Included in the Nikon yellow excitation fluorescence filter portfolio are two balanced combinations that contain exclusively bandpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide band of the orange, 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 yellow region.
The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the narrow bandpass emission (Y-2E/C; default) yellow 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 yellow 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 two filter combinations available for yellow 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 narrow and wide passband barrier (emission) filter sets.
The Nikon yellow excitation fluorescence filter combination group includes two carefully balanced sets that incorporate single bandpass (barrier) filters capable of selectively isolating fluorescence emission within the orange to red spectral region. These complementary filter combinations encompass an excitation wavelength range of 532 to 587 nanometers with passband widths of 40 and 55 nanometers. Both combinations employ the same longpass dichromatic mirror (595-nanometer cut-on). The two Nikon yellow excitation filter sets incorporate bandpass barrier filters having bandwidths of 60 and 75 nanometers.
Both of the Nikon yellow excitation filter sets incorporate bandpass emission (barrier) filters intended to reduce or eliminate interference from fluorophores emitting in the far red or near-infrared spectral regions, especially for specimens labeled with multiple probes. The two filter combinations cover the same general spectral bands, and are interchangeable in most applications. The Y-2E/C set combines a 40-nanometer excitation band (540-580 nanometers) with an emission bandpass of 60 nanometers (600-660 nanometers), which blocks much of the deep red to near-infrared spectral region.
The Texas Red HYQ combination (a member of the Nikon high-performance HYQ filter group) has similar optical specifications, although with wider excitation and emission bandpass regions (55 and 75 nanometers, respectively). The excitation passband of this set has a wavelength range from 532 to 587 nanometers, and is combined with a 75-nanometer emission passband (608 to 683 nanometers). The increased passband widths transmit greater excitation and emission energy, leading to brighter images, as well as extension of emission detection further into the deep red region. Each complementary filter combination utilizes the same dichromatic mirror, having a cut-on wavelength of 595 nanometers. Both of the sets provide performance specifically optimized for use with Texas Red, Alexa Fluor, and Cy3.5 fluorochromes, although they are suitable for use with a range of fluorophores excited by yellow wavelengths. Because of the wider bandpass windows, images produced using theTexas Red HYQ set reflect the detection of more red signal, and are somewhat brighter when compared to those acquired with the Y-2E/C combination.
Table 1 - Nikon Yellow Excitation Filter Combination Specifications
|Filter Set Description
|Excitation Filter (nm)
|Dichromatic Mirror (nm)
|Barrier Filter (nm)
|Medium Excitation Band Bandpass Barrier Filter
|Texas Red HYQ
|Wide Excitation Band Bandpass Barrier Filter
- Y-2E/C - The Y-2E/C filter combination for yellow wavelength excitation is designed as a specialty filter set for use with Texas Red and Cy3.5, although with an excitation bandwidth spanning most of the yellow wavelengths (overlapping a portion of the green region), the set can be utilized with a range of other fluorochromes. The bandpass barrier filter transmits signal from orange and orange-red emitting fluorophores, while eliminating most red and near-infrared emission.
- Texas Red HYQ - The Texas Red HYQ filter combination has a similar component profile to the Y-2E/C set, but with wider excitation and emission bandpass windows, which allow it to transmit more energy and produce comparatively brighter images. Resulting images also reflect additional red fluorescence signal due to the extension of the emission filter passband to longer wavelengths in the red spectral region.
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