Violet Excitation: Blue Fluorescent Protein (Bandpass Emission)

Ultraviolet and visible transmission spectral profiles for the high-performance Nikon blue fluorescent protein (BFP) filter combination are illustrated below in Figure 1. This filter set differs from the other two in the violet group by employing a bandpass emission filter, whose 50-nanometer passband (435-485 nanometers) restricts detection to those fluorochromes emitting in the blue spectral region. In addition, a narrow 22-nanometer excitation bandpass (extending to only 401 nanometers) minimizes autofluorescence, and is coupled with a lower dichromatic mirror cut-on wavelength (420 nanometers) than is used in the other violet filter sets. Although primarily designed for imaging of fluorescent proteins, this bandpass emission combination is useful for obtaining images of specimens having multiple labels when one of the fluorophores is efficiently excited in the violet spectral region.

Figure 1 - Blue Fluorescent Protein (Violet Excitation)

Violet Excitation Filter Block BFP Specifications

  • Excitation Filter Wavelengths: 379-401 nanometers (bandpass, 390 CWL)
  • Dichromatic Mirror Cut-on Wavelength: 420 nanometers (longpass, LP)
  • Barrier Filter Wavelengths: 435-485 nanometers (bandpass, 460 CWL)

The BFP filter block is designed for optimum performance with blue fluorescent protein, and by utilization of a bandpass emission filter, red and green fluorescence are excluded from detection in multiply-labeled specimens, producing images with a rich and deep blue color on a black background. The narrow bandwidth excitation filter is intended to minimize or eliminate excitation of endogenous nicotinamide adenine dinucleotide (NADH) at ultraviolet and violet wavelengths. The BFP filter combination is recommended when studying the following fluorophores: blue fluorescent protein, enhanced blue fluorescent protein, super glow blue fluorescent protein, Cascade Blue, catecholamine, fluorescamine, hydroxycoumarin, Leucophor SF, Pacific Blue, and TNS. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of violet absorbing fluorescence probes targeted at different intracellular locations.

Figure 2 - Nikon BFP Narrow Band Violet Excitation Bandpass Filter Set

Illustrated in Figures 2(a) and 2(e) are the emission intensities from a culture of Indian Muntjac deerskin fibroblast cells that were immunofluorescently labeled with either primary anti-bovine alpha-tubulin (Figure 2(a)) or anti-oxphos complex V inhibitor protein (Figure 2(e)) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Pacific Blue. The absorption maximum of Pacific Blue is 410 nanometers and the emission maximum occurs at 455 nanometers. Note the prominent staining of the intracellular microtubule (Figure 2(a)) and mitochondrial (Figure 2(e)) networks that extend throughout the cytoplasm in these specimens.

Figure 2(b) presents the fluorescence emission intensity from a thin section of mouse kidney stained with DAPI, Alexa Fluor 488 wheat germ agglutinin, a green fluorescent lectin that is specific to the glomeruli and convoluted tubules. The absorption maximum of DAPI is 358 nanometers and the emission maximum is 461 nanometers. In addition, the specimen was simultaneously stained with Alexa Fluor 568 phalloidin (filamentous actin). Note the absence of spectral bleed-through from the red and green fluorophores. Figure 2(d) presents bovine pulmonary artery cultured cells having the nuclei stained with DAPI. Although the DAPI fluorophore is commonly utilized with ultraviolet excitation filter combinations, many of the violet filter sets are able to produce excellent images as well.

Deep blue fluorescence emission intensity from a culture of rat kangaroo kidney epithelial cells (PtK2 line) is presented in Figure 2(c). The culture was immunofluorescently labeled with primary anti-cytokeratin (an intermediate filament protein) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Marina Blue. The absorption maximum of Marina Blue is 365 nanometers and the emission maximum occurs at 460 nanometers. In addition, the specimen was simultaneously stained for mitochondria with MitoTracker Red CMXRos. Note the absence of signal from the red fluorophore with this bandpass emission filter combination.

Autofluorescence in plant tissues (corn grain tissue section) is represented in Figure 2(f), and demonstrates the deep blue emission spectrum of endogenous fluorophores in these specimens. Note that images captured with the BFP filter combination exhibit far less background fluorescence than do the longpass emission (V-1A and V-2A) violet excitation filter sets.

Contributing Authors

Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.

John D. GriffinNathan S. ClaxtonMatthew J. Parry-HillThomas J. FellersKimberly M. VogtIan D. JohnsonShannon H. NeavesOmar AlvaradoLionel Parsons, Jr.Michael A. SoddersRichard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Violet Excitation: Blue Fluorescent Protein (Bandpass Emission)