Triple Band Excitation: DAPI-FITC-Texas Red
Ultraviolet and visible transmission spectral profiles for the Nikon DAPI-FITC-Texas Red filter combination are illustrated below in Figure 1. This filter set is designed for optimal detection of DAPI, FITC (fluorescein isothiocyanate), and Texas Red probes when applied in combination (triple) staining techniques. The DAPI-FITC-Texas Red set incorporates an excitation filter with narrow bandpass windows in the violet (395 to 410 nanometers), blue (490 to 505 nanometers), and green (560 to 580 nanometers) spectral regions.
Figure 1 - DAPI-FITC-Texas Red (Triple Band Excitation)
Three emission (barrier) filter bandpass regions enable detection of blue, green, and red emission from the three fluorochromes simultaneously. Sharp wavelength transitions between transmission and reflection bands for each filter, and application of a dichromatic mirror having multiple bandpass transmission regions chosen to be complementary to the emission and excitation wavelengths, permit triple-band signal detection with minimal crossover (spectral bleed-through), interference, and noise.
Triple Excitation Filter Block DAPI-FITC-Texas Red Specifications
- Excitation Filter Wavelengths: 395-410 nanometers (bandpass, 403 CWL), 490-505 nanometers (bandpass, 498 CWL), and 560-580 nanometers (bandpass, 570 CWL)
- Dichromatic Mirror Wavelengths: 445-480 nanometers (bandpass), 510-555 nanometers (bandpass), and 590-665 nanometers (bandpass)
- Barrier Filter Wavelengths: 450-470 nanometers (bandpass, 460 CWL), 515-545 nanometers (bandpass, 530 CWL), and 600-650 nanometers (bandpass, 625 CWL)
The DAPI-FITC-Texas Red triple band fluorescence filter combination is designed specifically for simultaneous detection of the fluorochromes DAPI, FITC, and Texas Red with minimal crossover between adjacent emission bands, and it can also be employed with other combinations of fluorescent probes that have similar spectral profiles. The short wavelength signal channel of the filter set selects narrowly defined spectral regions for violet excitation and blue emission detection; a second, longer wavelength channel corresponds to blue excitation and green emission detection, and green excitation coupled to red emission constitutes the third band of the filter set. The short wavelength excitation transmission profile is deliberately reduced in size to provide better balance of the blue emission with the other fluorochrome signals, and to minimize photobleaching. This filter set is recommended when studying various combinations of fluorophores chosen from the following three groups: DAPI, blue fluorescent protein (BFP), or Hoechst dyes (violet excitation); FITC, green fluorescent protein (GFP), Cy2, or Alexa Fluor 488 (blue excitation); Texas Red or Alexa Fluor 594 (green excitation). The images presented in Figure 2 demonstrate the performance of this filter set with a variety of fluorescence probe combinations targeted at different intracellular locations.
Figure 2 - Nikon DAPI-FITC-Texas Red Triple Band Excitation Filter Set
Figure 2(a) illustrates the fluorescence emission profile from a thin section of rabbit lung tissue stained for nuclei, actin filaments, and glycoproteins with DAPI, Alexa Fluor 546-phalloidin, and Oregon Green 488 wheat germ agglutinin, respectively. The visible light absorption maximum of Alexa Fluor 546 is 556 nanometers and the emission maximum occurs at 573 nanometers, while the corresponding values for Oregon Green 488 are 496 and 524 nanometers. DAPI (4',6-diamidino-2-phenylindole) is an ultraviolet light-absorbing dye with an absorption maximum at 358 and an emission maximum in the blue region at 461 nanometers. Note the presence of high signal levels from all three of the fluorophores employed to stain the specimen.
A culture of bovine pulmonary artery epithelial cells that were immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to BODIPY FL is illustrated in Figure 2(b). The cell culture was simultaneously stained with Texas Red phalloidin and DAPI, which target the cytoskeletal actin network and DNA in the nucleus, respectively. The visible light absorption maximum of BODIPY FL is 505 nanometers and the emission maximum occurs at 513 nanometers, while the corresponding values for Texas Red are 595 and 620 nanometers.
Presented in Figure 2(c) is a thin section of mouse intestine stained with Alexa Fluor 350 wheat germ agglutinin, a blue fluorescent lectin that is specific to the mucus of goblet cells. In addition, the specimen was simultaneously stained with Alexa Fluor 568 phalloidin (filamentous actin; 600 nanometer emission) and SYTOX Green (nuclei; 504 nanometer excitation and 523 nanometer emission). Note the presence of signal from the blue fluorophore (Alexa Fluor 350), as well as the bright green fluorescence of nuclei in the tissue specimen due to SYTOX Green, and the orange-red fluorescence (Alexa Fluor 568) from actin filaments in the brush border.
Fluorescence emission from a culture of albino Swiss mouse embryo cells (3T3 line) stained with MitoTracker Red CMXRos and Alexa Fluor 488 conjugated to phalloidin, which target the intracellular mitochondrial network and cytoskeletal actin filaments, respectively, is illustrated in Figure 2(d). The visible light absorption maximum of MitoTracker Red CMXRos is 579 nanometers and the emission maximum occurs at 599 nanometers, while the corresponding values for Alexa Fluor 488 are 495 and 519 nanometers. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; ultraviolet excitation with blue emission). Note the presence of high signal levels from all three of the fluorophores employed to stain the specimen.
Canine kidney epithelial cells (Madin-Darby; MDCK line) that were immunofluorescently labeled with primary anti-human golgin-97 mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein (through the isothiocyanate, FITC) are demonstrated in Figure 2(e). The visible light absorption maximum of FITC is 494 nanometers and the emission maximum occurs at 518 nanometers in the blue-green region of the spectrum. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; blue emission) and Alexa Fluor 568 conjugated to phalloidin (targeting the cytoskeletal actin network; orange-red emission). Anti-human golgin-97 antibodies recognize a 97-kiloDalton protein termed golgin-97, which is a member of the granin protein family and a peripheral membrane protein localized on the cytoplasmic face of the Golgi apparatus. Note the presence of green mitochondria in this specimen.
Figure 2(f) demonstrates autofluorescence in a thin section of Zamia (Zamia loddigesii) stem tissue. Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including lignins, chlorophyll, carotene, and xanthophyll. In the blue and green excitation regions, chlorophyll has an absorption band with a high extinction coefficient and produces a significant amount of fluorescence when excited with wavelengths between 450 and 550 nanometers. Other endogenous fluorophores are excited by ultraviolet light with this filter combination. For the Zamia stem tissue illustrated above, note the presence of autofluorescence emission intensity in the blue, green, and red spectral regions, which is characteristic of the Nikon DAPI-FITC-Texas Red fluorescence filter combination.
Additional Specimen Images with the DAPI-FITC-Texas Red Filter Combination
Rabbit Lung Tissue
Fluorescence emission intensity from a thin section of rabbit lung tissue stained for nuclei, actin filaments, and glycoproteins with DAPI, Alexa Fluor 546-phalloidin, and Oregon Green 488 wheat germ agglutinin, respectively.
Bovine Pulmonary Artery Cell Nuclei, Actin, and Tubulin
Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial cells that were immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to BODIPY FL.
Mouse Intestine Thin Section
Fluorescence emission intensity from a thin section of mouse intestine stained with Alexa Fluor 350 wheat germ agglutinin, a blue fluorescent lectin that is specific to the mucus of goblet cells.
Albino Swiss Mouse Embryo Cell Nuclei, Mitochondria, and Actin
Fluorescence emission intensity from a culture of albino Swiss mouse embryo cells (3T3line) stained with MitoTracker Red CMXRos and Alexa Fluor 488 conjugated to phalloidin, which target the intracellular mitochondrial network and cytoskeletal actin filaments, respectively.
Canine Kidney Cell Golgi Apparatus
Fluorescence emission intensity from a culture of canine kidney epithelial cells (Madin-Darby; MDCK line) that were immunofluorescently labeled with primary anti-human golgin-97 mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to fluorescein (through the isothiocyanate, FITC).
Zamia loddigesii Stem Thin Section
Autofluorescence emission intensity from a thin section of Zamia (Zamia loddigesii) stem tissue. Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including lignins, chlorophyll, carotene, and xanthophyll.
Human Osteosarcoma Cells with DAPI, MitoTracker Red CMXRos, and Alexa Fluor 488
Observe the fluorescence emission intensity from a culture of human osteosarcoma cells (U-2 line) stained with MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and DAPI, which target the cellular mitochondrial network, cytoskeletal actin filaments, and nucleus, respectively.
Rat Skin Hair Follicles with Tetramethylrhodamine, DAPI, and Alexa Fluor 488
Examine a thin section of rat epidermal skin tissue stained for DNA and glycoproteins with DAPI and tetramethylrhodamine (TMR) conjugated to wheat germ agglutinin, respectively. The visible light absorption maximum of TMR is 550 nanometers and the corresponding emission maximum occurs at 573 nanometers. In addition, the specimen was simultaneously labeled with Alexa Fluor 488 conjugated to phalloidin (targeting the actin network; green emission). Note the presence of both overlapping and discrete signals from all three of these fluorophores.
Rat Kangaroo Kidney Epithelial Cells with DAPI, Alexa Fluor 488, and Alexa Fluor 568
A culture of rat kangaroo kidney (PtK2 line) epithelial cells was immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 488. 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.
Bovine Pulmonary Artery Endothelial Cells with DAPI, BODIPY FL, and MitoTracker Red CMXRos
Examine the bright fluorescence emission intensity from a culture of bovine pulmonary artery epithelial cells stained with MitoTracker Red CMXRos, BODIPY FL conjugated to phallacidin, and DAPI, which target the intracellular mitochondrial network, cytoskeletal actin filaments, and nucleus, respectively. These combined probes all exhibit high signal levels with the Nikon DAPI-FITC-Texas Red triple band excitation filter combination.
Rabbit Bone Marrow Tissue with DAPI, Oregon Green 488, and Alexa Fluor 546
A thin section of rabbit bone marrow tissue was stained for DNA and glycoproteins with DAPI and Oregon Green 488 conjugated to wheat germ agglutinin, respectively. The visible light absorption maximum of Oregon Green is 496 nanometers and the emission maximum occurs at 524 nanometers. In addition, the specimen was simultaneously labeled with Alexa Fluor 546 conjugated to phalloidin (targeting the actin network; yellow-orange emission).
Human Lung Cells with DAPI, Alexa Fluor 488, and MitoTracker Red CMXRos
Observe the fluorescence emission from a culture of normal human lung cells (MRC-5 line) stained with MitoTracker Red CMXRos and Alexa Fluor 488 conjugated to phalloidin, which target the intracellular mitochondrial network and cytoskeletal actin filaments, respectively. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; ultraviolet excitation with blue emission).
Bovine Pulmonary Artery Endothelial Cells with DAPI, Cy3, and Alexa Fluor 488
A culture of bovine pulmonary artery endothelial (BPAE line) cells was immunofluorescently labeled with primary anti-vinculin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy3. The visible light absorption maximum of Cy3 is 552 nanometers and the emission maximum occurs at 570 nanometers. Note the prominent staining of the cellular attachment network in the central portion and periphery of these cells. 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 488 conjugated to phalloidin.
African Green Monkey Kidney Cells with DAPI, MitoTracker Red CMXRos, and Alexa Fluor 488
Observe the fluorescence emission from a culture of normal African green monkey kidney cells (CV-1 line) stained with MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and DAPI, which target the intracellular mitochondrial network, cytoskeletal actin filaments, and nucleus, respectively. Note the presence of high signal levels from all three of the fluorophores employed to stain the specimen, as well as the unusual morphology of these cells.
HeLa Cells with Hoechst 33258, Green Fluorescent Protein, and Alexa Fluor 546
HeLa carcinoma cells were transfected with an EGFP-peroxisomal targeting signal 1 (PTS1) fusion protein and stained with Alexa Fluor 546-phalloidin. These fluorescent probes target the peroxisomes and cytoskeletal actin filament network, respectively. The visible light absorption maximum of the EGFP-PTS1 chimera is 488 nanometers and the emission maximum occurs at 507 nanometers. In addition, the specimen was simultaneously stained with Hoechst 33258 (targeting the DNA in the nucleus; blue emission). Note the presence of signal from the blue fluorophore, as well as the bright orange-red fluorescence exhibited by the cytoskeletal actin filaments and the intense green emission from peroxisomes in the cytoplasm.
Canine Kidney Cells with DAPI, Alexa Fluor 488, and MitoTracker Red CMXRos
Examine the fluorescence emission intensity from a culture of canine kidney epithelial cells (Madin-Darby; MDCK line) stained with MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and DAPI. Note the presence of high signal levels from all three of the fluorophores employed to stain the specimen.
Corn Kernel Autofluorescence
View the emission intensity from a thin section of corn kernel (Zea mays) tissue using the Nikon DAPI-FITC-Texas Red filter set. For the corn kernel tissue illustrated in this section, note the presence of autofluorescence emission intensity in the blue, green, and red spectral regions (in some areas, mixed together to form white).
Indian Muntjac Deerskin Fibroblast Cells with DAPI, MitoTracker Red CMXRos, and Alexa Fluor 488
A culture of Indian Muntjac deerskin fibroblast cells was stained with MitoTracker Red CMXRos and Alexa Fluor 488, which target the intracellular mitochondrial network and cytoskeletal actin filaments, respectively. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; ultraviolet excitation and blue emission). Note the presence of high signal levels from the blue (DAPI) fluorophore, as well as the orange-red fluorescence exhibited by the tubular mitochondria and the green emission from actin filaments in the cytoplasm.
Rat Kangaroo Kidney Cells with Cy2, MitoTracker Red CMXRos, and DAPI
A culture of rat kangaroo kidney epithelial (PtK2 line) cells was immunofluorescently labeled with primary anti-cytokeratin mouse monoclonal antibodies 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. Note the prominent staining of the cytokeratin network throughout the cytoplasm in these cells. In addition, the specimen was simultaneously stained for DNA with the ultraviolet-absorbing probe DAPI, and for the mitochondrial network with MitoTracker Red CMXRos.
Normal Human Lung Fibroblast Cells with DAPI, Alexa Fluor 488, and Texas Red
Examine the fluorescence emission intensity from a culture of human lung cells (MRC-5 line) stained with wheat germ agglutinin conjugated to Texas Red, Alexa Fluor 488 conjugated to phalloidin, and DAPI, which target the intracellular Golgi network, cytoskeletal actin filaments, and nucleus, respectively. The visible light absorption maximum of Texas Red is 596 nanometers and the emission maximum occurs at 615 nanometers. Note the presence of significant signal levels from all three of the fluorophores employed to stain the specimen, and the high degree of localization for wheat germ agglutinin to the Golgi apparatus.
Contributing Authors
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
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