Included in the Nikon dual band excitation fluorescence filter portfolio are three carefully balanced combinations that each contain two bandpass emission regions capable of selectively isolating fluorescence emission through narrow bands of the blue, green, yellow, orange, and red spectral regions. This interactive tutorial explores how the variations in the excitation and emission filter spectral profiles, as well as those of the polychromatic mirrors, affect signal levels, spectral crossover, overall filter performance, and image contrast in combinations designed for dual excitation of multiply labeled fluorophores in the ultraviolet and blue or blue and green regions.
The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the ultraviolet-blue dual passband excitation filter combination (DAPI-FITC; default) spectral profile displayed on the Filter Set Spectral Profiles graph. Fluorophore absorption and emission spectral cross sections (the overlap region with filter transmission passbands) can be individually or collectively viewed by selecting the appropriate check box (Absorption or Emission) listed under the Spectral Cross Sections heading. When one or more check boxes are activated, the combined filter transmission and reflection spectra are superimposed over the absorption and emission spectra of the selected fluorophore(s) 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 black rectangular box in the lower portion 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 three filter combinations available for dual band 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 polychromatic 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 polychromatic 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 all 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.
Each of the Nikon dual band fluorescence sets is optimized for use with fluorescein isothiocyanate (FITC) in combination with either 4',6-diamidino-2-phenylindole (DAPI), tetramethylrhodamine isothiocyanate (TRITC), or Texas Red dyes. The relevant spectral regions corresponding to these fluorochrome pairs range from violet excitation and blue emission to green excitation and red emission. Other fluorescent probes with similar spectral characteristics are also suitable, singly or in pairs, for use with the Nikon dual excitation fluorescence filter combinations.
The Nikon DAPI-FITC dual band filter combination incorporates an excitation filter having one bandpass region for violet excitation (400 to 418 nanometers) coupled to a bandpass emission (barrier) filter transmitting blue fluorescence (450 to 465 nanometers) in one of its signal bands. The second excitation and emission wavelength combination for this filter set enables blue excitation in a narrow band of 478 to 495 nanometers, and detection of the resulting green fluorescence in the 510 to 555 nanometer range. The dual bands featured by this filter set provide optimal simultaneous detection of DAPI and FITC, when utilized in combination to specifically target different cellular components. The DAPI - Alexa Fluor 488 fluorochrome pair are suitable for this filter set as well, due to the close similarity of the Alexa Fluor probe and FITC spectral profiles in regards to absorption and emission characteristics.
In dual excitation filter cubes, the design of the polychromatic mirror is fundamental to the separation of the two fluorescence signals, ideally allowing their unique detection with minimal crosstalk and noise. In contrast to the standard longpass beamsplitter filters used in conventional combinations, multi-band filter sets employ modified dichromatic mirrors with multiple bandpass regions that are precisely located with respect to the multiple excitation and emission bands. The first (lower) cut-on wavelength value for the mirror is positioned a few nanometers higher than the short-wavelength excitation peak, and begins a transmission band that completely encompasses the corresponding emission peak, followed by an abrupt transmission cut-off. This design enables reflection of wavelengths in the second excitation band while simultaneously passing fluorescence emission created by the first band. At a wavelength just higher than the second excitation band, the mirror makes another sharp transition to a bandpass transmission region that corresponds to the second emission passband. The polychromatic mirror used in the DAPI-FITC filter combination has two bandpass transmission regions that are appropriately located with respect to each excitation-barrier filter complement, one having a cut-on wavelength of 435 nanometers, and another with a cut-on wavelength of 505 nanometers.
Application of thin-film interference technology in multi-band filter set fabrication techniques makes it possible to balance the fluorescence signal level of two (or more) fluorochromes and provide optimum imaging conditions. In many filter combinations, the transmission band profile of the shorter (usually ultraviolet or violet) wavelength excitation peak is reduced in order to balance the emission signal levels, as well as to minimize photobleaching and specimen damage. This is particularly important in sets designed for use with one fluorophore excited in the violet or ultraviolet spectral region, due to the high excitation efficiency of short wavelength radiation. The reduced intensity of the short-wavelength excitation peak is a notable feature in the transmission spectral profiles of filter combinations such as the Nikon DAPI-FITC set.
For the simultaneous detection of FITC (or a similar fluorophore) and a probe excited at longer wavelengths, such as TRITC, the FITC-TRITC dual band filter combination provides blue-region excitation with green emission detection, and green excitation with accompanying orange-red emission detection. In this filter set, the bandpass regions for the blue excitation-green emission fluorochrome (FITC, for example) are modified slightly from those employed in the DAPI-FITC set in order to minimize crosstalk with the green-absorbing fluorophore. The excitation filter utilized in this set has one bandpass region of 475 to 490 nanometers (blue excitation) coupled to an emission (barrier) filter passband of 503 to 530 nanometers (green emission), which is appropriate for FITC and similar fluorophores.
The second band of the FITC-TRITC filter combination enables excitation in the green wavelength range of 540 to 565 nanometers, and detection of corresponding orange-red emission in the region between 580 and 620 nanometers. Although optimized for the popular combination of FITC and TRITC fluorophores, probes with similar absorption and emission spectral characteristics can also be used. The bandpass regions of the polychromatic mirror (beamsplitter) are located in correspondence to the excitation and emission filter windows at cut-on wavelengths of 500 and 575 nanometers.
Simultaneous detection of FITC (or a similar probe) and a fluorochrome excited at wavelengths in the green and yellow-green spectral regions, with emission extending into the red, is afforded by the FITC-Texas Red filter set. Excitation bands for this set (blue and green spectral regions) range from 490 to 505 nanometers and from 560 to 580 nanometers. The corresponding dual green and red emission bands are 515 to 545 nanometers and 600 to 650 nanometers, respectively. The complementary polychromatic mirror cut-on wavelengths for the two signal transmission bands are located at 510 and 585 nanometers. Specifications for the polychromatic mirrors and filters for the three Nikon dual band filter combinations are listed in Table 1.
Table 1 - Nikon Dual Band Excitation Filter Combination Specifications
|Violet EX / Blue EM
Blue EX/ Green EM
|Blue EX / Green EM
Green EX / Orange-Red EM
|Blue EX /Green EM
Green EX / Red EM
- DAPI-FITC - The DAPI-FITC filter combination is designed to be utilized for simultaneously detecting emission from the fluorochromes DAPI and FITC, or other matched pairs of fluorescent probes that have similar spectral characteristics. Dual narrow excitation and emission bands correspond to specific regions of violet excitation with corresponding blue emission, and blue excitation coupled to green emission.
- FITC-TRITC - The FITC-TRITC filter combination is designed for simultaneous detection of emission from the fluorochromes FITC and TRITC, or probe pairs that are spectrally similar. Bandpass excitation and emission filters allow two signal channels, one corresponding to specific narrow regions of blue excitation and green emission, and the other to green excitation and orange-red emission.
- FITC-Texas Red - The FITC-Texas Red filter combination is designed with slightly different bandpass regions for blue excitation and green emission when compared to the FITC-TRITC set, allowing more efficient excitation of FITC. These shifts conform to the somewhat higher wavelength ranges for the Texas Red (green excitation; red emission) bands. The multiple bandpass regions provide optimal detection, with minimal crosstalk, for simultaneous visualization of FITC and Texas Red or of spectrally similar fluorophore pairs
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, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.