Included in the Nikon green excitation fluorescence filter portfolio are six carefully balanced combinations that contain either bandpass or longpass emission (barrier) filters capable of selectively isolating fluorescence emission through either a narrow or wide band of the yellow, 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 green region.
The tutorial initializes with a randomly selected fluorescent specimen appearing in the Specimen Image window and the bandpass emission (Cy3; default) green 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 green 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 green 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 green fluorescence filter combinations encompass an excitation wavelength range between 510 and 560 nanometers with passband width profiles of 10, 25, 30, and 50 nanometers (including narrow, medium, and wide excitation bands). Three of the combinations employ the same dichromatic mirror (565 nanometers), while the other three have mirrors with higher wavelength cut-on values (570 or 575 nanometers). Two of the six Nikon green excitation filter sets incorporate bandpass barrier filters having bandwidths of 60 and 75 nanometers.
Two of the Nikon green excitation filter sets incorporate bandpass emission (barrier) filters intended to reduce or eliminate interference from fluorophores emitting in the red or near-infrared spectral regions in specimens labeled with multiple probes. The G-2E/C set combines a narrow excitation band (25 nanometers) with a 60-nanometer emission bandpass. The Cy3 combination (a member of the Nikon HYQ filter group) incorporates similar components, but with wider-bandpass excitation and emission filters (30 and 75 nanometers, respectively), and with a dichromatic mirror cut-on wavelength of 570 nanometers compared to a 565-nanometer value for the G-2E/C set. Both of the sets utilizing bandpass emission filters are suitable for use with tetramethylrhodamine isothiocyanate (TRITC) and Cy3, having bandpass ranges of 590-650 nanometers for the G-2E/C set and 573-648 nanometers for the Cy3 combination. Images produced using the Cy3 set have a more yellow visual appearance, reflecting the extension of the emission bandpass to lower wavelengths, and the images are somewhat brighter due to the greater excitation and emission energy resulting from the wider bandpass values.
The G-1A and G-1B filter combinations have very similar optical components, and can be considered essentially interchangeable. Their primary application is with mercury arc-discharge lamps for selection of the 546-nanometer green emission line. Both are equipped with narrow 10-nanometer passband excitation filters (541-551 nanometers) to minimize autofluorescence, but differ somewhat in the cut-on wavelengths of their dichromatic mirrors and barrier (emission) filters. The G-1A set has a dichromatic mirror cut-on wavelength of 575 nanometers, coupled with a 580-nanometer (cut-on) emission filter. The G-1B filter set employs a lower wavelength dichromatic mirror (565 nanometers) in combination with a higher wavelength emission filter (590 nanometers). The increased wavelength separation between the mirror and emission filter wavelength values for the G-1B set compared to theG-1A can result in significant image differences with some fluorochromes. In addition, the higher emission filter cut-on excludes yellow wavelengths and results in a more reddish image appearance with the G-1B filter combination.
Considered the standard green-excitation filter set, the G-2A combination incorporates an excitation filter with a wide 50-nanometer passband (510-560 nanometers) that covers most of the green spectral region. The longpass barrier filter passes emission from a large range of fluorochromes that emit at wavelengths above the 590-nanometer cut-on (orange through near-infrared). The dichromatic mirror employed in the G-2A set has a cut-on wavelength of 565 nanometers. A similar filter set, the G-2B combination utilizes the same excitation filter (50-nanometer bandpass) as the G-2A, but has a 10-nanometer longer wavelength dichromatic mirror cut-on (575 nanometers), coupled with a longpass barrier filter that is shifted toward the red spectral region by 20 nanometers, to 610 nanometers. Images produced with the G-2B set have darker backgrounds than those from the G-2A, and also appear more reddish due to the increase in emission cut-on wavelength, which excludes more yellow-orange emission.
Table 1 - Nikon Green Excitation Filter Combination Specifications
|575 (LP)||580 (LP)||Narrow Excitation Band
Longpass Barrier Filter
|565 (LP)||590 (LP)||Narrow Excitation Band
Longpass Barrier Filter
|565 (LP)||590 (LP)||Standard G Set
Longpass Barrier Filter
|575 (LP)||610 (LP)||Darker Background
Longpass Barrier Filter
|Narrow Excitation Band
Bandpass Barrier Filter
|Medium Excitation Band
Bandpass Barrier Filter
- G-1A - The G-1A filter combination for green wavelength excitation is designed with a narrow excitation bandpass in order to reduce autofluorescence and minimize specimen radiation exposure. The longpass barrier (emission) filter is capable of transmitting signals from yellow, orange, and red-emitting fluorophores that have significant absorption in the central green wavelength region.
- G-1B - The G-1B filter combination has a similar component profile to the G-1Aset, incorporating the narrow excitation bandpass to reduce autofluorescence and specimen damage, but with different dichromatic mirror and emission filter cut-on wavelengths. The shift of the emission filter cut-on to longer wavelengths produces images that appear redder due to the increased blocking of yellow emission.
- G-2A - The G-2A filter combination is configured as the standard green filter block, with a wide excitation passband for application to a large number of fluorophores activated by green wavelengths. The longpass emission filter permits detection of all fluorescence wavelengths longer than the yellow spectral region.
- G-2B - The G-2B filter combination is designed with the same excitation characteristics as the G-2A filter block. However, with higher dichromatic mirror and emission filter cut-on wavelengths, the G-2B set produces images having darker backgrounds and a color shift toward the red.
- G-2E/C - The G-2E/C filter combination is designed for optimal performance with a number of popular fluorescent probes that are used in multiple labeling experiments. A narrow excitation bandpass provides selective excitation, while the bandpass emission filter reduces interference from fluorophores emitting in the red and near-infrared.
- Cy3 (HYQ) - The Cy3 green-excitation filter combination is similar in configuration to the G-2E/C set. However, with a wider excitation passband and broadening of the emission bandpass to encompass lower wavelengths, the images produced by the Cy3 filter block are somewhat brighter and reflect the detection of more yellow spectral region emission.
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, andMichael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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