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The excitation wavelength of Cy2 coupling group is 492 nm, and the emission is green visible light with the wavelength of 510 nm. Cy2 and FITC use the same filter. Because Cy2 is more stable in light than FITC. The use of blocking agent containing phosphorylated phenylenediamine should be avoided, because the reaction of this anti quenching agent with Cy2 will lead to weak fluorescence and diffusion after the storage of the dye sheet. Cy3 and Cy5 are brighter, more stable and weaker than other fluorescent probes. The maximum wavelength of Cy3 coupling group excitation is 550 nm, and the maximum emission is 570 nm. Because the wavelengths of excitation light and emission light are very close to TRITC, filter plates like TRITC can be used in fluorescence microscope.

Cy3 can be excited at 50% of the light intensity in argon lamp (514nm or 528nm), and about 75% in helium neon lamp (543nm) or mercury lamp (546nm). Cy3 can be double labeled with fluorescein. Cy3 and Cy5 can also be used as multiple markers in confocal microscopy. The maximum excitation wavelength of Cy5 coupling group is 650nm, and the maximum luminescence wavelength is 670nm. They can be excited 98% fluorescence in the krypton argon lamp (647nm), 63% in the helium neon lamp (633nm). Cy5 can be used in multiple labeling experiments with many other fluorescent groups. Because the maximum emission wavelength of Cy5 is 670 nm, it is difficult to observe Cy5 with naked eyes, and it can not be excited with mercury lamp. The confocal microscope with proper excitation and far-infrared detector is usually used to observe Cy5. Anti quenching agent should be added to the water phase sealing agent. Cy5 is not recommended for traditional surface fluorescence microscopy.