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.

Figure 1 - Green Excitation Fluorescence Filter Set Performance

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.

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.

Table 1 - Nikon Green Excitation Filter Combination Specifications

Filter Set DescriptionExcitation Filter (nm)Dichromatic Mirror (nm)Barrier Filter (nm)Remarks
G-1A546/10 (541-551)575 (LP)580 (LP)Narrow Excitation Band Longpass Barrier Filter
G-1B546/10 (541-551)565 (LP)590 (LP)Narrow Excitation Band Longpass Barrier Filter
G-2A535/50 (510-560)565 (LP)590 (LP)Standard G Set Longpass Barrier Filter
G-2B535/50 (510-560)575 (LP)610 (LP)Darker Background Longpass Barrier Filter
G-2E/C540/25 (528-553)565 (LP)620/60 (590-650)Narrow Excitation Band Bandpass Barrier Filter
Cy3 HYQ545/30 (530-560)570 (LP)610/75 (573-648)Medium Excitation Band Bandpass Barrier Filter
TRITC HYQ545/30 (530-560)570 (LP)620/60 (590-650)Medium Excitation Band Bandpass Barrier Filter
  • 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.

Table 2 - Fluorochromes with Green Excitation Spectral Profiles

FluorochromeExcitation Wavelength (Nanometers)Emission Wavelength (Nanometers)Recommended Filter Set(s)
AAD (Aminoactinomycin)546647All
Acid Fuchsin540630All
Acridine Red455-600560-680All
Alexa Fluor 532531554G-1A, G-2A G-1B, G-2E/C, Cy3
Alexa Fluor 546556573G-1A, G-2A G-1B, G-2E/C, Cy3
Alexa Fluor 555553568All
Alexa Fluor 568578603All
Alexa Fluor 594590617G-2A, G-2B Cy3
Alizarin Complexon530-560624-645All
Alizarin Red530-560580All
Astrazon Brilliant Red 4G500585G-2A, G-2B G-2EC, Cy3
Astrazon Red 6B520595All
BODIPY 530/550 (Difluoroboradiazaindacene)534554G-1A, G-2A G-1B, G-2E/C, Cy3
BODIPY 542/563543563All
BODIPY 558/568558569All
BODIPY 564/570564570All
BODIPY R542574All
BODIPY R6G528547G-2A, G-2B G-2E/C, Cy3
BODIPY TMR542574G-1A, G-2A G-1B, G-2E/C, Cy3
BODIPY TMR-X542574G-1A, G-2A G-1B, G-2E/C, Cy3
Calcium Orange549576G-1A, G-2A G-1B, G-2E/C, Cy3
Carbocyanine LDL520560G-1A, G-2A G-1B, G-2E/C, Cy3
Carboxynaphthofluorescein512563G-2A, G-1B G-2E/C
Carboxyrhodamine518543G-1A, G-2A G-1B, G-2E/C, Cy3
CellTracker Green BODIPY522529G-1A, G-2A G-1B, G-2E/C, Cy3
CellTracker Orange CMR541565G-1A, G-2A G-1B, G-2E/C, Cy3
CellTracker Yellow-Green CMEDA (5-Chloromethyleosin Diacetate)524544G-1A, G-2A G-1B, G-2E/C, Cy3
CoroNa Red551 (High Na) 547 (Low Na)576 (High Na) 570 (Low Na)G-1A, G-2A G-1B, G-2E/C, Cy3
Cy3552570G-1A, G-2A G-1B, G-2E/C, Cy3
Cy3.1.8554568G-1A, G-2A G-1B, G-2E/C, Cy3
Di-12-ANEPPQ (Aminonaphthylethenylpyridinium Dyes)519719G-2A, G-2B
Di-2-ANEPEQ517721G-2A, G-2B
Di-8-ANEPPQ516721G-2A, G-2B
Dil (Dioctadecylindocarbocyanine)549565G-1A, G-2A G-1B, G-2E/C, Cy3
DilC16(3)549565G-1A, G-2A G-1B, G-2E/C, Cy3
DilC18(3)550565G-1A, G-2A G-1B, G-2E/C, Cy3
DiSBAC2(3) (Dialkylthiobituric Acid)535560G-1A, G-2A G-1B, G-2E/C, Cy3
DsRed (DsRed Fluorescent Protein)558583All
Eosin524545G-1A, G-2A G-1B, G-2E/C, Cy3
Erythrosin529554G-1A, G-2A G-1B, G-2E/C, Cy3
Erythrosin ITC529555G-1A, G-2A G-1B, G-2E/C, Cy3
Ethidium Bromide510595All
Ethidium Homodimer528617All
Evans Blue550610All
FM5-95560734G-1A, G-2A G-1B, G-2B
Genacryl Brilliant Red B520590All
HEX (Hexachlorofluorescein)535556G-1A, G-2A G-1B, G-2E/C, Cy3
Hexidium Iodide518600All
JC-1514529-590G-2A, G-2B G-2E/C, Cy3
JOE (Dichlorodimethoxyfluorescein)520548G-1A, G-2A G-1B, G-2E/C, Cy3
JOJO-1, JO-PRO-1530545G-1A, G-2A G-1B, G-2E/C, Cy3
LDS 751-DNA (Laser Dye Styryl 751)543712G-1A, G-2A G-1B, G-2B
LOLO-1, LO-PRO-1565579G-2A, G-1B G-2E/C
LysoTracker Red577590G-1A, G-2A G-1B, G-2E/C, Cy3
LysoTracker Yellow534551G-1A, G-2A G-1B, G-2E/C, Cy3
Magdala Red (Phloxin B)524600All
Magnesium Orange550575G-1A, G-2A G-1B, G-2E/C, Cy3
Merocyanine555578G-1A, G-2A G-1B, G-2E/C, Cy3
MitoFluor Red 589588622G-2A, G-2B Cy3
MitoTracker Orange CMTMRos554576G-1A, G-2A G-1B, G-2E/C, Cy3
MitoTracker Red 580581644All
MitoTracker Red CMXRos579599All
NeuroTrace 530/615530615All
Neutral Red541640All
Nile Red552636All
Nuclear Fast Red289-530580All
Phycoerythrin B545576G-1A, G-2A G-1B, G-2E/C, Cy3
Phycoerythrin R480-565578G-1A, G-2A G-1B, G-2E/C, Cy3
PKH26551567G-1A, G-2A G-1B, G-2E/C, Cy3
Pontochrome Blue Black535-553605All
POPO-3, PO-PRO-3534570G-1A, G-2A G-1B, G-2E/C, Cy3
Propidium Iodide536617All
Pyronin Y555580All
Pyronin B540-590560-650All
RedoxSensor Red CC-1540600All
RH 237528782G-1A, G-2A G-1B, G-2B
RH 414532716G-1A, G-2A G-1B, G-2B
RH 421515704G-1A, G-2A G-1B, G-2B
RH 795530712G-1A, G-2A G-1B, G-2B
Rhod-2550571G-1A, G-2A G-1B, G-2E/C, Cy3
Rhodamine550573G-1A, G-2A G-1B, G-2E/C, Cy3
Rhodamine 6G525555G-1A, G-2A G-1B, G-2E/C, Cy3
Rhodamine B540625All
Rhodamine B 200523-557595All
Rhodamine BB540580All
Rhodamine B Extra550605All
Rhodamine BG540572G-1A, G-2A G-1B, G-2E/C, Cy3
Rhodamine Phalloidin542565G-1A, G-2A G-1B, G-2E/C, Cy3
Rhodamine WT530555G-1A, G-2A G-1B, G-2E/C, Cy3
Rose Bengal540550-560G-1A, G-2A G-1B, G-2E/C, Cy3
Sevron Brilliant Red 2B520595All
Sevron Brilliant Red B530590All
SNAFL-1540 (High pH) 508 (Low pH)623 (High pH) 543 (Low pH)G-2A
SNAFL-2543 (High pH) 514 (Low pH)630 (High pH) 546 (Low pH)G-2A
Spectrum Orange559588All
Sulphorhodamine B565586All
Sulphorhodamine B Can C520595All
SYPRO Red300/550630All
SYTO 14517549G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 15516546G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 25521556G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 80531545G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 81530544G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 82541560G-1A, G-2A G-1B, G-2E/C, Cy3
SYTO 83543559G-1A, G-2A G-1B, G-2E/C, Cy3
SYTOX Orange547570G-1A, G-2A G-1B, G-2E/C, Cy3
TAMRA (Carboxytetramethylrhodamine)542568G-2A, G-1B G-2E/C, Cy3
Tetrabromosulfonefluorescein524544G-1A, G-2A G-1B, G-2E/C, Cy3
Tetramethylrhodamine Ethyl549574G-1A, G-2A G-1B, G-2E/C, Cy3
Tetramethylrhodamine Methyl549573G-1A, G-2A G-1B, G-2E/C, Cy3
Texas Red596615G-1A, G-2B
Thiazine Red R510580All
TMR (Tetramethylrhodamine)550573G-1A, G-2A G-1B, G-2E/C, Cy3
TRITC (Tetramethylrhodamine Isothiocyanate)555580All
Xylene Orange546580All

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, 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. GriffinNathan S. ClaxtonMatthew J. Parry-HillThomas J. FellersKimberly M. VogtIan D. JohnsonShannon H. NeavesOmar AlvaradoLionel Parsons, Jr.Michael A. SoddersRichard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Green Excitation Filter Sets