Written and maintained by CASRAI Editorial Board
Last updated
Short answer. Cy5 is excited at a maximum of about 650 nm and emits at a maximum of about 670 nm, in the far-red. Using the same vendor’s values, Cy3 is 550/570 nm, FITC is 492/520 nm and DAPI (bound to DNA) is about 350/470 nm. Peak positions shift a few nanometres with the vendor, the conjugate and the environment, so treat any single pair as approximate.
Cy5, Cy3, FITC and DAPI at a glance
| Dye | Excitation max (nm) | Emission max (nm) | Stokes shift (nm)* | Source |
|---|---|---|---|---|
| FITC (fluorescein) | 492 | 520 | 28 | Jackson ImmunoResearch |
| Cy3 (indocarbocyanine) | 550 | 570 | 20 | Jackson ImmunoResearch |
| Cy5 (indodicarbocyanine) | 650 | 670 | 20 | Jackson ImmunoResearch |
| DAPI (bound to DNA) | about 350 | about 470 | about 120 | Thermo Fisher |
*Stokes shift here is simple arithmetic: emission maximum minus excitation maximum, calculated by CASRAI from the values in the table. The DAPI values are read from the vendor’s spectrum graphic, so they are approximate.
Cy5 excitation and emission
Jackson ImmunoResearch lists Cy5 with an excitation maximum of 650 nm and an emission maximum of 670 nm. For a close comparison with a spectrally similar dye, see Alexa Fluor 647, which this site lists at 650/668 nm.
The same vendor page reports how well common laser lines excite Cy5: the 647 nm line of a krypton or argon-krypton laser excites it to about 98% of its maximum, while the 633 nm helium-neon line reaches about 63%. The page also reports that Cy5 is brighter than other far-red dyes, including Alexa Fluor 647, in non-polar plastic mounting media. These are vendor statements about that vendor’s conjugates.
Cy3 excitation and emission
Cy3 is excited at about 550 nm and emits at about 570 nm (Jackson ImmunoResearch). Other vendor tables round the same dye to slightly different numbers, for example 554/568 nm, which is the normal spread between sources rather than a conflict. In the table above their emission maxima are 100 nm apart.
FITC excitation and emission
Jackson ImmunoResearch gives FITC (fluorescein) an excitation maximum of 492 nm and an emission maximum of 520 nm. Other references quote 498/517 nm, including the fluorophore listing in the fluorophores guide. Both describe the same dye; the difference is a few nanometres of measurement conditions. For FITC specifics see the FITC guide.
DAPI excitation and emission
Thermo Fisher’s DAPI page shows DNA-bound DAPI excited in the ultraviolet near 350 nm and emitting in the blue near 470 nm, and notes that in flow cytometry it is excited by the violet (405 nm) laser line. DAPI’s Stokes shift is far larger than those of the other three dyes, which makes it easy to separate from them. See DAPI in flow cytometry for its use as a DNA stain.
Matching dyes to laser lines and filters
Excitation peak and laser line rarely coincide exactly, so the useful question is how much of the maximum a given line delivers. The only figures sourced here are for Cy5 (98% at 647 nm, 63% at 633 nm) and for DAPI, which is used with a 405 nm line. For the other dyes, use the vendor’s spectrum for your exact conjugate or an interactive viewer such as FPbase.
Why published numbers differ
- Vendor and method. FITC appears as 492/520 nm in one source and 498/517 nm in another.
- Peak versus band. A maximum is a single wavelength; excitation and emission are broad curves, so neighbouring dyes overlap.
Common misunderstandings
- The filter must sit exactly on the peak. Filters and lasers select a band, and efficiency at that band matters more than the peak value.
- Cy5 and Alexa Fluor 647 are interchangeable. Their spectra are close, but the vendor reports differing brightness in some mounting media.
- A table value is a property of the dye alone. It also reflects the conjugate and the surroundings.
FAQ
What are the excitation and emission wavelengths of Cy5?
About 650 nm excitation and 670 nm emission maximum, per Jackson ImmunoResearch.
What are the Cy3 excitation and emission wavelengths?
About 550 nm and 570 nm (Jackson ImmunoResearch); other tables list 554/568 nm.
What are the FITC excitation and emission wavelengths?
492 nm and 520 nm in Jackson ImmunoResearch’s table; 498/517 nm in other references.
What are the DAPI excitation and emission wavelengths?
About 350 nm and 470 nm when bound to DNA; in flow cytometry it is excited with a 405 nm violet laser (Thermo Fisher).
Which laser excites Cy5?
The 647 nm krypton or argon-krypton line excites it to about 98% of maximum and the 633 nm helium-neon line to about 63% (Jackson ImmunoResearch).
Sources
- Jackson ImmunoResearch, Cy5 conjugate page and fluorophores page, read 2026-11-08.
- Thermo Fisher Scientific, DAPI stain page, read 2026-11-08.
