Fluorescence Filter Combinations

Red Excitation Filter Sets

The Nikon red excitation fluorescence filter combination category comprises a single carefully balanced set that incorporates a bandpass emission (barrier) filter capable of selectively isolating fluorescence emission occurring in the deep red to near-infrared spectral regions. The barrier filter passband is centered at 700 nanometers, with a bandwidth of 75 nanometers (663 to 738 nanometers). A wide 60-nanometer excitation band encompassing orange and red wavelengths covers the spectral range of 590 to 650 nanometers. The Cy5 HYQ combination employs a longpass dichromatic mirror having a cut-on wavelength of 660 nanometers, which is 10 nanometers higher than the excitation bandpass cut-off.

Performance of the Cy5 HYQ red excitation filter set can be judged by comparing images of fluorophores designed for excitation in the orange-red and emission in the deep red wavelength regions, as illustrated in Figure 1. The specimens are individual adherent monolayer cultures of Indian Muntjac deerskin fibroblast cells labeled with a variety of probes. Figure 1(a) illustrates the Muntjac cells with MitoTracker Deep Red 633, which has an absorption maximum of 644 nanometers and an emission maximum of 665 nanometers. Cellular nuclei from a different culture were labeled with the nucleic acid stain TO-PRO-3 (Figure 1(b); absorbance at 642 nanometers and emission at 661 nanometers). The filamentous actin cytoskeletal network is presented in Figure 1(c), which features the far red probe Alexa Fluor 633 conjugated to phalloidin. Alexa Fluor 633 has an absorption maximum at 621 nanometers and a corresponding fluorescence emission peak centered at 639 nanometers. All of the images in Figure 1 were captured in 12-bit grayscale mode, but subsequently pseudocolored by converting to the RGB color palette and setting the green and blue channels to zero (to produce a red image).

Because most of the emission resulting from excitation by red wavelengths occurs in the deep red to near-infrared spectral region, the fluorescence signal is, in general, only faintly visible or is invisible to the human eye. Consequently, detection using photomultiplier tubes or imaging by CCD cameras without an infrared filter is required for full utilization of the generated fluorescence signal. Although autofluorescence is typically a less significant problem in the far-red and near-infrared regions than at shorter wavelengths, the bandpass emission filter of the Cy5 HYQ set restricts emission at wavelengths longer than approximately 740 nanometers.

The Cy5 HYQ filter combination for red wavelength excitation shares the design characteristic of other filter sets in the Nikon HYQ series of wider passbands with sharp transitions for both the excitation and emission filters. The wide passbands provide increased energy for generating and acquiring fluorescence signal, and with the excitation window spanning wavelengths from yellow-orange into the red, the Cy5 HYQ set can be utilized with a considerable range of fluorochromes, in addition to Cy5, for which it is primarily designed. The bandpass barrier filter transmits signal ranging from visible far-red wavelengths into the invisible near-infrared spectral region.

Nikon Red Excitation Filter Combination Specifications
Filter Set
Description
Excitation
Filter (nm)
Dichromatic
Mirror (nm)
Barrier
Filter (nm)
Remarks
CY5 HYQ 620/60
(590-650)
660 (LP) 700/75
(663-738)
Wide Excitation Band
Bandpass Barrier Filter
Table 1
  • Cy5 HYQ - The Cy5 HYQ filter combination for red-wavelength excitation is designed to provide high-energy excitation of the fluorophore Cy5, although the wide excitation range (yellow-orange to red) allows the set to be utilized with a variety of fluorochromes. Sharp filter transitions between reflection and transmission regions enable maintaining signal separation in combination with the high brightness level resulting from the use of wide passbands. The bandpass barrier filter transmits signal from the far-red into the near-infrared, a wavelength range that is most efficiently detected using electronic detectors, such as photomultipliers and CCD cameras without an infrared filter.

A wide array of fluorophores has been developed for investigations using excitation wavelengths spanning the orange to red wavelength regions. Catalogued in Table 2 are many of the popular dyes and fluorescent probes that can be visualized with the Nikon Cy5 HYQ red excitation filter combination. The localized environment significantly influences fluorophore absorption and emission spectra maximum (peak) wavelengths, so the values presented in Table 2 may vary with experimental conditions. This list is intended to serve only as a guide for filter and fluorophore selection and should not be considered a comprehensive or exhaustive compilation. Many of the fluorescent probes included in the table are proprietary and have been developed to minimize photobleaching while ensuring a maximum overlap between the fluorochrome absorption and emission spectra and common fluorescence filter combinations. Note that due to broad absorption and emission bands, several of the fluorescent probes listed in Table 2 are also suitable for use with filter combinations having other excitation wavelength passbands, particularly in the yellow spectral region.

Fluorochromes with Red Excitation Spectral Profiles
Fluorochrome Excitation
Wavelength
(Nanometers)
Emission
Wavelength
(Nanometers)
Recommended
Filter Set(s)
Alexa Fluor 610 612 628 CY5
Alexa Fluor 633 621 639 CY5
Alexa Fluor 647 649 666 CY5
Alexa Fluor 660 663 691 CY5
Alexa Fluor 680 679 702 CY5
APC (Allophycocyanin) 630 660 CY5
APC-Cy7 625-650 755 CY5
BODIPY 650/665 (Difluoroboradiazaindacene) 646 660 CY5
BODIPY 665/676 665 676 CY5
Carboxynaphthofluorescein 598 (High pH)
512 (Low pH)
668 (High pH)
563 (Low pH)
CY5
Cy5 649 670 CY5
Cy5.1 8 649 666 CY5
Cy5.5 675 695 CY5
DiD (Indodicarbocyanine) 644 665 CY5
DiIC1(5) 638 658 CY5
DiSC3 (Thiadicarbocyanine) 651 675 CY5
MitoFluor Red 594 598 663 CY5
MitoTracker Deep Red 633 644 665 CY5
MRFP-1 Naphthofluorescein 602 672 CY5
NeuroTrace 640/660 640 660 CY5
Nile Blue 631 660 CY5
QDot 655 450-650 655 CY5
QDot 705 450-650 705 CY5
SpectrumFRed 655 675 CY5
SYTO 60 652 678 CY5
SYTO 62 652 676 CY5
SYTO 63 657 673 CY5
TOTO-3, TO-PRO-3 642 661 CY5
Ultralite 656 678 CY5
Table 2

Although the Cy5 HYQ filter combination described in this section adequately serves for a majority of the investigations with red excitation wavelengths, several additional specialized filter sets are available from the aftermarket manufacturers. Some of these combinations incorporate orange or red-band excitation with non-standard dichromatic mirrors and barrier filters, which may be chosen to match particular detector characteristics. In other variations, a narrow excitation bandpass may be applied to selectively isolate specific emission lines for sources such as lasers or mercury arc-discharge lamps, which occur in discrete spectral regions. If no strong excitation line for the fluorochrome of interest exists, a wider excitation filter passband may be required in order to collect sufficient signal, and a similar approach is necessary when broadband low-intensity illumination sources, such as tungsten-halogen lamps, must be used.

One minor variation of the Nikon Cy5 HYQ filter set, available from specialty manufacturers, is a longpass version that utilizes the same excitation filter and dichromatic mirror, but incorporates a longpass emission filter for use in investigations in which detection of the longer wavelengths is desired. Other specialized combinations are configured with narrower excitation and/or emission bandpass windows for applications in which signal discrimination is more important than a high signal level. Some of these sets employ very narrow passbands designed for specific fluorophores that have unusually small Stokes shift values. A number of the fluorochromes in the red excitation category are efficiently excited by the 633-nanometer line of the helium-neon laser, and special filter sets are available that have extremely narrow excitation passbands of only a few nanometers width, centered on that wavelength.


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Contributing Authors

Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.

John D. Griffin, Nathan S. Claxton, Matthew J. Parry-Hill, Thomas J. Fellers, Kimberly M. Vogt, Ian D. Johnson, Shannon H. Neaves, Omar Alvarado, Lionel Parsons, Jr., Michael A. Sodders, Richard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.