BV-1A, BV-2A BV-2B|
|Pacific Blue||410||455||BV-2A, BV-2B|
|POPO-1, POPRO-1||434||456||BV-1A, BV-2A BV-2B|
|Primuline O||410||550||BV-2A, BV-2B|
|PyMPO (pyridyloxazole)||415||570||BV-2A, BV-2B|
|QDots 525-655||350-450||525-655||BV-1A, BV-2A BV-2B|
|Quinacrine Mustard||440||510||BV-1A, BV-2A BV-2B|
|Sevron Orange||440||530||BV-1A, BV-2A BV-2B|
|Sevron Yellow L||430||490||All|
|Spectrum Blue||400||450||BV-2A, BV-2B|
|SYTO 40||420||441||BV-1A, BV-2A BV-2B|
|SYTO 41||430||454||BV-1A, BV-2A BV-2B|
|Tetracycline||390-425||525-560||BV-1A, BV-2A BV-2B|
|Thioflavin S||430||550||BV-1A, BV-2A BV-2B|
|Uranine B||420||520||BV-1A, BV-2A BV-2B|
Although the four filter combinations described in this section serve adequately in a majority of the fluorescence investigations with blue-violet excitation wavelengths, several additional special filter sets are available from the aftermarket manufacturers. Some of these combinations incorporate blue-violet band excitation with non-standard dichromatic mirrors and barrier filters, which may be selected to match particular detector characteristics. Filter sets designed for xenon arc-discharge lamp excitation generally employ wide bandpass excitation filters (approximately 100 nanometers) centered in the 400-440 nanometer (blue-violet) spectral region. In other variations, a narrow excitation bandpass may be designed to selectively isolate specific emission lines of sources such as mercury lamps, which occur in the appropriate spectral region, or if no strong excitation line for the fluorochrome of interest is available, wider emission filter bandpass may be required in order to collect sufficient signal.
Other specialized filter sets intended for ratiometric analysis of various environment-sensitive probes include two emission filters with distinct bandpass regions. Sets for ion-sensitive probes, such as beta-lactamase, 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 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 for specific fluorophores have been developed, some of which are designed to allow rapid dual imaging with a single emission filter and two excitation filters.
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