The Nikon B-1A fluorescence filter set is designed with a narrow passband excitation range (20 nanometers) in order to minimize autofluorescence and photobleaching. Ultraviolet, visible, and near-infrared transmission spectral profiles for the filter combination are illustrated below in Figure 1. The longpass barrier (emission) filter is capable of transmitting signals from green, yellow, and red fluorophores that have significant absorption in the upper blue wavelength region. Similar to other filters in this series, the B-1A has a longpass dichromatic mirror with a cut-on wavelength of 505 nanometers.

Blue Excitation Filter Block B-1A Specifications

• Excitation Filter Wavelengths: 470-490 nanometers (bandpass, 480 CWL)
• Dichromatic Mirror Cut-on Wavelength: 505 nanometers (longpass, LP)
• Barrier Filter Wavelengths: 520 nanometer cut-on (longpass, LP)

The longpass emission filter contained in the B-1A combination transmits significantly more signal than any of the E-series filter sets, which utilize bandpass emission filters, but the 20-nanometer excitation bandwidth is the narrowest in the Nikon blue excitation/longpass emission filter collection. As a result, images from the B-1A filter combination are not as bright as those produced with the B-2A and B-3A sets. In general, however, blue-excitation filter combinations having longpass emission filters produce images that are considerably brighter than their bandpass (emission filter) counterparts, but usually at the expense of much lower signal-to-noise ratios (leading to lighter backgrounds). Fluorescence emission in the yellow, orange, and red regions is often visible in images collected using the B-1A filtercombination (See Figure 2).

The B-1A filter combination is designed to maximize the emission of popular dyes excited by blue light (including fluorescein and Alexa Fluor 488) when employed in combination with a counterstain such as propidium iodide (PI) or tetramethylrhodamine isothiocyanate (TRITC). This set also is recommended when studying the following fluorophores: Acridine Orange, Acridine Yellow, Auramine O, 5-carboxyfluorescein (5-FAM), Alexa Fluor 488, BODIPY FL, Calcein, Calcein Green-1, coriphosphine O, Cy2, DiO, fluorescein isothiocyanate (FITC), Fluo-3, FluoroJade, FluorX, FM 1-43, LysoTracker Green, MitoTracker Green, Oregon Green derivatives, oxacarbocyanine dyes (such as DiO), RH 414, Rhodamine 110, Spectrum Green, SYTO, SYTOX Green, YOYO-1, and YO-PRO-1. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of blue absorbing fluorescence probes targeted at different intracellular locations.

Illustrated in Figure 2(a) is the fluorescence emission intensity from a culture of rat kangaroo (PtK2) epithelial cells that were immunofluorescently labeled with primary anti-bovine alpha-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 488. The absorption maximum of Alexa Fluor 488 is 495 nanometers and the emission maximum occurs at 519 nanometers. Note the prominent staining of the intracellular microtubular network that extends throughout the cytoplasm. In addition, the specimen was simultaneously stained for the nuclear protein cdc6 (conjugated to Pacific Blue), and for F-actin with phalloidin conjugated to Alexa Fluor 568. Note the presence of signal from the red (Alexa Fluor 568) fluorophore, but the absence of fluorescence emission from the blue (Pacific Blue) probe, which is not efficiently excited at the wavelengths utilized by this filter combination.

Figure 2(b) demonstrates the performance of the B-1A filter combination using a culture of Indian Muntjac cells that were labeled with SYTOX Green to stain chromatin in the nuclei. The absorption maximum of SYTOX Green is 504 nanometers and the emission maximum occurs at 523 nanometers. In addition, the specimen was simultaneously labeled with primary anti-human OxPhos Complex V inhibitor protein mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Pacific Blue (absorption maximum at 410 nanometers). The actin cytoskeletal network was also labeled with phalloidin conjugated to Alexa Fluor 568. Note the presence of weak signal levels from the red (Alexa Fluor 568) fluorophore.

A thin section of mouse intestine the specimen stained with Alexa Fluor 568 phalloidin (filamentous actin; 600 nanometer emission) and SYTOX Green (nuclei; 504 nanometer excitation and 523 nanometer emission) is presented in Figure 2(c). Note the low level of background noise in comparison to the other Nikon longpass filter combinations (B-2A and B-3A), and the significant amount of red signal arising from Alexa Fluor 568 that appears in the image. Figure 2(d) illustrates HeLa epithelial cells that 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. The absorption maximum of Alexa Fluor 488 is 495 nanometers and the emission maximum occurs at 519 nanometers. In addition, the specimen was simultaneously stained for F-actin with Alexa Fluor 568 (red) conjugated to phalloidin, and for DNA with 4',6-diamidino-2-phenylindole (DAPI). Note the presence of signal from both the red and green fluorophores, but the absence of any significant emission intensity from DAPI, which is not efficiently excited in the blue wavelength region.

Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial cells stained with BODIPY FL phallacidin, which binds to the intracellular filamentous actin network, is depicted in Figure 2(e). The absorption maximum of BODIPY FL is 503 nanometers and the emission maximum occurs at 512 nanometers. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; blue emission) and MitoTracker Red CMXRos (targeting mitochondria; red emission). Note the presence of signal from the red fluorophore (MitoTracker), but the absence of blue fluorescence from DAPI, which is not efficiently excited in this spectral region.

Illustrated in Figure 2(f) is autofluorescence emission intensity from a thin section of wheat grain (Triticum aestivum) tissue. Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including chlorophyll, carotene, and xanthophyll. In the blue region, chlorophyll has an absorption band with a high extinction coefficient and produces a significant amount of fluorescence when excited with wavelengths between 420 and 460 nanometers. Note the presence of spectral bleed-through from autofluorescence emission in the yellow and red spectral regions with the B-1A filter combination.