Blue Excitation

Included in the Nikon blue excitation fluorescence filter portfolio are six carefully balanced combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide band of the green, yellow, red, and near-infrared spectral regions. This interactive tutorial explores how the variations in the excitation and emission filter spectral profiles, as well as those of the dichromatic mirrors, affect signal levels, overall filter performance, and image contrast in combinations designed for excitation of fluorophores in the blue region.

The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the bandpass emission (B-2E; default) blue excitation filter combination spectral profile displayed on the Filter Set Spectral Profiles graph. The combined filter transmission and reflection spectra are superimposed over the absorption and emission spectra of the blue absorbing fluorophore utilized to label the specimen (fluorophore spectral profiles are not included for autofluorescent plant specimens). Fluorophore absorption spectra are presented in the tutorial using a brown fill, while the corresponding emission spectra are represented with a gray fill. Wavelength characteristics for the filter combination indicated by the Filter Set slider are displayed in the yellow box in the lower right-hand corner of the tutorial. These values are constantly updated as the slider is translated from left to right.

In order to operate the tutorial, use the Filter Set slider to transition between the various filter combinations available for blue excitation. As the slider is translated from left to right, the spectral profiles of the excitation and barrier filters, as well as that of the dichromatic mirror, are modified to simulate changes to the spectral profiles. Note that the continuously changing spectral profiles do not imply that any filter combination is possible, nor are the individual filter sets variable (without physically changing filters) in regards to the spectral profiles. Alterations of the spectral profiles between selected filter sets are simply intended to help establish the relationship between the filter combinations used in each optical block.

Individual Filter Spectra (excitation, emission, and dichromatic mirror) can be added or removed from the Filter Set Spectral Profiles graph by selecting or deselecting the appropriate check boxes beneath the graph. In addition, the fluorophore absorption and emission spectra can be added or removed with a similar set of check boxes (Spectral Cross Sections). The specimen image changes simultaneously with the filter profiles to reflect variations in contrast and signal levels produced by the alterations to the filter combinations produced by translation of the slider. A new specimen can be selected at any time using the Choose A Specimen pull-down menu, and the fluorophores utilized to label the selected specimen are listed directly beneath the menu box. In many cases, the specimens are stained with two or more fluorescent probes to demonstrate the selective isolation of fluorescence with bandpass and longpass barrier (emission) filter sets.

The Nikon blue fluorescence filter combinations cover an excitation wavelength range between 420 and 495 nanometers with bandpass width profiles of 20, 30, 40, and 70 nanometers. Five of the combinations employ the same dichromatic mirror, while the sixth set has a mirror with a lower wavelength cut-on to increase the acquired signal. Bandpass barrier filters for the Nikon blue excitation filter combinations all have a spectral width of 40 nanometers. One of the filters (B-3A) is designed to be employed with tungsten-halogen illumination.

Sets having bandpass emission filters in the Nikon blue fluorescence excitation series, the B-1E, B-2E, and B-2E/C, produce images at different levels of contrast with a deep green color on a jet-black background, and are ideal for use in multi-color fluorescence imaging with other excitation filter combinations. The bandpass emission combinations are designed to eliminate, or at least significantly reduce (depending upon spectral characteristics), fluorescence from yellow, red, and near-infrared fluorophores in specimens labeled with multiple probes. Generally, filter combinations utilizing bandpass emission components produce higher signal-to-noise ratios than those employing longpass filters, and the images exhibit visually darker backgrounds, although with less overall brightness.

All of the three filter combinations having bandpass emission filters utilize the same dichromatic mirror (505-nanometer cut-on) and similar 40-nanometer bandwidth emission filters. The B-1E set is equipped with a narrow-bandpass excitation filter centered on 480 nanometers, while the B-2E set differs only in having a wide excitation band centered at 470 nanometers. The emission passband of the B-2E/C combination is shifted by 5 nanometers to shorter wavelengths in order to optimize detection of popular fluorophores (FITC and others). The medium bandwidth of the excitation filter in the B-2E/C set is designed to exclude crossover detection of yellow and orange fluorochromes utilized in specific multiple labeling or counterstaining techniques.

The B-1A filter combination incorporates a narrow-bandpass excitation filter (20-nanometers) that is intended to minimize photobleaching as well as specimen autofluorescence when excited by the shorter wavelengths in the blue spectral region. The dichromatic mirror in the B-1A filter set has a cut-on wavelength of 505 nanometers, as is used in most of the blue excitation sets. The longpass barrier filter passes emission above the 520-nanometer cut-on, allowing detection of a wide range of fluorochromes emitting in the green, yellow, orange, and red wavelength regions. Containing similar components, but with a wider 40-nanometer excitation passband, the B-2A filter set is considered the standard Nikon blue-excitation set. The wide excitation band, in the center of the blue spectral region, is combined with a 5-nanometer lower dichromatic mirror cut-on wavelength (500 nanometers) than the other blue-excitation filter blocks. This combination provides a brighter emission signal than is observed with the B-1A set for many popular fluorochromes.

The B-3A filter set combines the same dichromatic mirror and emission filter as the B-1A set, but with a very wide 70-nanometer excitation passband (420-490 nanometers) to maximize the energy available to fluorochromes absorbing at essentially any wavelength in the blue spectral region. The set is designed to be utilized primarily with tungsten-halogen illumination, although it may also be useful with arc-discharge lamps when employing probes having a very weak emission signal. The dichromatic mirror cut-on wavelength of 505 nanometers, coupled with a longpass emission filter (520-nanometer cut-on), enables detection of a large range of fluorochromes emitting green, yellow, orange, and red wavelengths. Specifications for the dichromatic mirrors and filters from the various Nikon blue filter combinations are listed in Table 1.

Table 1 - Nikon Blue Excitation Filter Combination Specifications

Filter Set
Filter (nm)
Mirror (nm)
Filter (nm)
B-1A 480/20
505 (LP) 520 (LP) Narrow Excitation Band
Longpass Emission Filter
B-2A 470/40
500 (LP) 515 (LP) Wide Excitation Band
Longpass Emission Filter
B-3A 455/70
505 (LP) 520 (LP) Very Wide Excitation Band
Halogen Illumination
B-1E 480/20
505 (LP) 540/40
Narrow Excitation Band
Bandpass Emission Filter
B-2E 470/40
505 (LP) 540/40
Wide Excitation Band
Bandpass Emission Filter
B-2E/C 480/30
505 (LP) 535/40
Medium Excitation Band
Bandpass Emission Filter
  • B-1A - The B-1A filter combination is designed with a narrow excitation passband in order to reduce autofluorescence and photobleaching. The longpass barrier (emission) filter is capable of transmitting signals from green, yellow, orange, and red fluorophores that have significant absorption in the upper blue wavelength region corresponding to the excitation bandpass window.
  • B-2A - The B-2A filter combination is the standard filter set in the Nikon blue excitation group. Its design incorporates a wide excitation bandpass in order to provide an expanded absorption window for fluorophores compared to the B-1Acombination, and the set also has lower dichromatic mirror and barrier filter cut-on wavelengths, producing enhanced image brightness when compared to the B-1A filter set.
  • B-3A - The B-3A filter combination utilizes the same dichromatic mirror and longpass emission filter as the B-1A set, but employs a very wide excitation band that makes it suitable for tungsten-halogen illumination. This combination enables the transmission of a significant amount of the signal from green, yellow, orange, and red fluorophores that have absorption bands in the blue wavelength region.
  • B-1E - The B-1E filter combination is one of three sets in the blue fluorescence excitation series that utilize bandpass, as opposed to longpass, barrier filters, and which differ primarily in the width of the excitation band. Each of the bandpass barrier filter sets provides performance suitable for imaging specimens labeled with fluorescein isothiocyanate (FITC) (green emission), while blocking yellow, orange, and red emission. The B-1E combination uses a narrow-bandpass excitation filter in conjunction with the bandpass emission (barrier) filter.
  • B-2E - The B-2E filter combination employs a wide excitation bandpass, extending 20 nanometers lower in wavelength than the excitation band of the B-1E set. The dichromatic mirror and bandpass barrier filter specifications are the same in the B-1E and B-2E filter combinations.
  • B-2E/C - The B-2E/C filter combination employs a medium-width excitation band in conjunction with a slightly lowered emission bandpass wavelength region compared to the B-1E and B-2E sets. The performance is optimized for detection of a number of popular fluorophores used in multiple-labeling experiments, while providing improved exclusion of yellow to red wavelengths.

Contributing Authors

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

Matthew J. Parry-Hill, Thomas J. Fellers, Lionel Parsons, Jr., Kimberly M. Vogt, Ian D. Johnson, andMichael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Blue Excitation