The Nikon yellow fluorescent protein fluorescence filter category comprises a single high-performance balanced combination, which effectively extends the fluorescent protein detection capabilities afforded by the three green fluorescent protein (GFP) filter sets to the longer wavelength enhanced yellow chromatic variants of GFP (YFP and EYFP). The YFP HYQ filter combination employs relatively narrow passband excitation and emission (barrier) filters, which are designed to correspond with the specific spectral characteristics of enhanced yellow fluorescent protein, and enable the fluorescence emission from YFP derivatives to be evaluated separately from that of other fluorescent proteins. In the Nikon YFP HYQ block, excitation is limited to a 20-nanometer band of wavelengths in the blue-green spectral region (490 to 510 nanometers), and is coupled to an emission filter transmitting yellowish-green fluorescence in the range of 520 to 550 nanometers. The longpass dichromatic mirror has a cut-on wavelength of 515 nanometers.
Performance of the yellow fluorescent protein filter set can be judged by comparing images from a series of cell cultures transfected with EYFP chimeric vectors targeting specific subcellular locations, as illustrated in Figure 1. The specimens are three individual adherent cultures of albino Swiss mouse embryo fibroblasts (3T3 line), which were transfected at 60 to 80-percent confluence with EYFP vectors targeting either the mitochondria (Figure 1(a)), nucleus (Figure 1(b)), or filamentous actin in the cytoskeletal network (Figure 1(c)). In each case, expression of the fluorescent protein domain is restricted to subcellular locations dictated by the targeting sequence. Mitochondria were localized using a chimera of EYFP fused to the mitochondrial targeting nucleotide sequence from subunit VIII of human cytochrome C oxidase. Likewise, the nucleus and actin were localized with vectors containing three tandem repeats of the nuclear localization signal (NLS) from simian virus 40 (SV40) large T-antigen and a functional copy of human cytoplasmic beta-actin, respectively. In addition to applications involving the examination of EYFP and YFP transfectants, the YFP HYQ filter combination is useful with a wide variety of synthetic fluorophores (see Table 2) that are excited by light in the blue-green portion of the visible spectrum.
The Nikon YFP HYQ filter set incorporates a narrow-band excitation filter, centered in the blue-green spectral region, in conjunction with a bandpass emission filter, which together are capable of detecting fluorescence from yellow fluorescent protein with minimal interference from other fluorescent proteins or from autofluorescence occurring at longer wavelengths. The growing number of applications that utilize green fluorescent protein derivatives is due in large part to the development of enhanced wild type GFP mutants containing red-shifted excitation peaks and significantly enhanced fluorescence emission intensity, as well as to the construction of several different color variants. Placed in order from shortest to longest emission wavelength, the chromatic variants of wild type GFP are enhanced blue, cyan, green, and yellow fluorescent protein (EBFP, ECFP, EGFP, and EYFP). A red fluorescent protein engineered for expression studies is also commercially available (DsRed; Discosoma, various species), but was derived from a different organism than wild type GFP. The Nikon YFP HYQ combination for enhanced yellow fluorescent protein (sometimes referred to simply as YFP, or yellow-emitting GFP) is one member of a continuum of filter sets that allows multiple labeling of specimens expressing several different fluorescent proteins, which can subsequently be monitored separately in living systems.
The bandpass ranges of the YFP HYQ filter set are designed to enable detection of enhanced YFP, and are sufficiently narrow that enhanced cyan fluorescent protein (ECFP) will typically not be detected, making the filter set ideal for discriminating between YFP and CFP in dual labeling strategies. Because the emission passband (centered at 535 nanometers) overlaps the green spectral region, fluorescence from YFP appears visually to be green, rather than yellow. Although it employs a relatively narrow 20-nanometer excitation passband, the Nikon YFP HYQ filter combination can be utilized to detect a considerable range of fluorochromes excited by the longer wavelength blue-to-green spectral region, in addition to YFP, for which it is primarily designed. When used with yellow fluorescent protein (or other fluorophores), the filter set can also take advantage of the 488-nanometer emission line of popular argon-ion and krypton-argon confocal microscopy lasers for excitation.
Table 1 - Yellow Fluorescent Protein Filter Combination Specifications
|Filter Set Description||Excitation Filter (nm)||Dichromatic Mirror (nm)||Barrier Filter (nm)||Remarks|
|YFP HYQ||500/20 (490-510)||515 (LP)||535/30 (520-550)||Narrow Excitation Band Bandpass Barrier Filter|
- YFP HYQ - The YFP HYQ filter combination is designed for detection of emission from yellow fluorescent protein, which appears greenish within the relatively narrow passband, and is applicable with a range of other fluorochromes excited in the blue-green spectral region. The sharp passband transitions enable discrimination, in most cases, from cyan fluorescent protein when co-expressed in living or fixed cells with YFP.
The wide range of synthetic and natural fluorophores that has been developed includes many that are suitable for investigations using excitation wavelengths corresponding to the bandpass region of the YFP HYQ filter combination (blue-green excitation). Although this filter set is designed to provide specific performance advantages in applications using the enhanced yellow fluorescent protein variant of GFP, its application can be extended to a number of probes with suitable spectral profiles, particularly those that are typically utilized with standard blue-excitation filter sets.
Catalogued in Table 2 are some of the most popular dyes and fluorescent probes that can be visualized with the Nikon yellow fluorescent protein filter combination. The localized environment significantly influences fluorophore absorption and emission spectra 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 the table 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 listed fluorescent probes are also suitable for use with filter combinations in other excitation wavelength regions, including blue and green.
Table 2 - Fluorochromes with Blue-Green (YFP) Excitation Spectral Profiles
|Fluorochrome||Excitation Wavelength (Nanometers)||Emission Wavelength (Nanometers)|
|Acridine Orange (Bound to DNA)||500||526|
|Alexa Fluor 488||495||519|
|Alexa Fluor 500||503||525|
|Astrazon Orange R||470||540|
|BODIPY 492/515 (Difluoroboradiazaindacene)||490||515|
|CellTracker Green CMFDA (Chloromethylfluorescein Diacetate)||492||517|
|CFDA (Carboxyfluorescein Diacetate)||495||520|
|Cl-NERF||514 (High pH)||540 (High pH)|
|DiBAC4(3) (Bisdibutylbarbituric Acid)||493||516|
|DiO (Dipentyloxacarbocyanine Iodide)||484||502|
|DM-NERF||510 (High pH) 497 (Low pH)||536 (High pH) 527 (Low pH)|
|DTAF (Fluorescein Dichlorotriazine)||494||520|
|FDA (Fluorescein Diacetate)||495||520|
|FITC (Fluorescein Isothiocyanate)||494||518|
|FlCRhR (Cyclic AMP Fluorosensor)||500||517|
|Fluorescein Sulfonic Acid||476||519|
|LysoTracker Green DND-26||504||511|
|MitoTracker Green FM||490||516|
|Newport Green DCF||506||535|
|Newport Green PDX||490||518|
|Nonyl Acridine Orange||495||519|
|Oregon Green 488||496||524|
|Oregon Green 500||503||522|
|Oregon Green 514||511||530|
|PFB-F (Pentafluorobenzoyl Fluorescein)||492||516|
|Phen Green FL||492||517|
|Phen Green SK||507||532|
|SYBR Green I||498||522|
|SYBR Green II||492||513|
|YFP/EYFP (Enhanced Yellow Fluorescent Protein)||512||529|
Although the filter combination described in this section adequately serves in a majority of the fluorescence investigations that employ enhanced yellow fluorescent protein variants, and for many applications using synthetic blue-green excited fluorochromes, a number of additional filter sets are available from aftermarket manufacturers. In addition, certain filter sets originally intended for other fluorochromes are useful for yellow fluorescent protein studies, although at some compromise in performance compared to the specialized YFP combination. For example, earlier longpass and bandpass designs for fluorescein (FITC; including the Nikon FITC HYQ set) are applicable, as are many of the current blue excitation sets. A longpass version of the YFP HYQ set is available, which has the same excitation filter and dichromatic mirror, but incorporates a longpass emission filter to allow longer wavelength signal detection. In general, however, application of a longpass emission filter results in decreased signal to noise ratio, compared to that obtained with bandpass filters.
Included among the specialized combinations are filter set pairs designed for use in combination with each other for dual imaging of two fluorescent proteins, such as enhanced green fluorescent protein and enhanced yellow fluorescent protein. Filter sets are available that are specifically designed for fluorescence resonance energy transfer (FRET) techniques utilizing matched pairs of different fluorescent protein variants. Cyan and yellow fluorescent proteins (as donor and acceptor, respectively) are considered the best pair for FRET experiments in living cells. Special filter sets are available that provide for CFP excitation and detection of the resulting YFP emission, which occurs if the proteins are in close enough proximity for energy transfer between the donor (cyan) and acceptor (yellow) macromolecules to occur.
Other specialized filter sets incorporate dual excitation filters for individual excitation of two proteins for sequential image capture. Dual-band filter sets are available for simultaneous visualization of fluorescent protein combinations, including cyan and yellow fluorescent protein varieties. Two excitation and two emission filters are employed in some sets, in combination with filter wheels on both excitation and emission light paths, for sequential capture of emission from two fluorescent proteins, such as enhanced GFP and enhanced YFP. Similar specialized sets for protein pairs, including CFP-YFP and BFP-YFP, are configured in different variations having excitation filter bandpass properties optimized for use with either mercury or xenon light sources. Finally, complex filter assemblies are produced having triple, and even quadruple, excitation and emission filters for sequentially imaging fluorescence from various combinations of fluorescent proteins or from fluorescent proteins used in multiple labeling schemes with other fluorophores, such as Cy5 and Texas Red.
Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Nathan S. Claxton, John D. Griffin, Matthew J. Parry-Hill, Thomas J. Fellers