The Nikon triple band fluorescence filter portfolio includes two carefully balanced combinations 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.

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.

Performance of the triple band 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 an adherent culture of albino Swiss mouse embryo cells that was fixed and stained with a combination of three fluorophores. The nuclei were stained with the nucleic acid specific fluorophore DAPI (violet excitation and blue emission), while the filamentous actin network was labeled with Alexa Fluor 488 (blue excitation and green emission) conjugated to phalloidin. Before fixation, the cells were incubated with MitoTracker Red CMXRos (green excitation and orange-red emission) targeted at the active mitochondria in living cells.

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. Figure 1(b) illustrates imaging of Alexa Fluor 488 in combination with DAPI and MitoTracker Red CMXRos, a fluorophore with absorption and emission spectral profiles that lie midway between Texas Red and TRITC. Note the color shift observed when images of the MitoTracker probe are compared between the two triple band excitation filter blocks (Figures 1(a) and 1(b)). 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.

• 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.

The wide range of probes developed for fluorescence microscopy includes many that are suitable for investigations using excitation wavelengths corresponding to the bandpass regions of the triple band excitation filters. The Nikon triple band filter combinations are designed for applications with specific fluorochrome combinations, although their use can be extended to a range of fluorophores with suitable spectral profiles. Many fluorochromes that are typically utilized in single-labeling investigations can be combined for multiple simultaneous detection provided that their excitation and emission wavelengths fall within the bandpass regions of one of the multiband excitation filter sets. Examples include the use of the DAPI-FITC-TRITC filter set with DAPI, Alexa Fluor 488, and Alexa Fluor 546, or with a combination of a Hoechst dye, Cy2, and Cy3. Similarly, the DAPI-FITC-Texas Red filter combination can be used effectively with DAPI, GFP (green fluorescent protein), and Alexa Fluor 594, among other probe combinations.

Although the two filter combinations described above serve in a large number of investigations requiring simultaneous detection of fluorescence from three fluorophores, additional specialized filter sets in the triple band excitation category are available from the aftermarket manufacturers. For example, a number of filter sets designed for the same excitation and emission wavelengths are available in different versions, which are optimized either for real-time visual observation, or with reduced excitation intensities for imaging with color film or CCD detectors. Other combinations are available in different versions, which have identical spectral characteristics, but include a mechanism for attenuating one emission signal to balance its intensity with emission from the other fluorochromes. Available DAPI-FITC-Propidium Iodide (PI) and DAPI-FITC-Texas Red filter combinations are essentially identical, except for design features to attenuate the typical brightness of the propidium iodide emission for better balance with the FITC signal.

As a further example, a filter set for DAPI-FITC-TRITC can be used with DAPI, FITC, and Cy3, although the latter combination is better visualized with a special set providing attenuation of the Cy3 emission. Strategies for balancing emission levels often rely on narrowing of emission bandpass regions corresponding to fluorochromes requiring signal attenuation, and are particularly important for simultaneous visual detection of certain fluorophore combinations, because of the much greater relative sensitivity of the eye to some wavelength ranges. Some filter sets and fluorochrome combinations do not allow direct real-time visualization, and require application of a detector such as an infrared-sensitive color CCD camera for signal recording. Filter sets designed to include detection of Cy5 fall into this category because the Cy5 emission is not visible to the human eye.