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.

In order to maintain two separate fluorescence signal bands, these specialized filter sets also incorporate dichromatic mirrors with multiple passband characteristics (often termed polychroic or polychromatic mirrors or beamsplitters), having transmission and reflection regions that are complementary to the specific excitation and emission (barrier) filters employed. Each of the Nikon dual band fluorescence sets is optimized for use with fluorescein isothiocyanate (FITC) in combination with either 4',6-diamidino-2-phenylindole (DAPI), tetramethylrhodamine isothiocyanate (TRITC), or Texas Red dyes. The relevant spectral regions corresponding to these fluorochrome pairs range from violet excitation and blue emission to green excitation and red emission. Other fluorescent probes with similar spectral characteristics are also suitable, singly or in pairs, for use with the Nikon dual excitation fluorescence filter combinations.

Performance of the dual 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 Indian Muntjac deer skin fibroblast cells that were 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 cytoskeletal network was labeled with Alexa Fluor 488 (similar spectral characteristics to fluorescein; blue excitation and green emission) conjugated to phalloidin. Before fixation, the cells were incubated with MitoTracker Red CMXRos targeted at the actively respiring mitochondria. The absorption and emission spectral profiles of MitoTracker Red CMXRos (green excitation and orange-red emission) are centrally positioned in wavelength range between TRITC and Texas Red.

The Nikon DAPI-FITC dual band filter combination incorporates an excitation filter having one bandpass region for violet excitation (400 to 418 nanometers) coupled to a bandpass emission (barrier) filter transmitting blue fluorescence (450 to 465 nanometers) in one of its signal bands. The second excitation and emission wavelength combination for this filter set enables blue excitation in a narrow band of 478 to 495 nanometers, and detection of the resulting green fluorescence in the 510 to 555 nanometer range. The dual bands featured by this filter set provide optimal simultaneous detection of DAPI and FITC, when utilized in combination to specifically target different cellular components. The DAPI - Alexa Fluor 488 fluorochrome pair are suitable for this filter set as well, due to the close similarity of the Alexa Fluor probe and FITC spectral profiles (see Figure 1(a)) in regards to absorption and emission characteristics.

In dual excitation filter cubes, the design of the polychromatic mirror is fundamental to the separation of the two fluorescence signals, ideally allowing their unique detection with minimal crosstalk and noise. In contrast to the standard longpass beamsplitter filters used in conventional combinations, multi-band filter sets employ modified dichromatic mirrors with multiple bandpass regions that are precisely located with respect to the multiple excitation and emission bands. The first (lower) cut-on wavelength value for the mirror is positioned a few nanometers higher than the short-wavelength excitation peak, and begins a transmission band that completely encompasses the corresponding emission peak, followed by an abrupt transmission cut-off. This design enables reflection of wavelengths in the second excitation band while simultaneously passing fluorescence emission created by the first band. At a wavelength just higher than the second excitation band, the mirror makes another sharp transition to a bandpass transmission region that corresponds to the second emission passband. The polychromatic mirror used in the DAPI-FITC filter combination has two bandpass transmission regions that are appropriately located with respect to each excitation-barrier filter complement, one having a cut-on wavelength of 435 nanometers, and another with a cut-on wavelength of 505 nanometers.

Application of thin-film interference technology in multi-band filter set fabrication techniques makes it possible to balance the fluorescence signal level of two (or more) fluorochromes and provide optimum imaging conditions. In many filter combinations, the transmission band profile of the shorter (usually ultraviolet or violet) wavelength excitation peak is reduced in order to balance the emission signal levels, 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 short wavelength radiation. The reduced intensity of the short-wavelength excitation peak is a notable feature in the transmission spectral profiles of filter combinations such as the Nikon DAPI-FITC set.

For the simultaneous detection of FITC (or a similar fluorophore) and a probe excited at longer wavelengths, such as TRITC, the FITC-TRITC dual band filter combination provides blue-region excitation with green emission detection, and green excitation with accompanying orange-red emission detection. In this filter set, the bandpass regions for the blue excitation-green emission fluorochrome (FITC, for example) are modified slightly from those employed in the DAPI-FITC set in order to minimize crosstalk with the green-absorbing fluorophore. The excitation filter utilized in this set has one bandpass region of 475 to 490 nanometers (blue excitation) coupled to an emission (barrier) filter passband of 503 to 530 nanometers (green emission), which is appropriate for FITC and similar fluorophores.

The second band of the FITC-TRITC filter combination enables excitation in the green wavelength range of 540 to 565 nanometers, and detection of corresponding orange-red emission in the region between 580 and 620 nanometers. Although optimized for the popular combination of FITC and TRITC fluorophores, probes with similar absorption and emission spectral characteristics can also be used. Figure 1(b) illustrates fluorescence of Alexa Fluor 488 in combination with MitoTracker Red CMXRos, a fluorophore with green light absorption and orange-red emission, similar to TRITC. The bandpass regions of the polychromatic mirror (beamsplitter) are located in correspondence to the excitation and emission filter windows at cut-on wavelengths of 500 and 575 nanometers.

Simultaneous detection of FITC (or a similar probe) and a fluorochrome excited at wavelengths in the green and yellow-green spectral regions, with emission extending into the red, is afforded by the FITC-Texas Red filter set (as illustrated in Figure 1(c)). Excitation bands for this set (blue and green spectral regions) range from 490 to 505 nanometers and from 560 to 580 nanometers. The corresponding dual green and red emission bands are 515 to 545 nanometers and 600 to 650 nanometers, respectively. The complementary polychromatic mirror cut-on wavelengths for the two signal transmission bands are located at 510 and 585 nanometers. Specifications for the polychromatic mirrors and filters for the three Nikon dual band filter combinations are listed in Table 1.

• 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.

The wide range of fluorophores currently available for fluorescence microscopy includes many that are suitable for investigations using excitation wavelengths corresponding to the bandpass regions of dual band excitation filters. The three Nikon dual band filter combinations are designed for applications with specific popular fluorochrome pairs, although their use can be extended to a much larger range of probes with suitable spectral profiles. Many fluorochromes that are typically utilized in single-labeling investigations can be combined for dual simultaneous detection, provided that their excitation and emission wavelengths fall within or very close to the bandpass regions featured by one or more of the dual excitation sets. Some typical examples include the use of the DAPI-FITC filter set with blue fluorescent protein (BFP) in combination with green fluorescent protein (GFP), and use of the FITC-TRITC set for investigating Cy2 or GFP paired with Cy3 or Alexa Fluor 546. Similarly the FITC-Texas Red filter combination can be used effectively with GFP and Alexa Fluor 594.

Although the three filter combinations described above serve in a large number of investigations requiring simultaneous detection of fluorescence from two fluorophores, additional specialized filter sets in the dual band excitation category are available from the aftermarket manufacturers. One useful combination is optimized for DAPI-TRITC, and employs a narrow bandpass region covering the 546-nanometer line of a mercury arc-discharge lamp for TRITC excitation. Other sets are available for the DAPI-Texas Red fluorochrome pair, as well as pairs including the cyanine dyes (Cy2, Cy3, and Cy5), fluorescent proteins, and other traditional probes such as Lucifer Yellow, DiI, and rhodamine. Filter combinations designed to image propidium iodide with another fluorophore typically are designed to attenuate the relatively high brightness level of the propidium iodide signal for better balance. For example, FITC-Texas Red and FITC-propidium iodide sets are essentially identical, but follow a similar attenuation strategy to balance the typical brightness of the propidium iodide signal with the FITC emission.

A number of filter sets designed for the same excitation and emission wavelengths are available in different versions, which are optimized to balance the emission from specific fluorochrome pairs, typically either by narrowing emission bandpass regions for fluorochromes requiring signal attenuation, or by incorporating additional absorbing components in the filter construction. For example, a filter combination for DAPI-TRITC can be used with DAPI-Cy3, but the latter pair is better visualized with a special set providing attenuation of the Cy3 fluorophore emission. 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 optimized for Cy3-Cy5 and FITC-Cy5 fall into this category because the Cy5 emission (above 650 nanometers) is not visible to the human eye.