Aminomethylcoumarin (AMCA) conjugates absorb light maximally around 350 nm and fluoresce maximally around 450 nm (Figure 2 and Table 1). For fluorescence microscopy, AMCA can be excited with a mercury lamp and observed using a UV filter set. Since blue fluorescence is not well detected by the human eye, AMCA-conjugated secondary antibodies should be used only with the most abundant antigens in multiple-labeling experiments. Ways of improving the visibility of AMCA include dark adapting the eyes, using fluorite instead of glass objectives, avoiding mounting media that absorb UV light (such as plastic-based media), and capturing photographic images with blue-sensitive film or CCD cameras. AMCA fades rapidly in conventional epifluorescence and confocal microscopy, and therefore it should be used with mounting media containing an anti-fading agent such as n-propyl gallate.
For flow cytometry, AMCA can be excited with a mercury lamp or with a water-cooled argon ion laser which emits some lines in the UV. AMCA has been used mostly for multiple labeling since there is minimal fluorescence overlap with green-fluorescing dyes and little or no overlap with longer wavelength-emitting fluorophores. Applications for multiple labeling with this probe include both immunofluorescence microscopy and flow cytometry. AMCA is not suggested for single labeling in one-photon microscopy because of its relatively weak signal and rapid fading. However, AMCA has been found to be a bright and photostable dye for 2-photon microscopy. In one-photon microscopy DyLight 405 is a better choice for multiple labeling.
|Excitation Peak||Emission Peak (nm)|