Opal 690 Bleed-Through - Has Anyone Else Seen This?

Probes & Prep
1

Hello everyone,

I’m hoping to get some help on an issue we’re having with Opal 690 in multiplex immunofluorescence.

A user of ours is using Opal 690 for a multiplex panel on a confocal system. Even though we are using sequential acquisition to minimize crosstalk, we are observing significant bleed-through of the Opal 690 signal into the Near-Infrared (NIR) channel (730 laser, 740-850 filter).

Single-plex controls have been done(staining a sample with only Opal 690), and the issue is confirmed: the Opal 690 fluorophore is clearly visible in the NIR channel, with some weaker, but still present, signal in other channels as well.

This was a new one for me, but the user then pointed me to this paper: Cell Press: STAR Protocols (figure 4)

My questions are:

  1. Has anyone else encountered this specific bleed-through with Opal 690?

  2. Are you successfully using Opal 690 in your multiplex panels?

  3. Does this align with the general understanding of Opal 690’s emission profile being broader than the standard spectra sheets might suggest?

Any insights, shared experiences, or suggested workarounds would be greatly appreciated!

Thanks in advance.