The Nikon yellow fluorescent protein YFP HYQ filter set is designed to transmit excitation illumination over a narrow (20-nanometer) band of wavelengths in the blue-green spectral region, coupled with green to yellow emission detection within a 30-nanometer bandpass region. Ultraviolet, visible, and near-infrared transmission spectral profiles for the filter combination are illustrated below in Figure 1. The block is optimized for imaging YFP, while maintaining discrimination of cyan fluorescent protein in dual staining techniques. The combination incorporates a 20-nanometer excitation passband (490 to 510 nanometers), longpass dichromatic mirror (515-nanometer cut-on), and bandpass emission filter centered on 535 nanometers (520 to 550 nanometers). The restricted detection range efficiently transmits YFP emission, while limiting interference from autofluorescence and signal from fluorophores emitting at longer wavelengths.

Yellow Fluorescent Protein (Blue-Green Excitation) Filter Block YFP HYQ Specifications

• Excitation Filter Wavelengths: 490-510 nanometers (bandpass, 500 CWL)
• Dichromatic Mirror Cut-on Wavelength: 515 nanometers (longpass, LP)
• Barrier Filter Wavelengths: 520-550 nanometers (bandpass, 535 CWL)

The YFP HYQ yellow fluorescent protein filter combination is designed to provide optimal excitation for enhanced yellow variants of GFP. It employs a 30-nanometer bandpass emission filter that enables imaging of YFP while limiting interference from autofluorescence at higher wavelengths, as well as maintaining discrimination of cyan fluorescent protein (CFP) emission occurring at shorter wavelengths. In specimens that exhibit high autofluorescence, the use of a bandpass emission filter significantly increases the image signal-to-noise ratio compared to that from longpass sets.

The spectral characteristics of the Nikon YFP HYQ filter combination make it the primary choice for detection of yellow fluorescent protein alone or in various fluorophore combinations, particularly in dual separate staining experiments with cyan fluorescent protein. In addition to the design application (YFP), this set is suitable for application with a number of other fluorophores having absorption profiles that lie within its blue-green excitation wavelength range. The YFP HYQ filter combination is recommended when studying the following fluorophores: enhanced YFP (EYFP), Acridine Orange (bound to DNA), Alexa Fluor 488 and 500, Astrazon Orange R, Aurophosphine, BODIPY derivatives, Calcium Green, CellTracker Green CMFDA, DiBAC4(3), DiO, DiOC, DM-NERF, DTAF, FAM (carboxyfluorescein), Fluo derivatives (3, 4, 4FF, 5F, 5N), fluorescein (FITC), FluoZin, Magnesium Green, MitoTracker Green FM, NeuroTrace 500/525 and 515/535, Oregon Green (488, 500, 514), PKH67, Rhodamine (110, 123, Green), SYBR, SYTO, TAMRA (carboxytetramethylrhodamine), YO-PRO-1, and YO-YO-1. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of enhanced yellow fluorescent protein probes targeted at different intracellular locations.

The collection of specimens illustrated in Figure 2 demonstrates the effectiveness of the Nikon YFP HYQ filter combination with a series of cell lines transfected by EYFP plasmid vectors that express a fluorescent fusion protein targeted at very specific subcellular locations. Susceptible adherent cell cultures were transfected with the appropriate vector using proprietary lipophilic reagents, and were then cultured for a period of at least 24 hours in nutrient medium supplemented with fetal bovine serum to allow full expression of the fluorescent fusion protein. The links outlined below lead to sections that feature a variety of cell lines transfected with localized (subcellular organelle) yellow fluorescent protein fusion products.

Actin Filament Subcellular Localization - The network of fibrous actin filaments (also commonly referred to as microfilaments) found in most mammalian cell lines appears as a complex interconnected organization of linear bundles aligned in two-dimensional arrays or as a three-dimensional matrix. These protein fibers are easily labeled with synthetic or natural fluorophores and subsequently visualized using fluorescence microscopy. The pEYFP vector gene product illustrated in Figure 2(a) contains a fusion nucleotide sequence of the enhanced yellow fluorescent protein domain with a fully functional copy of human cytoplasmic beta-actin. When expressed in living cells, the gene product is incorporated into growing actin filaments without significant structural interference from the EYFP protein moiety. The fluorescence excitation maximum of EYFP is 513 nanometers and the corresponding emission maximum occurs at 527 nanometers with a high fluorescence quantum yield.

Nuclear Protein Subcellular Localization - Localization of specific proteins and complexes to the nucleus in mammalian cells is generally accomplished through the use of peptide signals that mediate transport to the organelle. Recombinant plasmids have been constructed that contain a fusion protein consisting of the yellow-green variant (referred to as enhanced yellow fluorescent protein; EYFP) of the Aequorea victoria green fluorescent protein (GFP) coupled to multiple copies of nuclear localization signal peptides. Upon transcription and translation of the plasmid in transfected mammalian hosts, the nuclear localization signals are responsible for transport of the fluorescent protein chimera to the cell nucleus. The nucleus can be subsequently visualized using fluorescence microscopy (see Figure 2(b)). The single bandpass emission filter featured by the Nikon YFP HYQ optical block, which was employed to capture these images, produces sharp contrast with little interference from autofluorescence or other fluorescent species.

Mitochondria Subcellular Localization - Localization of specific peptides, proteins, and macromolecular complexes to the mitochondria in mammalian cells is generally accomplished through the use of peptide signals that mediate transport to the organelle. Plasmid pEYFP-Mitochondria vector gene product expression in various cell types (from both transiently and stably transfected clones) occurs due to the efficient intracellular translation of a fusion nucleotide sequence combining the enhanced yellow fluorescent protein domain with the mitochondria targeting sequence from subunit VII of human cytochrome C oxidase. The human lung mitochondria specimen illustrated in Figure 2(c) demonstrates the effectiveness of the Nikon YFP HYQ filter combination for imaging cell lines containing the chimeric EYFP plasmid that expresses a fluorescent fusion protein targeted at the intracellular mitochondrial network. The fusion protein is transported in vivo to the mitochondria and readily enables visualization of the subcellular structure in living and fixed cells.

Golgi Apparatus Subcellular Localization - The Golgi apparatus (often referred to as the Golgi complex) consists of stacked and flattened vesicles in eukaryotic cells, which are often located near the nucleus, but can also be distributed widely throughout the cytoplasmic matrix. Golgi vesicles are important in the synthesis of complex carbohydrates, and in the processing and packaging of secretory proteins produced by the endoplasmic reticulum. A recombinant plasmid containing a fusion protein consisting of the EYFP domain coupled to a sequence encoding the N-terminal 81 amino acids of human beta-1,4-galactosyltransferase was employed to target the Golgi complex (see Figure 2(d)). This region of the glycoprotein contains a membrane-anchoring signal peptide that targets the plasmid fusion protein to the trans-medial region of the Golgi apparatus. Upon transcription and translation of the plasmid in transfected mammalian hosts, the fused glycoprotein domain is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular Golgi network.

Tubulin Subcellular Localization - Microtubules are cylindrical biopolymeric filaments that play an important structural and functional role in the dynamics of the cellular cytoskeleton. These hollow, but rigid, helical tubes are constructed with several variations of a protein known as tubulin. Among the many biological tasks assigned to microtubules are segregation of the chromosomes during mitosis, formation of cilia and flagella on the surface of a cell, and transport of materials through axons. The human alpha-tubulin protein can be efficiently incorporated into the microtubules from a variety of mammalian cell lines, and such networks, when labeled with tubulin monomer subunits fused to fluorescent protein domains (Figure 2(e)), can be readily visualized using fluorescence microscopy.

Endosomal Subcellular Localization - In eukaryotic cells, endosomes constitute a large network of cytoplasmic vesicles that are formed through the fusion of smaller internalized vesicles arising from receptor-mediated endocytosis. Often, the endosomal vesicles ultimately transfer their contents to the lysosomes for processing, but this is not always the case. Recombinant plasmids have been constructed that contain a fusion protein consisting of the EYFP fluorescent protein domain coupled to human RhoB GTPase, an enzyme that is localized in early endosomes, recycling endosomes, and multivesicular bodies. Upon transcription and translation of the plasmid in transfected mammalian hosts, the fused RhoB domain is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular endosomal network. The human cervical carcinoma (HeLa) cell illustrated in Figure 2(f) is an example of this highly specific targeted intracellular distribution.