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.

Two of the HYQ fluorescence filter sets transmit green excitation light (TRITC HYQ and Cy3 HYQ), while one provides yellow-region excitation (Texas Red HYQ) and one is designed for orange to red excitation (Cy5 HYQ). HYQ filter combinations are typically characterized by wider-bandpass excitation and emission filters, compared to many standard sets, in order to provide increased energy transmission and enhanced fluorescence signal level. As a group, the HYQ filter combinations provide spectral coverage that includes excitation wavelengths ranging from 530 nanometers (green) to 650 nanometers (red), and emission detection that ranges from 573 nanometers (yellow) to 738 nanometers (infrared). Excitation filter passbands are 30, 55, or 60 nanometers in width, and are combined with emission filter passbands of 60 or 75 nanometers.

Performance of the individual HYQ filter sets can be judged by examining selected images of specimens labeled with fluorophores having spectral profiles that match passbands and cut-on wavelengths of the filter combinations, as illustrated in Figure 1. The marsupial (rat kangaroo; PtK2 cell line) cytokeratin intermediate filament network presented in Figure 1(a) was immunofluorescently stained with anti-cytokeratin antibodies conjugated to Cy3, while the mitochondria (Indian Muntjac deerskin fibroblasts) in Figure 1(b) were labeled with MitoTracker Red CMXRos. Likewise, fluorescence from the actin filament network (Indian Muntjac) illustrated in Figure 1(c) originates from Alexa Fluor 568 conjugated to phalloidin. All of these probes are useful in combination with Nikon HYQ filter sets. In the deep red fluorescence emission region, Alexa Fluor 647 performs well (HeLa cell nuclei in Figure 1(d)) in combination with the Cy5 HYQ filter combination.

The high-performance design features of the HYQ filter sets include implementation of wider filter passbands while maintaining high signal-to-noise ratio, with minimal crossover (spectral bleedthrough) between signal bands. These objectives are accomplished through the use of filter fabrication technology that incorporates a greater number of interference cavities in both the excitation and emission filters to produce very steep cut-on and cut-off transitions in the filter transmission profiles. These sharp boundaries between reflection and transmission wavelength regions allow precise positioning of passbands in close proximity to each other with little performance degradation from signal crossover. The result is to provide the highest excitation efficiency and maximum fluorescence detection, while maintaining separation of signal among several fluorophores when they are combined in multiple-labeling techniques.

The two HYQ filter sets designed for green-wavelength excitation, the TRITC HYQ and Cy3 HYQ, employ identical bandpass excitation filters (530 to 560 nanometers) and dichromatic mirrors (570-nanometer cut-on wavelength). Both detect fluorescence in the orange to red spectral regions, but differ in that the wider passband of the Cy3 HYQ emission filter extends further toward lower wavelengths, into the yellow region. The 75-nanometer emission passband of the Cy3 HYQ combination ranges from 573 to 648 nanometers; the TRITC HYQ set restricts emission to a more narrow 60-nanometer range of 590 to 650 nanometers.

For excitation of fluorophores at longer wavelengths than those provided by the two green-excitation HYQ combinations, the Texas Red HYQ filter set incorporates an excitation filter with a passband that extends from the green through the yellow region (532 to 587 nanometers) into the lower orange wavelengths. The combination of a 595-nanometer dichromatic mirror cut-on wavelength and an emission filter passband extending from the orange into the far-red (608 to 683 nanometers) accommodates a wide range of fluorochromes, in addition to Texas Red. This filter combination is similar to the Y-2E/C yellow excitation set, with wider excitation and emission bandpass windows, allowing it to transmit more energy and produce comparatively brighter images. The wide transmission passbands of 55 and 75 nanometers for the excitation and emission filters, respectively, and the sharp cut-on and cut-off transitions result in the increased performance that characterizes filter sets with the HYQ designation.

The Cy5 HYQ filter set is the longest-wavelength HYQ combination and provides a wide 60-nanometer excitation passband spanning the entire orange spectral region and extending into the red (590 to 650 nanometers). Fluorescence emission detection is possible within a 75-nanometer band of wavelengths from the deep red into the invisible near-infrared region, and in many applications requires use of a CCD or other infrared-sensitive detector for full utilization. The Cy5 HYQ wide passbands with sharp transitions for both the excitation and emission filters, providing increased energy for signal generation and detection, are typical of filter combinations in the Nikon HYQ series. The emission passband of the Cy5 HYQ set is centered on 700 nanometers, and extends from 663 to 738 nanometers. The lower (cut-on) wavelength is positioned only 3 nanometers above the 660-nanometer cut-on of the longpass dichromatic mirror.

Nikon HYQ Filter Combination Specifications

Filter Set Description Excitation Filter (nm) Dichromatic Mirror (nm) Barrier Filter (nm) Remarks TRITC HYQ 545/30 (530-560) 570 (LP) 620/60 (590-650) Medium Green Excitation Band Bandpass Barrier Filter Cy3 HYQ 545/30 (530-560) 570 (LP) 610/75 (573-648) Medium Green Excitation Band Bandpass Barrier Filter Texas Red HYQ 560/55 (532-587) 595 (LP) 645/75 (608-683) Wide Yellow Excitation Band Bandpass Barrier Filter Cy5 HYQ 620/60 (590-650) 660 (LP) 700/75 (663-738) Wide Red Excitation Band Bandpass Barrier Filter

Table 1

• 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.

The four Nikon HYQ filter combinations employ excitation wavelengths ranging from 530 nanometers (green) to 650 nanometers (red). A wide array of fluorophores has been developed for investigations using excitation wavelengths within the green, yellow, and red spectral regions for which the various HYQ sets are designed. Each filter group page for the relevant excitation wavelength category (green excitation filter sets for the TRITC HYQ and Cy3 HYQ combinations, yellow excitation sets for the Texas Red HYQ, and red excitation sets for the Cy5 HYQ) includes a table that catalogues many of the popular dyes and fluorescent probes, and the filter combinations with which they can be visualized. The localized environment significantly influences fluorophore absorption and emission spectra maximum (peak) wavelengths, and consequently, the values presented in the tables may vary with experimental conditions. The lists are 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 the tables 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, a number of the fluorescent probes listed in each table are also suitable for use with filter combinations in other excitation wavelength regions.