Included in the Nikon blue-violet excitation fluorescence filter portfolio are four combinations that include either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide region of the cyan, green, and red wavelengths.
The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the bandpass emission (Cyan GFP; default) blue-violet 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-violet 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-violet 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-violet fluorescence filter combinations cover an excitation wavelength range between 400 and 446 nanometers with bandpass width profiles of 10, 20, and 40 nanometers. Three of the combinations employ the same dichromatic mirror, while the fourth set has a mirror with a higher wavelength cut-on (5 nanometers) to conform with its other components.
Of the four filter blocks constituting the Nikon blue-violet excitation series, only the CFP set incorporates a bandpass emission filter. This combination has been designed for applications with cyan fluorescent proteins, particularly in dual and triple fluorophore techniques, and produces images with an intense cyan color on a jet-black background. The bandpass emission filter in the CFP combination eliminates fluorescence from red, yellow, and green fluorophores in specimens labeled with multiple probes. This filter set typically excludes signal from yellow fluorescent protein, but not from green fluorescent protein. TheBV-1A filter combination passes wavelengths from a wide range of fluorochromes that emit in the cyan-blue, green and red spectral regions (greater than 470 nanometers), and has a narrow 10-nanometer excitation band to minimize autofluorescence. The dichromatic mirror in the BV-1A filter set has a cut-on wavelength of 455 nanometers.
Commonly specified as the standard blue-violet filter set, the BV-2A combination is equipped with a 40-nanometer bandpass excitation filter that covers the entire blue-violet wavelength region. Coupled with a 455-nanometer cut-on dichromatic mirror and a longpass barrier filter (470-nanometer cut-on), the BV-2A produces the brightest images of any filter combination in the Nikon blue-violet group. The BV-2B filter set is similar in configuration to the BV-2A, except that each component is red-shifted by 5 nanometers in center or cut-on wavelength specification. The result is that images acquired under the same conditions have darker backgrounds and improved overall contrast compared to those using the BV-2A filter block. Specifications for the dichromatic mirrors, excitation, and barrier (emission) filters from the various Nikon blue-violet filter combinations are listed in Table 1.
Table 1 - Nikon Blue-Violet Filter Combination Specifications
|455 (LP)||470 (LP)||Narrow Excitation Band
Longpass Barrier Filter
|455 (LP)||470 (LP)||Standard BV Cube
Longpass Barrier Filter
|460 (LP)||475 (LP)||Darker Background
Longpass Barrier Filter
|Narrow Excitation Band
Bandpass Barrier Filter
- BV-1A - The BV-1A filter combination is designed as a filter block for blue-violet fluorescence excitation with a very narrow bandwidth to minimize specimen autofluorescence. The longpass emission filter allows detection of a wide range of fluorochrome wavelengths.
- BV-2A - The BV-2A combination is designed as the standard blue-violet fluorescence filter block. With a medium excitation filter bandpass and longpass emission filter, the images produced with this combination are the brightest of the Nikon blue-violet filter group.
- BV-2B - The BV-2B combination is designed as a filter block for blue-violet fluorescence that provides a darker background and higher contrast than the standard BV-2A filter combination.
- CFP (Cyan Fluorescent Protein) - The CFP filter block combines a narrow excitation bandwidth with a bandpass emission filter to limit autofluorescence as well as to exclude detection of red, yellow, and many green-emitting fluorochromes.
Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.
Matthew J. Parry-Hill, Lionel Parsons, Jr., Kimberly M. Vogt, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.