Green Excitation: G-1A (Longpass Emission)

The Nikon G-1A filter set is designed with a narrow passband (10 nanometers) excitation filter, centered at 546 nanometers (a wavelength referred to as the e-line of the mercury arc-discharge lamp). The combination includes a dichromatic mirror with a cut-on wavelength of 575 nanometers, and a longpass emission or barrier filter (having a 580-nanometer cut-on wavelength), thus enabling transmission of yellow, orange, and red emission from fluorophores absorbing in the central portion of the green spectral region. Ultraviolet, visible, and near-infrared transmission spectral profiles for the Nikon G-1A filter combination are illustrated below in Figure 1. Note the sharp profile exhibited by the excitation filter and the close proximity of the cut-on wavelength values for the dichromatic mirror and emission filter.

Figure 1 - G-1A (Narrow Band Green Excitation)

Green Excitation Filter Block G-1A Specifications

  • Excitation Filter Wavelengths: 541-551 nanometers (bandpass, 546 CWL)
  • Dichromatic Mirror Cut-on Wavelength: 575 nanometers (longpass, LP)
  • Barrier Filter Wavelengths: 580 nanometer cut-on (longpass, LP)

The G-1A filter combination is equipped with a narrow bandpass (10 nanometers) excitation filter, which minimizes autofluorescence and photobleaching. The center wavelength of the filter is positioned to correspond to the 546-nanometer emission line of a mercury arc-discharge illumination source, which is the recommended application for using the G-1A set. The longpass emission (barrier) filter employed by this set is designed to collect fluorescence signals at wavelengths exceeding 580 nanometers, permitting visualization of yellow, orange, and red emission. The G-1A filter combination is recommended when investigating the following fluorophores: Alexa Fluors (532, 546, 555, 568, and 594), dichlorodimethoxyfluorescein (JOE), Alizarin Red, BODIPY probes, Calcium Orange, Cy3, Cy3.1.8, dioctadecyl tetramethylindocarbocyanine (DiI), ethidium bromide, FluoroRuby, hexachlorofluorescein (HEX), LDS 751-DNA, MitoTracker Orange and Red, R and B-phycoerythrin, POPO-3, PO-PRO-3, propidium iodide (PI), Pyronin B, RedoxSensor Red CC-1, RH probes (237, 414, 421, 795), many rhodamine derivatives, Sevron Brilliant Red, SYTO derivatives, SYTOX Orange, and Xylene Orange. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of green-absorbing fluorescence probes targeted at different intracellular locations.

Figure 2 - Nikon G-1A Green Excitation Longpass Emission Filter Set

Figure 2(a) illustrates fluorescence emission intensity from a culture of HeLa cells that were immunofluorescently labeled with primary anti-histone (pan) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 546. The absorption maximum of Alexa Fluor 546 is 556 nanometers and the emission maximum occurs at 573 nanometers (in the yellow region of the visible light spectrum). The primary antibody, produced from purified nuclear fractions of HeLa cells, binds specifically to an antigen determinant that is present on all five histone proteins (H1, H2A, H2B, H3, and H4). Note the strong yellow-orange fluorescence intensity generated by the sister chromatids in the nucleus.

A culture of bovine pulmonary artery endothelial cells stained with MitoTracker Red CMXRos, which targets the intracellular mitochondrial network, is shown in Figure 2(b). The absorption maximum of MitoTracker Red CMXRos is 579 nanometers and the emission maximum occurs at 599 nanometers. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; blue emission) and BODIPY FL-phallacidin (targeting actin; green emission). Note the absence of signal from the blue (DAPI) and green (BODIPY) fluorophores, but the bright yellow-orange fluorescence exhibited by the tubular mitochondria.

Figure 2(c) presents a thin section of mouse intestine stained with Alexa Fluor 350 wheat germ agglutinin, a blue fluorescent lectin that is specific to the mucus of goblet cells. In addition, the specimen was simultaneously stained with Alexa Fluor 568 phalloidin (filamentous actin; 600 nanometer emission) and SYTOX Green (nuclei; 504 nanometer excitation and 523 nanometer emission). Note the low level of background noise in comparison to the other Nikon longpass filter combinations (G-2A and G-2B), and the significant amount of yellow-red signal arising from Alexa Fluor 568 that appears in the image.

Bovine pulmonary artery endothelial cells that were immunofluorescently labeled with primary anti-cytokeratin mouse monoclonal antibodies followed by goat anti-mouse Fabfragments conjugated to Alexa Fluor 546 are illustrated in Figure 2(d). Keratins are a class of water-insoluble proteins that polymerize to form monofilaments, a class of intermediate filaments that forms part of the cytoskeletal complex in many epithelial tissues. Because the composition of keratin filaments varies widely with species and cell type, the microfilament network is not always clearly delineated when labeled with a specific antibody, as occurs with the specimen presented in Figure 2(d).

A culture of Indian Muntjac deerskin fibroblast cells stained with Alexa Fluor 568 conjugated to phalloidin, which binds to the intracellular filamentous actin network, is presented in Figure 2(e). In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; blue emission) and MitoTracker Green FM (targeting mitochondria; green emission). Note the absence of signal from the green (MitoTracker) and blue (DAPI) fluorophores, which are not efficiently excited at green wavelengths, but the obvious presence of bright orange-red fluorescence exhibited by the actin filaments.

Autofluorescence emission from a thin section of caster bean (Ricinus communis) tissue is demonstrated in Figure 2(f). Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including chlorophyll, carotene, and xanthophyll. In the green excitation region, chlorophyll has an absorption band with a low extinction coefficient, but still produces a detectable level of fluorescence at emission wavelengths of 550 nanometers and above (green, yellow, orange, and red). For the caster bean thin section illustrated above, note the presence of autofluorescence emission intensity in the orange and red spectral regions, but the absence of intensity at lower (green and blue) wavelengths.

Additional Specimen Images with the G-1A Filter Combination

HeLa Cell Histones

Fluorescence emission intensity from a culture of HeLa cells that were immunofluorescently labeled with primary anti-histone (pan) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 546.

Bovine Pulmonary Artery Cell Mitochondria

Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial cells stained with MitoTracker Red CMXRos, which targets the intracellular mitochondrial network.

Mouse Intestine Thin Section

Fluorescence emission intensity from a thin section of mouse intestine stained with Alexa Fluor 350 wheat germ agglutinin, a blue fluorescent lectin that is specific to the mucus of goblet cells.

Bovine Pulmonary Artery Keratin Network

Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial cells that were immunofluorescently labeled with primary anti-cytokeratin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 546.

Indian Muntjac Cell Actin Cytoskeleton

Fluorescence emission intensity from a culture of Indian Muntjac deerskin fibroblast cells stained with Alexa Fluor 568 conjugated to phalloidin, which binds to the intracellular filamentous actin network.

Caster Bean Thin Section

Autofluorescence emission intensity from a thin section of caster bean (Ricinus communis) tissue. Endogenous autofluorescence in plant tissues arises from a variety of biomolecules, including chlorophyll, carotene, and xanthophyll.

Pig Kidney Cell Mitochondria

Examine the fluorescence emission from a culture of normal pig kidney (LLC-PK1 line) epithelial cells that were transfected with a pDsRed-Mitochondria plasmid subcellular localization vector. Cells were transiently transfected and cultured in nutrient medium for a minimum of 24 hours before recording images. The absorption maximum of the DsRed-Mitochondria fusion gene product is 558 nanometers, while the corresponding emission maximum occurs at 583 nanometers, values that closely match the spectral profiles of the Nikon G-1A filter combination.

Contributing Authors

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

John D. GriffinNathan S. ClaxtonMatthew J. Parry-HillThomas J. FellersKimberly M. VogtIan D. JohnsonShannon H. NeavesOmar AlvaradoLionel Parsons, Jr.Michael A. SoddersRichard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Green Excitation: G-1A (Longpass Emission)

Introduction