Fluorescence Filter Combinations

Yellow Fluorescent Protein Excitation: YFP HYQ

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.

The enhanced yellow fluorescent protein gene used in these studies contains four amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by 18 nanometers, or from approximately 509 to 527 nanometers. The gene is optimized with human codons and features a consensus Kozak translation initiation signal to achieve higher expression in mammalian cell cultures. Vectors targeted at specific subcellular organelles contain a fusion gene segment, which couples the YFP gene to a peptide sequence or complete protein that is localized to the region of interest in living cells.

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.

Additional Images with the YFP HYQ Filter Combination

Normal African Green Monkey Kidney Epithelial Cell Actin with Alexa Fluor 488 - Examine the fluorescence emission intensity observed in a culture of normal African green monkey kidney cells labeled with phalloidin conjugated to Alexa Fluor 488. The visible light absorption maximum of Alexa Fluor 488 is 495 nanometers and the emission maximum occurs at 519 nanometers, values that fall within the useful range of the Nikon YFP HYQ filter combination.

Human Lung Carcinoma Cell Mitochondria with Cy2 - A culture of human lung carcinoma epithelial cells (A-549 line) was immunofluorescently labeled with primary anti-oxphos complex V inhibitor protein monoclonal antibodies (mouse) followed by goat anti-mouse Fab fragments conjugated to Cy2. The visible light absorption maximum of Cy2 is 489 nanometers and the emission maximum occurs at 506 nanometers.

Indian Muntjac Skin Cell Tubulin with Fluorescein - Indian Muntjac deerskin fibroblast cells were immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein. The visible light absorption maximum of fluorescein is 490 nanometers and the emission maximum occurs at 525 nanometers.

HeLa Cellular Endosomes with EGFP - Observe the fluorescence emission intensity from a culture of human cervical adenocarcinoma (HeLa line) epithelial cells that were transfected with a pEGFP-Endosome plasmid subcellular localization vector. The enhanced green fluorescent protein gene used in these studies contains several important amino acid substitutions that shift the absorption and emission maxima of the wild type protein to 488 and 507 nanometers, respectively.

Rat Kangaroo Cell Intermediate Filaments with Cy2 - Rat kangaroo kidney (PtK2 line) epithelial cells were immunofluorescently labeled with primary anti-keratin (an intermediate filament protein) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to the cyanine dye, Cy2. Note the high level of green fluorescence observed from the keratin intermediate filament network that extends throughout the cytoplasm.

Normal Human Lung Fibroblast Golgi with Oregon Green - A culture of normal human lung fibroblast cells (MRC-5 line) was stained with wheat germ agglutinin conjugated to Oregon Green 488, Alexa Fluor 568 conjugated to phalloidin, and DAPI, which target the intracellular Golgi network, cytoskeletal actin filaments, and nucleus, respectively. Note the presence of high signal levels from the green fluorophore, but the absence of fluorescence intensity from the red and blue (Alexa Fluor 568 and DAPI, respectively) probes.

Rat Skeletal Muscle Nuclei with SYTOX Green - Examine the fluorescence emission intensity from a culture of rat skeletal muscle tissue cells (L6 cell line; myoblasts) that were labeled with SYTOX Green for nuclear DNA. The visible light absorption maximum of SYTOX Green, when bound to DNA, is 504 nanometers and the emission maximum occurs at 523 nanometers.

Human Lung Carcinoma Mitochondria with Alexa Fluor 488 - Human lung carcinoma epithelial cells (A-549 line) were immunofluorescently labeled with primary anti-oxphos complex V inhibitor protein monoclonal antibodies (mouse) followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 488. Note the high level of green fluorescence from the labeled intracellular mitochondria, which are distributed throughout the cytoplasm.

African Green Monkey Kidney Cell Cadherin with Cy2 - Normal (non-transformed) African green monkey kidney fibroblast (CV-1 line) cells were immunofluorescently labeled with primary anti-cadherin monoclonal antibodies (mouse) followed by goat anti-mouse Fab fragments conjugated to Cy2. In addition, the specimen was simultaneously stained for DNA with the ultraviolet-absorbing probe Hoechst 33258, and for the cytoskeletal filamentous actin network with Alexa Fluor 568 conjugated to phalloidin.

HeLa Human Cervical Carcinoma Cellular Actin with EGFP - This section reviews fluorescence emission intensity observed with the Nikon YFP HYQ blue-green excitation filter combination in a culture of HeLa cells labeled with a chimera of enhanced green fluorescent protein (EGFP) and human cytoplasmic beta-actin, a protein that targets the actin cytoskeletal network.

Rat Kangaroo Cell Intermediate Filaments with Fluorescein - Observe the fluorescence emission intensity from a culture of rat kangaroo kidney (PtK2 line) epithelial cells that were immunofluorescently labeled with primary anti-vimentin (an intermediate filament protein) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein. Vimentins represent a class of water-insoluble proteins, along with desmin, glial fibrillary acidic protein (GFAP), and peripherin, which form intermediate filaments and help to mechanically stabilize the cell.

Rat Liver Thin Section with SYTOX Green - A thin section of rat liver was stained with SYTOX Green for nuclear DNA. In addition, the specimen was simultaneously stained for filamentous actin with Alexa Fluor 546 conjugated to phalloidin, and with Alexa Fluor 350 wheat germ agglutinin (WGA), a blue fluorescent lectin conjugate that is specific to the mucus of goblet cells. Note the strong green fluorescence emission from labeled DNA in the cell nuclei, while no signal is observed from the blue and yellow fluorophores (Alexa Fluor 350 and Alexa Fluor 546) targeting glycoproteins and actin, respectively.

Albino Swiss Mouse Embryo Cell Tubulin with Cy2 - Swiss mouse embryo cells (3T3 line) were immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy2. In addition, the specimen was simultaneously stained for DNA with the ultraviolet-absorbing probe DAPI, and for the cytoskeletal filamentous actin network with Alexa Fluor 568 conjugated to phalloidin. Note the high level of green fluorescence observed from the intracellular microtubule network that extends throughout the cytoplasm, but an absence of signal from the blue and orange fluorophores (DAPI and Alexa Fluor 568) targeting the nuclei and actin, respectively.

African Green Monkey Kidney Cells with QDot 525 - A culture of African green monkey (COS-1 line) cells were immunofluorescently labeled with primary anti-cytokeratin mouse monoclonal antibodies followed by goat anti-mouse F(ab')2 fragments conjugated to biotin. The secondary antibody was subsequently stained with quantum dots (QDot 525) conjugated to streptavidin. QDot 525 conjugates exhibit absorption over a broad wavelength range extending from the ultraviolet to near their maximum emission wavelength of approximately 525 nanometers, values that fall within the useful range of the Nikon YFP HYQ filter combination.

Rabbit Heart Tissue with Oregon Green 488 - Examine the fluorescence emission from a rabbit heart tissue thin section stained with wheat germ agglutinin conjugated to Oregon Green 488. In addition, the specimen was simultaneously stained for filamentous actin with Alexa Fluor 568 conjugated to phalloidin, and for DNA with the ultraviolet-absorbing probe DAPI. Note the strong green fluorescence emission from the green fluorophore, while no signal is observed from the blue and orange probes, DAPI and Alexa Fluor 568, respectively.

Bovine Pulmonary Artery Cell Vinculin with Cy2 - The cell culture was immunofluorescently labeled with primary anti-vinculin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy2. In addition, the specimen was simultaneously stained for DNA with the ultraviolet-absorbing probe Hoechst 33258, and for the cytoskeletal filamentous actin network with Alexa Fluor 568 conjugated to phalloidin.

Canine Kidney Cell Mitochondria with Fluorescein - Review the fluorescence emission intensity observed with the Nikon YFP HYQ blue-green excitation filter combination in a culture of canine kidney epithelial (MDCK line) cells immunofluorescently labeled with fluorescein for intracellular mitochondria, Alexa Fluor 568 conjugated to phalloidin for actin, and stained with DAPI for the cell nuclei. Note the high level of green fluorescence from the labeled intracellular mitochondria, but an absence of detected emission from the blue and orange fluorophores (DAPI and Alexa Fluor 568) targeting the nuclei and actin, respectively.

HeLa Cell Histones with Fluorescein - An adherent culture of HeLa cells was immunofluorescently labeled with primary anti-histone (pan) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein (FITC). The primary antibody, produced from purified nuclear fractions of HeLa cells, binds specifically to an antigen determinant that is present on all five histone proteins (H1, H2A, H2B, H3, and H4). Note the strong green fluorescence emission exhibited by both the interphase nuclei and those in various stages of mitosis.


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Contributing Authors

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, Kimberly M. Vogt, Ian D. Johnson, Shannon H. Neaves, Omar Alvarado, Lionel Parsons, Jr., Michael A. Sodders, Richard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.