Ultraviolet and visible transmission spectral profiles for the Nikon longpass emission UV-1A filter combination are illustrated below in Figure 1. This set, intended for applications using a mercury arc-discharge lamp, is designed to significantly reduce specimen autofluorescence and photobleaching by incorporating a very narrow bandwidth (10-nanometer) ultraviolet excitation cross section with a 40-nanometer gap between the dichromatic mirror and longpass emission filter.
Ultraviolet Excitation Filter Block UV-1A Specifications
- Excitation Filter Wavelengths: 360-370 nanometers (bandpass, 365 CWL)
- Dichromatic Mirror Cut-on Wavelength: 380 nanometers (longpass, LP)
- Barrier Filter Wavelengths: 420 nanometer cut-on (longpass, LP)
The UV-1A fluorescence filter combination is designed to minimize autofluorescence partially by utilizing only the i-line region of the mercury ultraviolet emission spectrum (centered at 365 nanometers). Consequently, this filter combination is recommended primarily for microscopes equipped with a mercury arc-discharge lamp. The longpass emission (barrier) filter used in the UV-1A combination is designed to collect signals at wavelengths exceeding 420 nanometers, enabling visualization of red, orange, yellow, green, and blue emission from fluorophores excited in the ultraviolet. The UV-1A filter combination is recommended when studying the following fluorophores: 7-amino-4-methylcoumarin-3-acetic acid (AMCA), anthroyl stearate, bisbenzamide, Calcein Blue, Cascade Blue, 4',6-diamidino-2-phenylindole (DAPI), FluoroGold, and Hoechst 33342/33258, among many others. The images presented in Figure 2 demonstrate the performance of this filter combination with a variety of ultraviolet absorbing fluorescence probes targeted at different intracellular locations.
Illustrated in Figure 2(a) is the 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. The ultraviolet absorption maximum of Alexa Fluor 350 is 346 nanometers and the emission maximum is 442 nanometers. In addition, the specimen was simultaneously stained with Alexa Fluor 568 phalloidin (filamentous actin) and SYTOX Green (nuclei). Note the presence of spectral bleed-through from the red and green fluorophores.
Figure 2(b) shows emission intensity from a culture of Indian Muntjac deerskin fibroblast cells stained with Alexa Fluor 350 phalloidin, which targets actin, while Figures 2(d) and 2(e) present bovine pulmonary artery cultured cells and a mouse kidney thin section, respectively, both having the nuclei stained with DAPI.
Figure 2(c) illustrates culture of Indian Muntjac deerskin fibroblasts stained with Hoechst 33258, which specifically binds to DNA and stains the nuclei. The ultraviolet absorption maximum of Hoechst 33258 is 346 nanometers and the emission maximum is centered at 460 nanometers. Autofluorescence in plant tissues (fern sporangium) is represented in Figure 2(f) and demonstrates the wide emission spectrum of endogenous fluorophores in these specimens. Note that images captured with the UV-1A filter combination have less contrast than images from the UV-2E/C set and less emission intensity than related combinations in the UV-2A and UV-2B filter blocks.
Additional Specimen Images with the UV-1A Filter Combination
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.
Indian Muntjac Actin
Fluorescence emission intensity from a culture of Indian Muntjac deerskin fibroblasts stained with Alexa Fluor 350 phalloidin, which targets actin.
Indian Muntjac Nuclei
Fluorescence emission intensity from a culture of Indian Muntjac deerskin fibroblasts stained with Hoechst 33258, which specifically binds to DNA and stains the nuclei.
Bovine Pulmonary Artery Cell Nuclei
Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial cells stained with DAPI, which targets DNA in the cell nucleus.
Mouse Kidney Tissue
Fluorescence emission intensity from a thin section of mouse kidney stained with multiple fluorophores.
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