Long Stokes Shift Dyes

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.

DyeλexλexEC (M-1 cm-1)QYVendors
Dy480XL50063050000Dyomics
Cytodiagnostics (Cyto500LSS)
Promokine (PromoFluor 500-LSS)
Dy481XL51565050000Dyomics
Cytodiagnostics (Cyto514LSS)
Promokine (PromoFluor 514-LSS)
Dy485XL48556050000Dyomics
Promokine (PromoFluor 488-LSS)
Dy510XL50959050000Dyomics
Cytodiagnostics (Cyto520LSS)
Promokine (PromoFluor 520-LSS)
Dy521XL52366850000Dyomics
STAR440SX437515227000.68Aberrior
STAR470SX477627227000.80Aberrior
STAR470SXP472623290000.12Aberrior
Atto430LS433547320000.65Atto-Tec
Atto490LS496661400000.30Atto-Tec
Chromeo49449462855000Active Motif

Here are links to the vendors:

4 thoughts on “Long Stokes Shift Dyes

  1. Hi Kurt,

    I’ve used Dyomics 485XL, and 521XL (or 520XL) in oligonucleotide FISH to detect individual transcripts. This works, but my impression is that the dyes are not as bright as more conventional dyes (eg. Cy3 / Cy5). That was using epi-fluorescent wide field microscopy (Deltavision) as the signal isn’t really strong enough to see by confocal (not just for the mega-stokes dyes, but true for other dyes as well). But, I don’t feel very confident to comment on the absolute brightness of the dyes as such as our filters were not very well suited to the task. Incidentally I used the dyes for exactly the same reason you’re alluding to: to increase the number of channels. I did try some experiments with DAPI, Alexa 488, Dy 485XL, Cy3, Dy521XL, and Cy5. If your signal is strong enough, it shouldn’t be too difficult to distinguish these from each other, esp if you can either choose your ex / em windows freely, or use linear unmixing. I had some trouble as I was also using combinatorially detected transcripts which appeared to give rise to a FRET signal that was difficult to distinguish from the Megastoke dyes’ signals. That should be possible to extract by post-processing, but combined with some bleeding through of the dyes and other difficulties it just seemed a bit too much trouble at the time.

    cheers,

    Martin

  2. Dyes with larger stokes shift inherently have lower brightness, ass their emission and absorbtion profiles are broad. The emission energy is thus spread out over a larger range of wavelengths, lowering intensity.

    • I don’t think there is any intrinsic reason that long Stokes shift dyes can’t have narrow excitation or emission linewidths. In fact, some inorganic systems, such as lanthanide phosphors have very large Stokes shifts and very narrow emission linewidths.

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