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:
Has anyone else encountered this specific bleed-through with Opal 690?
Are you successfully using Opal 690 in your multiplex panels?
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!
You’re not alone! We’ve had this issue several times with a similar setup to yours, usually noticing bleed-through into the FITC channel for RNAScope FISH. We’ve replicated the problem on several instruments and with different targets. We’ve found that we get the best results with Opal 690 when it is paired with a low expressor with a high expressor in the FITC channel.
I don’t have a single solution, but what we’ve found is that the bleed-through is sometimes weak enough (and the signal in other channels strong enough) that the MaxEntropy thresholding algorithm can pick apart the signal from the background. It’s not at all an ideal solution, though. Our lab is exploring other options for TSA labelling, and I’ll report back here if we find an improvement in bleed-through from the far-red to the other channels.
I’ve asked around and it seems Opal690 is problematic with bleed through in NIR channel. Same goes for Cy5.5 as both have a long ‘emission tail’ overlapping with Opal 780 and Opal780 emission yield is relative low. If possible substitute for Cy5 which more blue shifted and has much shorter emission tail. The Cy5 emission is not picked up with a narrow band emission filter for Opal620 detection. You can check this best with tools like FPbase Fluorescence Spectra Viewer
Thanks for your reply. I know the Opal 690 has a tail in the NiR, but it shouldn’t get excited with our NiR laser (730). I know about FP base and, according to that, there is only a 2 percentage excitation of Opal 690 with that laser that can’t justified the amount of signal we see in the NiR bleedthrough. We do everything sequencial. They can’t changed the fluorophores as this is part of a kit for RNAscope.
With FPbase.org and the specific emission filter you use in your detection system you can further calculate the theoretical bleed through by normalizing to the excitation wavelength. For example, a Chroma 750LP filter and 730nm excitation shows a detection efficiency of 17.4%/ 94.3% for Opal 690 / Opal 780, that is a 1:5 ratio! If you have abundant epitopes labeled with Opal 690 you will surely see this bleeding through, even at a relatively poor 2% excitation efficiency.
An essential missing factor is the extinction coefficient and quantum yield of the Opal dyes, so the calculation in this case may not be very meaningful. These values are not published but perhaps Opal 690 has a high QY making things problematic.
Please see paper that substituted to TSA-Cyanine 5: Rao et al., DOI:10.1016/j.xpro.2024.103388 “Protocol for detection of glial complement expression in relation to amyloid plaques in mouse brain with combined FITC and IHC.