Included in the Nikon violet excitation fluorescence filter portfolio are three carefully balanced combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide region of the blue, green, and red visible wavelengths. 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 violet region.
The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the bandpass emission (Blue GFP; default) 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 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 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 violet fluorescence filter combinations cover an excitation wavelength range between 379 and 420 nanometers with bandpass width profiles of 10, 22, and 40 nanometers. Two of the combinations employ the same dichromatic mirror, while the third set has a mirror with a lower wavelength cut-on to coincide with its shorter-wavelength excitation band.
The lone bandpass emission filter in the Nikon violet excitation series is incorporated in the BFP set, which has been designed for utilization with blue fluorescent protein derivatives, and produces images with a deep blue color on a jet black background. The bandpass emission filter in the BFP combination eliminates fluorescence from red and green fluorophores in specimens labeled with multiple probes. In addition, the narrow excitation band avoids autofluorescence resulting from long-wavelength ultraviolet excitation of nicotinamide adenine dinucleotide (NADH). The V-1A filter combination contains a very narrow excitation band (10 nanometers) that is intended to minimize autofluorescence, while simultaneously passing all emission wavelengths exceeding 435 nanometers. The dichromatic mirror in the V-1A filter set has a cut-on wavelength of 430 nanometers.
Commonly specified as the standard violet filter set, the V-2A combination is equipped with a 40-nanometer bandpass excitation filter that covers the majority of the violet wavelength region. Coupled with a 430-nanometer cut-on dichromatic mirror and a longpass emission filter, the V-2A produces the brightest images of any filter combination in the Nikon violet group. Specifications for the dichromatic mirrors and filters from the various Nikon violet filter combinations are listed in Table 1.
Table 1 - Nikon Violet Filter Combination Specifications
|430 (LP)||435 (LP)||Narrow Excitation Band
Longpass Emission Filter
|430 (LP)||450 (LP)||Medium Excitation Band
Longpass Emission Filter
|Narrow Excitation Band
Bandpass Emission Filter
- V-1A - The V-1A filter combination is designed as a filter block for violet fluorescence excitation with a narrow bandpass region to minimize autofluorescence. The longpass emission filter allows detection of a wide range of fluorochrome wavelengths.
- V-2A - The V-2A combination is designed as a standard violet fluorescence filter block having the greatest degree of versatility. With a medium excitation bandpass and longpass emission filter, the images produced are the brightest of the Nikon violet filter group.
- BFP (Blue Fluorescent Protein) - The BFP filter block combines a narrow excitation band with a bandpass emission filter to limit autofluorescence as well as to exclude detection of emission from red and green fluorophores. This filter combination is excellent for imaging blue fluorescent protein derivatives
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.