Included in the Nikon ultraviolet excitation fluorescence filter portfolio are four 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 ultraviolet region.
The tutorial initializes with a randomly selected fluorescent specimen appearing in theSpecimen Image window and the bandpass emission (UV-2E/C; default) ultraviolet 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 ultraviolet-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 ultraviolet 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 ultraviolet excitation fluorescence filter combinations cover an excitation wavelength range between 330-380 nanometers with bandpass width profiles of 10, 40, and 50 nanometers. Three of the combinations employ the same dichromatic mirror, while the fourth set has a mirror with a lower wavelength cut-on to coincide with its narrower excitation band. The ultraviolet filter sets contain either longpass or single bandpass emission filters.
The lone bandpass emission filter in the Nikon ultraviolet excitation series, the UV-2E/C, produces images with a deep blue color on a jet black background, and is ideal for use in multi-color fluorescence imaging with other excitation filter combinations. The bandpass emission filter in the UV-2E/C combination eliminates fluorescence from red and green fluorophores in specimens labeled with multiple probes. The UV-1A filter combination contains a very narrow excitation band (10 nanometers) that coincides with the wavelength of the mercury i-line produced by common arc-discharge lamps. This filter combination is designed to minimize autofluorescence through a narrow bandpass excitation region, while simultaneously passing all emission wavelengths exceeding 420 nanometers. The dichromatic mirror in the UV-1A filter set has the shortest cut-on wavelength (380 nanometers) in the ultraviolet series.
Often referred to as the standard ultraviolet set, the UV-2A combination is equipped with a 50-nanometer bandpass excitation filter that covers most of the longer wavelength ultraviolet region. Coupled to a 400-nanometer cut-off dichromatic mirror and a longpass emission filter, the UV-2A produces the brightest images of any filter combination in the Nikon ultraviolet set. The UV-2B is similar in profile to the UV-2A, but is equipped with a longer cut-on wavelength (red-shifted by 15 nanometers) barrier filter in order to reduce background fluorescence. When compared to the UV-2A, the UV-2B combination produces a much darker background with superior overall image contrast. Specifications for the dichromatic mirrors and filters (excitation and barrier) from the various Nikon ultraviolet filter combinations are listed in Table 1.
Table 1 - Nikon Ultraviolet Filter Combination Specifications
|380 (LP)||420 (LP)||Narrow Excitation Band
Longpass Barrier Filter
|400 (LP)||420 (LP)||Standard UV Cube
Longpass Barrier Filter
|400 (LP)||435 (LP)||Darker Background
Longpass Barrier Filter
|Medium Excitation Band
Bandpass Barrier Filter
- UV-2E/C - The UV-2E/C filter combination is designed as a sharp cutoff filter block for ultraviolet fluorescence. Filters are the soft-coated type intended to generate a high signal/noise ratio. The narrow band pass barrier filter utilized in this combination is designed to dramatically reduce or completely eliminate green and red visible wavelengths.
- UV-1A - The UV-1A combination is designed as a filter block for ultraviolet fluorescence with a narrow excitation band pass (only the i-line of the mercury spectrum is utilized) and a narrow dichromatic mirror band pass, which minimizes autofluorescence and photobleaching.
- UV-2A - The UV-2A filter combination is designed as a standard filter block for ultraviolet fluorescence and is the brightest ultraviolet filter set in the Nikon palette.
- UV-2B - The UV-2B combination is designed as a filter block for ultraviolet fluorescence that provides a darker background and better contrast than the UV-2A filter combination.
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, Kimberly M. Vogt, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.