I’ve recently had a request from a group trying to do five-color imaging on our four-laser confocal system. On a widefield system, one way to do this would be to add an infrared dye to the usual combination of DAPI / FITC / Cy3 / Cy5 (or equivalents) but on a confocal system the expense of adding a new laser is considerable. So instead, I’ve recommended to them to try using a long Stokes shift dye. This is a dye whose emission wavelength is unusually far red-shifted from its excitation wavelength, and potentially allows reuse of the existing excitation lasers and emission filters on the microscope. For example, a dye that is excited at 488 nm and emits at 700 nm could be excited with the 488 nm laser and detected with the Cy5 emission filter, or one that is excited at 488 nm and emits at 610 nm could be excited with the 488 nm laser and detected with the Cy3 emission filter. Assuming there isn’t too much excitation of the Cy3- or Cy5-like dyes at 488 nm, adding either of these dyes would allow imaging a fifth channel.
A number of companies sell long Stokes shift dyes, but not much information is available about them. To help simplify choosing a long stokes dyes, I’ve summarized the information available from the manufacturers of several below. In general the available data is poor; several manufacturers don’t provide quantum yield information, and I’m suspicious that the five Dyomics dyes all provide the same quantum yield of 50,000. Structures are available for the molecules from Dyomics, Cytodiagnostics, and Promokine and these show that the sell the same dye; whether Cytodiagnostics and Promokine are reselling the Dyomics dyes or make them independently is not clear. The structures are not available for dyes from the other vendors, but the Dyomics dyes all share a common scaffold, a coumarin with additional conjugation through the 3- position.
If anyone has experience with these dyes I’d like to hear about it.
|Dye||λex||λex||EC (M-1 cm-1)||QY||Vendors|
Promokine (PromoFluor 500-LSS)
Promokine (PromoFluor 514-LSS)
Promokine (PromoFluor 488-LSS)
Promokine (PromoFluor 520-LSS)
Here are links to the vendors: