Fluorescence Filter Combinations

Epi-fluorescence interference and absorption filter combinations are housed in a filter cube (or optical block) and include an excitation filter, dichromatic beamsplitter (often referred to as a mirror), and a barrier (or emission) filter, as illustrated in Figure 1(a). Use this guide in selecting the appropriate filter set to match the spectral excitation and emission characteristics of chromophores used in widefield fluorescence microscopy investigations. The spectral profiles of a typical high-performance bandpass emission blue excitation filter combination are presented as an example in Figure 1(b). Nikon fluorescence filter combinations are provided in narrow, medium, and wide passband excitation versions with corresponding emission filters available with either bandpass or longpass spectral characteristics.

Ultraviolet Excitation - Included in the Nikon ultraviolet excitation fluorescence filter portfolio are four carefully balanced combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide region of the blue, green, and red visible wavelengths. These filter combinations cover an excitation wavelength range between 330 and 380 nanometers with passband width profiles of 10, 40, and 50 nanometers. Three of the combinations employ the same dichromatic mirror, while the fourth set has a mirror with a lower wavelength cut-on to coincide with its narrower excitation band. The ultraviolet filter sets contain either longpass or single bandpass emission filters.

Violet Excitation - The Nikon violet excitation fluorescence filter series includes three combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide region of the blue, green, and red wavelengths. These combinations cover an excitation wavelength range between 379-420 nanometers with bandpass width profiles of 10, 22, and 40 nanometers. Two of the combinations employ the same dichromatic mirror, while the third set has a mirror with a lower wavelength cut-on to coincide with its shorter-wavelength excitation band.

Blue-Violet Excitation - 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 complementary filter sets 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.

Blue Excitation - Nikon blue excitation fluorescence filter complements consist of six 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 sets 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.

Green Excitation - The Nikon green excitation fluorescence filter combination series includes six blocks 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.

Yellow Excitation - Yellow excitation fluorescence filter combinations from Nikon include two balanced sets that incorporate single bandpass (barrier) filters capable of selectively isolating fluorescence emission within the orange to red spectral region. These complementary filter combinations encompass an excitation wavelength range of 532 to 587 nanometers with passband widths of 40 and 55 nanometers. Both combinations employ the same longpass dichromatic mirror (595-nanometer cut-on). The two Nikon yellow excitation filter sets incorporate bandpass barrier filters having bandwidths of 60 and 75 nanometers..

Red Excitation - The Nikon red excitation fluorescence filter combination category comprises a single block that incorporates a bandpass emission (barrier) filter capable of selectively isolating fluorescence emission occurring in the deep red to near-infrared spectral regions. The barrier filter passband is centered at 700 nanometers, with a bandwidth of 75 nanometers (663 to 738 nanometers). A wide 60-nanometer excitation band encompassing orange and red wavelengths covers the spectral range of 590 to 650 nanometers. The Cy5 HYQ combination employs a longpass dichromatic mirror having a cut-on wavelength of 660 nanometers, which is 10 nanometers higher than the excitation bandpass cut-off.

Yellow Fluorescent Protein (YFP) Excitation - The Nikon yellow fluorescent protein fluorescence filter category comprises a single high-performance balanced combination, which effectively extends the fluorescent protein detection capabilities afforded by the three green fluorescent protein (GFP) filter sets to the longer wavelength enhanced yellow chromatic variants of GFP (YFP and EYFP). The YFP HYQ filter combination employs relatively narrow passband excitation and emission (barrier) filters, which are designed to correspond with the specific spectral characteristics of enhanced yellow fluorescent protein, and enable the fluorescence emission from YFP derivatives to be evaluated separately from that of other fluorescent proteins.

Dual Band Excitation - The Nikon dual band fluorescence filter portfolio includes three carefully balanced combinations that contain dual bandpass excitation and emission (barrier) filters, which are incorporated into a single element capable of selectively isolating fluorescence emission from two fluorophores simultaneously. Each of the filter sets is designed for optimal performance with a specific fluorochrome pair, although they are equally effective with alternate pairs of fluorescent probes that have similar absorption and emission spectral profiles. Utilizing precise wavelength band selection, with steep bandpass transitions between reflection and transmission regions, the multiple excitation and emission signals are separated with minimal crossover interference.

Triple Band Excitation - Triple excitation band fluorescence filters offered by Nikon include two balanced blocks that contain triple bandpass excitation and emission (barrier) filters capable of selectively isolating fluorescence emission from three fluorophores simultaneously. Each of the filter sets is designed for optimal performance with a specific three-fluorochrome suite, although they are equally effective with alternate probe combinations that have similar absorption and emission spectral profiles. Utilizing precise wavelength band selection, with steep bandpass transitions between reflection and transmission regions, the multiple excitation and emission signals are separated with minimal interference.The triple cubes.

HYQ Cubes - The Nikon HYQ fluorescence filter combination group encompasses four carefully balanced high performance sets, all of which incorporate bandpass emission (barrier) filters for selective isolation of fluorescence within a limited wavelength range. The nomenclature for each HYQ filter set reflects the name of the fluorochrome for which it is primarily designed, but within its respective excitation range, each combination can be applied to a variety of appropriate fluorochromes.

Nikon Fluorescence Filter Set Master List

The filter nomenclature employed by Nikon derives from a mixture of terms dating back to the early 1990s. At that time, all of the Nikon complementary filter combinations were produced using the hard coat sputter technique, but many of the currently available filters take advantage of newer softer coating methods. Although soft coats are more susceptible to humidity and heat degradation, and must be handled more carefully than hard coat filters, they exhibit higher blocking value optical densities and provide greater ease of fine-tuning specific wavelength bands. Understanding the Nikon filter combination code nomenclature provides a mechanism to quickly determine whether a particular set will perform adequately for a specific fluorophore.

The first letter in the Nikon proprietary alphanumeric filter designation code indicates the wavelength excitation spectral region (for example, UV, V, B, and G, which are simple abbreviations for ultraviolet, violet, blue, and green, respectively). The number following the excitation code relates to the excitation filter passband width: 1 for narrow band excitation, 2 for medium and wide band excitation, and 3 for very wide band excitation. Finally, one or more letters following the excitation bandpass size number identifies the barrier filter characteristics. The code letter A indicates a standard longpass barrier filter with the lowest cut-on wavelength, while B designates a higher cut-on wavelength value for a longpass emission filter. Bandpass emission filters are identified with the letter E (referring to the term "enhanced") to indicate their superior performance with regard to eliminating crossover. The E/C filters are soft coat interference combinations designed for best performance with specific probes, such as DAPI, FITC, TRITC, and Texas Red.

Ultraviolet Excitation

UV-2E/C - The UV-2E/C filter combination is designed as a sharp cutoff filter block for ultraviolet fluorescence. Filters are the soft-coated type intended to generate a high signal/noise ratio. The narrow bandpass barrier filter utilized in this combination is designed to dramatically reduce or completely eliminate green and red visible wavelengths.

UV-1A - The UV-1A combination is designed as a filter block for ultraviolet fluorescence with a narrow excitation bandpass (only the i-line of the mercury spectrum is utilized) and a narrow dichromatic mirror passband, which minimizes autofluorescence and photobleaching.

UV-2A - The UV-2A filter combination is designed as a standard filter block for ultraviolet fluorescence and is the brightest ultraviolet filter set in the Nikon palette.

UV-2B - The UV-2B combination is designed as a general purpose filter block for ultraviolet fluorescence that provides a darker background and better contrast than the UV-2A filter combination.

Violet Excitation

V-1A - The V-1A filter combination is designed as a filter block for violet fluorescence excitation with a narrow bandpass region to minimize autofluorescence. The longpass emission filter allows detection of a wide range of fluorochrome wavelengths.

V-2A - The V-2A combination is designed as a standard violet fluorescence filter block having the greatest degree of versatility. With a medium excitation bandpass and longpass emission filter, the images produced are the brightest of the Nikon violet filter group.

BFP (Blue Fluorescent Protein) - The BFP filter block combines a narrow excitation band with a bandpass emission filter to limit autofluorescence as well as to exclude detection of emission from red and green fluorophores. This filter combination is excellent for imaging blue fluorescent protein derivatives.

Blue-Violet Excitation

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.

Blue Excitation

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 wide 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.

Green Excitation

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.

Yellow Excitation

Y-2E/C - The Y-2E/C filter combination for yellow wavelength excitation is designed as a specialty filter set for use with Texas Red and Cy3.5, although with an excitation bandwidth spanning most of the yellow wavelengths (overlapping a portion of the green region), the set can be utilized with a range of other fluorochromes. The bandpass barrier filter transmits signal from orange and orange-red emitting fluorophores, while eliminating most red and near-infrared emission.

Texas Red HYQ - The Texas Red HYQ filter combination has a similar component profile to the Y-2E/C set, but with wider excitation and emission bandpass windows, which allow it to transmit more energy and produce comparatively brighter images. Resulting images also reflect additional red fluorescence signal due to the extension of the emission filter passband to longer wavelengths in the red spectral region.

Red Excitation

Cy5 HYQ - The Cy5 HYQ filter combination for red-wavelength excitation is designed to provide high-energy excitation of the fluorophore Cy5, although the wide excitation range (yellow-orange to red) allows the set to be utilized with a variety of fluorochromes. Sharp filter transitions between reflection and transmission regions enable maintaining signal separation in combination with the high brightness level resulting from the use of wide passbands. The bandpass barrier filter transmits signal from the far-red into the near-infrared, a wavelength range that is most efficiently detected using electronic detectors, such as photomultipliers and CCD cameras without an infrared filter.

Dual Band Excitation

DAPI-FITC - The DAPI-FITC filter combination is designed to be utilized for simultaneously detecting emission from the fluorochromes DAPI and FITC, or other matched pairs of fluorescent probes that have similar spectral characteristics. Dual narrow excitation and emission bands correspond to specific regions of violet excitation with corresponding blue emission, and blue excitation coupled to green emission.

FITC-TRITC - The FITC-TRITC filter combination is designed for simultaneous detection of emission from the fluorochromes FITC and TRITC, or probe pairs that are spectrally similar. Bandpass excitation and emission filters allow two signal channels, one corresponding to specific narrow regions of blue excitation and green emission, and the other to green excitation and orange-red emission.

FITC-Texas Red - The FITC-Texas Red filter combination is designed with slightly different bandpass regions for blue excitation and green emission when compared to the FITC-TRITC set, allowing more efficient excitation of FITC. These shifts conform to the somewhat higher wavelength ranges for the Texas Red (green excitation; red emission) bands. The multiple bandpass regions provide optimal detection, with minimal crosstalk, for simultaneous visualization of FITC and Texas Red or of spectrally similar fluorophore pairs.

Triple Band Excitation

DAPI-FITC-TRITC - The DAPI-FITC-TRITC filter combination is designed to be utilized for simultaneously detecting emission from the fluorochromes DAPI, FITC, and TRITC or other probe combinations that have similar spectral characteristics. Three narrow excitation and emission bands correspond to specific regions of violet excitation with corresponding blue emission, blue excitation coupled to green emission, and green excitation with orange-red emission.

DAPI-FITC-Texas Red - The DAPI-FITC-Texas Red filter combination is designed with slightly different passband regions for violet excitation - blue emission, and for blue excitation - green emission, when compared to the standard DAPI-FITC-TRITC set. This design is intended to better conform with the higher excitation and emission wavelengths necessary for Texas Red, relative to the values for TRITC. The multiple bandpass regions provide optimal detection, with minimal crossover and noise, for simultaneous visualization of DAPI, FITC, and Texas Red, or of spectrally similar fluorophore combinations.

HYQ Filter Combinations

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 a higher signal level for these fluorophores, and allow improved detection of DsRed fluorescent protein variants and the far-red fluorescing HcRed proteins.

Cy3 HYQ - The Cy3 HYQ green excitation filter combination has a similar component profile to the G-2E/C set, although it generates increased excitation and emission energy through expanded passband windows. In addition, the emission passband extends to lower wavelengths (into the yellow), resulting in images that are both brighter and shifted toward yellowish-orange hues. This is the recommended filter combination for detecting the cyanine fluorescent probe Cy3, especially in multiple-labeling techniques, in which it reduces interference from fluorophores emitting in the far-red and near-infrared.

Texas Red HYQ - The Texas Red HYQ filter combination is optimized for use with the fluorescent probes Texas Red and Cy3.5, having wider excitation and emission passbands than the similar Y-2E/C set. Because of the resulting higher signal level, and the extension of the emission passband to longer wavelengths, compared to the Y-2E/C set, this combination produces images that are brighter and more reddish in hue. The Texas Red HYQ combination provides improved performance for probes emitting at wavelengths beyond the upper limits of the Y-2E/C emission band, while limiting interference from fluorophores emitting in the deep red and near-infrared.

Cy5 HYQ - The Cy5 HYQ filter combination is the only Nikon set designed for high-energy red-wavelength excitation, and although optimized for use with the fluorophore Cy5, the wide yellow-orange to red excitation range is suitable for applications employing a variety of fluorochromes. Fluorescence generated at far-red to near-infrared wavelengths, corresponding to the emission filter passband, is largely invisible to the human eye, and the most efficient detection requires use of photomultiplier tubes or CCD cameras. Wide filter bandpass regions, combined with steep cut-on and cut-off transitions, maintains maximum signal separation and signal-to-noise ratio, characteristic of the HYQ filter series.

Fluorescent Protein Filter Combinations

Endow GFP Longpass Emission (FGP(R)-LP) - The Endow GFP longpass emission filter combination is a longpass version of the Endow GFP bandpass filter set and was designed by Dr. Sharyn Endow of Duke University. This filter set is useful for fluorescence studies utilizing wtGFP and EGFP. Along with other filters in this series, the set is designed to increase secondary fluorescence intensity (brightness) without a consequential increase in noise levels. Steeper passbands are created by increasing the number of interference cavities in both the excitation and emission filters, allowing close proximity between filter spectra. The resulting wider and steeper filter spectra allow the set to provide more excitation and capture more fluorescence intensity.

Endow GFP Bandpass Emission (FGP(R)-BP) - The Endow GFP Bandpass Emission (FGP(R)-BP) filter combination is a bandpass version of the Endow GFP longpass filter set and was designed by Dr. Sharyn Endow of Duke University. This filter set is useful for fluorescence studies utilizing wtGFP and EGFP, and is one of the most widely distributed GFP filter sets.

Piston GFP Bandpass Emission - The Piston GFP Bandpass Emission filter combination is a bandpass filter set designed by Dr. David Piston of Vanderbilt University. The emission filter has a narrow bandpass that is useful to quantitate EGFP (Endow Green Fluorescence Protein) fluorescence while avoiding autofluorescence at wavelengths exceeding 540 nanometers.

Yellow Fluorescent Protein Bandpass Emission (YFP HYQ) - The Nikon yellow fluorescent protein YFP HYQ filter set is designed to transmit excitation illumination over a narrow (20-nanometer) band of wavelengths in the blue-green spectral region, coupled with green to yellow emission detection within a 30-nanometer bandpass region. The block is optimized for imaging YFP, while maintaining discrimination of cyan fluorescent protein in dual staining techniques.


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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.