The Nikon BV-2A fluorescence filter combination includes a medium-width bandpass excitation filter (400-440 nanometers) coupled with a longpass barrier filter (470-nanometer cut-on wavelength) to enable signal collection from a wide range of fluorescent probes. Ultraviolet, visible, and near-infrared transmission spectral profiles for this complement of filters are illustrated below in Figure 1. The BV-2A filter set utilizes a dichromatic mirror with the same 455-nanometer cut-on wavelength specification as the BV-1A block, providing a 15-nanometer separation from the barrier filter cut-on.

Blue-Violet Excitation Filter Block BV-2A Specifications

• Excitation Filter Wavelengths: 400-440 nanometers (bandpass, 420 CWL)
• Dichromatic Mirror Cut-on Wavelength: 455 nanometers (longpass, LP)
• Barrier Filter Wavelengths: 470 nanometer cut-on (longpass, LP)

The BV-2A fluorescence filter set is designed to perform with a wide range of fluorochromes excited by blue-violet wavelengths, and is the standard Nikon blue-violet filter block. By employing a medium-width excitation passband in conjunction with a longpass barrier filter, this filter set (along with the BV-2B set) produces the brightest images of the blue-violet group. The BV-2A filter combination is recommended when studying the following fluorophores: aminochloromethoxyacridine (ACMA), acridine homodimer, acriflavin, aminopyrene trisulfate (APTS), Alexa Fluor 430, Astrazon Yellow, atabrine, berberine sulfate, beta-lactamase (non-ratiometric), catecholamine, cyan fluorescent protein (CFP), Calcofluor White, Chromomycin A, chlorotetracycline (CTC), Lucifer Yellow, mithramycin, quinacrine mustard, Sevron Yellow, and thioflavine S. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of blue-violet-absorbing fluorescence probes targeted at different intracellular locations.

Illustrated in Figures 2(a) and 2(b) is the fluorescence emission intensity from a culture of rat thoracic aorta (muscle) cells that were immunofluorescently labeled with either primary anti-cdc6 (human) protein (Figure 2(a)) or anti-oxphos complex V inhibitor protein (Figure 2(b)) monoclonal antibodies (mouse) followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 430 and Pacific Blue, respectively. The absorption maximum of Alexa Fluor 430 is 431 nanometers and the emission maximum occurs at 541 nanometers, while the corresponding excitation and emission values for Pacific Blue are 410 and 455 nanometers. The specimens were simultaneously stained for F-actin with Alexa Fluor 488 conjugated to phalloidin, and for mitochondria with MitoTracker Red CMXRos (Figure 2(a)) or DNA with SYTOX Orange (Figure 2(b)). Note the cyan fluorescence scattered throughout the cytoplasm due to Alexa Fluor 430 (Figure 2(a)) and Pacific Blue (Figure 2(b)), as well as the presence of substantial signal levels from the red (MitoTracker in Figure 2(a)) and green (Alexa Fluor 488) probes. Much of the SYTOX Orange signal, which appears throughout the nuclear region, appears green in Figure 2(b).

A thin section of mouse kidney stained with multiple (3) fluorophores is presented in Figure 2(c). Nuclei in the tissue section were targeted with the nucleic acid probe DAPI, which has an excitation maximum at 358 nanometers and an emission maximum at 461 nanometers when bound to DNA in cell cultures and tissue sections. In addition, the cryostat section was also simultaneously stained with Alexa Fluor 488 wheat germ agglutinin (glomeruli and convoluted tubules) and Alexa Fluor 568 phalloidin (filamentous actin and the brush border). Note the absence of signal from the red (Alexa Fluor 568) fluorophore, but the significant amount of fluorescence from the green (Alexa Fluor 488) and blue (DAPI) probes.

Fluorescence emission intensity from a culture of rat skeletal muscle tissue cells (L6 cell line; myoblasts) that were immunofluorescently labeled with primary anti-cdc6 (human) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Marina Blue is demonstrated in Figure 2(d). The absorption maximum of Marina Blue is 365 nanometers and the emission maximum occurs at 460 nanometers. Cdc6 is expressed in actively replicating cells to function during eukaryotic replication initiation, and is essential for DNA synthesis. In addition, the specimen was simultaneously stained for F-actin with Alexa Fluor 488 conjugated to phalloidin, and for mitochondria with MitoTracker Red CMXRos. Note the presence of substantial signal levels from the red (MitoTracker) and green (Alexa Fluor 488) probes, as well as the cyan cdc6 signal that is scattered throughout the cytoplasm.

A tissue 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 is presented in Figure 2(e). The absorption maximum of Alexa Fluor 350 is 346 nanometers and the emission maximum is 442 nanometers. In addition, the specimen was simultaneously stained with Alexa Fluor 568 phalloidin (filamentous actin; 600 nanometer emission) and SYTOX Green (nuclei; 523 nanometer emission). Note the presence of high signal levels from the green fluorophore, but the very weak fluorescence emission from Alexa Fluor 350 (blue emission), which is not efficiently excited by the BV-2A filter combination. In many cases, as is evident in the image, SYTOX Green stains other cytoplasmic components in addition to DNA. Signal from the red fluorophore (Alexa Fluor 568) is also not observed in this specimen.

Autofluorescence emission intensity from a fern (Holly; Cyrtomium caryotideum) sorus indusium is illustrated in Figure 2(f). Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including lignins, chlorophyll, carotene, and xanthophyll. In the blue-violet region, chlorophyll has an absorption band with a high extinction coefficient and produces a significant amount of fluorescence when excited with wavelengths between 400 and 440 nanometers. Note the presence of autofluorescence emission intensity in the green and red spectral regions, which is strongly reminiscent of a darkfield image.