The Nikon yellow excitation fluorescence filter combination group includes two carefully 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.
Performance of the yellow 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 a monolayer culture of bovine pulmonary artery endothelial cells labeled with MitoTracker Red CMXRos for the mitochondrial network. The absorption maximum wavelength of MitoTracker Red is 579, while the fluorescence emission curve has a peak at 599 nanometers, in the orange-red portion of the visible spectral region. In addition, the specimen was labeled with DODIPY FL conjugated to phallacidin to target F-actin in the cytoskeleton and DAPI for DNA in the nucleus. Neither of the latter two probes is efficiently excited by light in the yellow spectral range. Note the very slight variation in emission color and intensity exhibited by this specimen using the yellow excitation filter combinations.
Both of the Nikon yellow excitation filter sets incorporate bandpass emission (barrier) filters intended to reduce or eliminate interference from fluorophores emitting in the far red or near-infrared spectral regions, especially for specimens labeled with multiple probes. The two filter combinations cover the same general spectral bands, and are interchangeable in most applications. The Y-2E/C set combines a 40-nanometer excitation band (540-580 nanometers) with an emission bandpass of 60 nanometers (600-660 nanometers), which blocks much of the deep red to near-infrared spectral region.
The Texas Red HYQ combination (a member of the Nikon high-performance HYQ filter group) has similar optical specifications, although with wider excitation and emission bandpass regions (55 and 75 nanometers, respectively). The excitation passband of this set has a wavelength range from 532 to 587 nanometers, and is combined with a 75-nanometer emission passband (608 to 683 nanometers). The increased passband widths transmit greater excitation and emission energy, leading to brighter images, as well as extension of emission detection further into the deep red region. Each complementary filter combination utilizes the same dichromatic mirror, having a cut-on wavelength of 595 nanometers. Both of the sets provide performance specifically optimized for use with Texas Red, Alexa Fluor, and Cy3.5 fluorochromes, although they are suitable for use with a range of fluorophores excited by yellow wavelengths. Because of the wider bandpass windows, images produced using the Texas Red HYQ set reflect the detection of more red signal (as illustrated in Figure 1(b)), and are somewhat brighter when compared to those acquired with the Y-2E/C combination (Figure 1(a)).
Table 1 - Nikon Yellow Excitation Filter Combination Specifications
|Filter Set Description||Excitation Filter (nm)||Dichromatic Mirror (nm)||Barrier Filter (nm)||Remarks|
|Y-2E/C||560/40 (540-580)||595 (LP)||630/60 (600-660)||Medium Excitation Band Bandpass Barrier Filter|
|Texas Red HYQ||560/55 (532-587)||595 (LP)||645/75 (608-683)||Wide Excitation Band Bandpass Barrier Filter|
- 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.
A wide array of fluorophores has been developed for investigations using excitation wavelengths spanning the yellow (540-590 nanometers) wavelength region. Catalogued in Table 2 are many of the popular dyes and fluorescent probes that can be visualized with the Nikon yellow excitation filter combinations. The localized environment significantly influences fluorophore absorption and emission spectra maximum (peak) wavelengths, so the values presented in Table 2 may vary with experimental conditions. This list is 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 Table 2 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, several of the fluorescent probes listed in Table 2 are also suitable for use with filter combinations having other excitation wavelength passbands, particularly in the green and red spectral regions.
Table 2 - Fluorochromes with Yellow Excitation Spectral Profiles
|Fluorochrome||Excitation Wavelength (Nanometers)||Emission Wavelength (Nanometers)||Recommended Filter Set(s)|
|AAD (Aminoactinomycin D)||546||647||All|
|Alexa Fluor 555||553||568||Y-2E/C|
|Alexa Fluor 568||578||603||All|
|Alexa Fluor 594||590||617||All|
|DiBAC4(5) (Dibutylbarbituric Acid)||590||616||All|
|LDS 751-RNA (Laser Dye Styryl 751)||590||607||All|
|LysoTracker Red DND-99||577||592||All|
|MitoFluor Red 589||588||622||All|
|MitoTracker Red 580||581||644||All|
|MitoTracker Red 589||588||622||All|
|MitoTracker Red CMXRos||579||599||All|
|mRFP-1 (Monomeric Red FP)||584||607||All|
|Naphthofluorescein||602||672||Texas Red HYQ|
|NeuroTrace 530/615||530||615||Texas Red HYQ|
|Pontochrome Blue Black||535-553||605||All|
|RedoxSensor Red CC-1||540||600||All|
|Rhodamine B 200||523-557||595||All|
|Rhodamine B Extra||550||605||All|
|Sevron Brilliant Red 2B||520||595||All|
|Sevron Brilliant Red B||530||590||All|
|TRITC (TMR Isothiocyanate)||555||580||Y-2E/C|
|XRITC (X-Rhodamine Isothiocyanate)||580||605||All|
Although the two filter combinations described in this section adequately serve for a majority of the investigations with yellow excitation wavelengths, several additional specialized filter sets are available from the aftermarket manufacturers. Some of these combinations incorporate yellow-band excitation with non-standard dichromatic mirrors and barrier filters, which may be chosen to match particular detector characteristics. In other variations, a narrow excitation bandpass may be designed to selectively isolate specific emission lines for sources such as mercury arc-discharge lamps, which occur in the appropriate spectral region. If no strong excitation line for the fluorochrome of interest exists, a wider excitation filter passband may be required in order to collect sufficient signal, and a similar approach is necessary when broadband low-intensity illumination sources, such as tungsten-halogen lamps, must be used.
Other specialized filter sets intended for ratiometric analysis of probes that exhibit environment-sensitive fluorescence emission include two emission filters with distinct bandpass regions. Sets for ion-sensitive probes, such as SNARF, can be configured in several variations, each having a single excitation filter and dual emission filters, while utilizing different dichromatic mirror complements. This fluorochrome can be excited at 515 or 534 nanometers in the green spectral region, or at 546 nanometers (yellow), and applicable filter sets are available with different excitation filters. As configured for simultaneous ratiometric imaging of both emission wavelengths with an emission-splitting system, two dichromatic mirrors are included in the filter set. Only one dichromatic mirror is utilized for sequential ratiometric imaging with an emission filter wheel. In addition, filter combinations are tailored for specific fluorophores whose excitation profile is ion dependent.
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