Triple Band Excitation

Included in the Nikon triple band excitation fluorescence filter portfolio are two carefully balanced combinations that each contain three bandpass emission regions capable of selectively isolating fluorescence emission through narrow bands of the blue, green, yellow, orange, and red spectral regions. This interactive tutorial explores how the variations in the excitation and emission filter spectral profiles, as well as those of the polychromatic mirrors, affect signal levels, spectral crossover, overall filter performance, and image contrast in combinations designed for triple excitation of multiply labeled fluorophores in the ultraviolet, blue, and green regions.

The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the ultraviolet-blue-green triple passband excitation filter combination (DAPI-FITC-TRITC; default) spectral profile displayed on the Filter Set Spectral Profiles graph. Fluorophore absorption and emission spectral cross sections (the overlap region with filter transmission passbands) can be individually or collectively viewed by selecting the appropriate check box (Absorption or Emission) listed under the Spectral Cross Sections heading. When one or more check boxes are activated, the combined filter transmission and reflection spectra are superimposed over the absorption and emission spectra of the selected fluorophore(s) utilized to label the specimen (fluorophore spectral profiles are not included for autofluorescent plant specimens). Fluorophore absorption spectra are presented in the tutorial using a brown fill, while the corresponding emission spectra are represented with a gray fill. Wavelength characteristics for the filter combination indicated by the Filter Set slider are displayed in the black rectangular box in the lower portion of the tutorial. These values are constantly updated as the slider is translated from left to right.

In order to operate the tutorial, use the Filter Set slider to transition between the two filter combinations available for triple band excitation. As the slider is translated from left to right, the spectral profiles of the excitation and barrier filters, as well as that of the polychromatic mirror, are modified to simulate changes to the spectral profiles. Note that the continuously changing spectral profiles do not imply that any filter combination is possible, nor are the individual filter sets variable (without physically changing filters) in regards to the spectral profiles. Alterations of the spectral profiles between selected filter sets are simply intended to help establish the relationship between the filter combinations used in each optical block.

Individual Filter Spectra (excitation, emission, and polychromatic mirror) can be added or removed from the Filter Set Spectral Profiles graph by selecting or deselecting the appropriate check boxes beneath the graph. In addition, the fluorophore absorption and emission spectra can be added or removed with a similar set of check boxes (Spectral Cross Sections). The specimen image changes simultaneously with the filter profiles to reflect variations in contrast and signal levels produced by the alterations to the filter combinations produced by translation of the slider. A new specimen can be selected at any time using the Choose A Specimen pull-down menu, and the fluorophores utilized to label the selected specimen are listed directly beneath the menu box. In all cases, the specimens are stained with two or more fluorescent probes to demonstrate the selective isolation of fluorescence with narrow and wide passband barrier (emission) filter sets.

The Nikon triple band fluorescence sets are primarily 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. In order to maintain three separate fluorescence signal bands, these specialized filter sets also incorporate dichromatic mirrors with multiple bandpass characteristics, having transmission and reflection regions that are complementary to the specific excitation and emission filters employed. Each Nikon triple band fluorescence set is optimized for use with DAPI and FITC (fluorescein isothiocyanate) in combination with either TRITC (tetramethylrhodamine isothiocyanate) or Texas Red probes. The relevant spectral regions corresponding to these fluorochrome combinations range from violet excitation and blue emission to green excitation and red emission.

The Nikon DAPI-FITC-TRITC triple band filter combination incorporates an excitation filter having one passband for violet-region excitation (385 to 400 nanometers) coupled to a bandpass emission (barrier) filter transmitting blue fluorescence (450 to 465 nanometers) in its first (shortest-wavelength) signal band. The second excitation-emission wavelength combination for this filter set allows blue excitation in a narrow band of 475 to 490 nanometers, and detection of the resulting green fluorescence in the 505 to 535 nanometer range. A third fluorescence signal is produced by green-region excitation (545 to 565 nanometers), which results in orange-red emission, detected in a wavelength range of 580 to 620 nanometers. The triple fluorescence bands of this set provide optimal simultaneous detection of DAPI, FITC, and TRITC, specifically targeting different cellular components; the DAPI - Alexa Fluor 488 - MitoTracker Red CMXRos fluorochrome combination is suitable for this filter set as well, due to the similarity of the Alexa Fluor 488 and MitoTracker Red CMXRos spectral profiles to those of FITC and TRITC.

In triple excitation filter blocks, as in other multiband sets, the design of the dichromatic mirror is fundamental to the separation of the multiple fluorescence signals, allowing their detection with minimal crossover (spectral bleedthrough) and noise. In contrast to the longpass beamsplitters often used in conventional filter combinations, multiband sets employ dichromatic mirrors with several bandpass regions that are precisely located with respect to the multiple excitation and emission bands. Typically, the first (lower) cut-on wavelength value for the mirror is positioned just a few nanometers above the short-wavelength excitation peak, beginning a transmission band that completely encompasses the corresponding emission peak, followed by a steep transmission cut-off, which allows reflection of the second excitation band. At a designated wavelength just higher than the second excitation band, the mirror makes another sharp transition to a transmission region that corresponds with the second emission bandpass. In triple-band filter sets, this transmission-reflection pattern is repeated once more for the third signal channel. The dichromatic mirror used in the DAPI-FITC-TRITC combination has multiple bandpass transmission regions that are appropriately located with respect to the various excitation and emission filter complements. The cut-on wavelengths occur at 435, 500, and 570 nanometers.

Application of thin-film interference technology in multiband filter set fabrication makes it possible to balance the fluorescence signal level of two (or more) fluorochromes in order to provide optimum imaging characteristics. In many filter combinations, the transmission profile of the short-wavelength excitation peak is reduced in size to balance the two or three emission signals, as well as to minimize photobleaching and specimen damage. This is particularly important in sets designed for use with one fluorophore excited in the violet or ultraviolet spectral region, due to the high excitation efficiency of shorter wavelengths. The significantly reduced intensity of the short wavelength excitation peak is illustrated in the transmission spectra of both Nikon triple band combinations. It may also be noted by examination of the filter set transmission spectra that certain components are common to more than one filter block. For example, the dual band FITC-Texas Red and the triple band DAPI-FITC-Texas Red sets utilize the same dichromatic mirror and emission filter, differing only in the excitation filter component, where a third short-wavelength passband is added for DAPI excitation in the triple set.

Simultaneous detection of DAPI, FITC, and Texas Red (or spectrally similar fluorophores) can be easily accomplished with the Nikon DAPI-FITC-Texas Red triple excitation band filter combination. The violet-excited, blue emission filter band, and the blue-excited, green emission band (corresponding to DAPI and FITC, respectively) have similar characteristics to those of the DAPI-FITC-TRITC set, although with slight modifications for better integration with the spectral characteristics of Texas Red, whose green-excited red emission occurs at higher wavelengths compared to TRITC. The excitation filter used in this set has one bandpass region of 395 to 410 nanometers (violet excitation) coupled to an emission (barrier) filter passband of 450 to 470 nanometers (blue emission), which is appropriate for DAPI and similar fluorophores. The second band of the filter set provides excitation in the blue wavelength range of 490 to 505 nanometers, and detection of corresponding green emission in the range between 515 and 545 nanometers (for FITC and similar fluorochromes). The signal channel optimized for Texas Red fluorescence has an excitation range of 560 to 580 nanometers (green excitation) coupled to an emission band in the red spectral region of 600 to 650 nanometers.

Although optimized for the triple combination of DAPI, FITC, and Texas Red fluorophores, probes with similar absorption and emission spectral characteristics can also be used with this filter set. The DAPI-FITC-TRITC block, which has a red emission passband shifted 15 nanometers to lower wavelengths when compared to the DAPI-FITC-Texas Red block, produces images that appear more orange in hue. The bandpass regions of the dichromatic mirror (beamsplitter) in the DAPI-FITC-Texas Red filter combination are strategically located to correspond with the excitation and emission filter windows at cut-on wavelengths of 445, 510, and 590 nanometers. Specifications for the dichromatic mirrors and filters for the two Nikon triple band filter combinations are listed in Table 1.

Table 1 - Nikon Triple Band Excitation Filter Combination Specifications

Filter Set
Filter (nm)
Mirror (nm)
Filter (nm)
Violet EX / Blue EM
Blue EX / Green EM
Green EX / Orange-Red EM
Texas Red
Violet EX/ Blue EM
Blue EX / Green EM
Green EX / Red EM
  • 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.

Contributing Authors

Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.

Matthew J. Parry-Hill, Thomas J. Fellers, Lionel Parsons, Jr., Kimberly M. Vogt, Ian D. Johnson, andMichael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Triple Band Excitation