Opal probes with confocal and spectral unmixing

[Cammer, Michael]

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1. We were unable to find spectral info for emission of the new probes so we did a quick scan and have posted the results here:  http://microscopynotes.com/880/opals/

2.  If anyone has successfully done spectral unmixing  with these probes (six Opals plus dapi) using a Zeiss 880, we would be very interested in tips on how to do this.
We have lasers at 405, 440 (or 445), 458, 488, 514, 561, 594, 633 nm and a detector we could use for emission from 690 to 750 nm.  Therefore, we expect this is really a problem of spectral unmixing of five probes and adding in the dapi and Opal 690 as separate sequential detections.

Cheers--

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
C: 914-309-3270  [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/


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[Douglas Richardson]

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Hi Michael,

Haven't done it with Opal dyes, but we have done it with similar Alexa dyes.

You should be able to simultaneously excite all of the dyes in the sample
with 405, 488 (or 514 although you need a custom MBS filter - 514/561/633),
561 and 633 .

The tricky part is because some dyes won't be excited as well (probably
540, 620, 690) you will need to adjust your labeling conditions to increase
the labeling density of these dyes (or decrease the others).

Here's an example with Alexa 514, 555, 568, 594, 633, 647 and 660 on an
880: https://www.pnas.org/content/pnas/115/34/8633.full.pdf

-Doug

On Tue, Jan 29, 2019 at 3:30 PM Cammer, Michael <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 1. We were unable to find spectral info for emission of the new probes so
> we did a quick scan and have posted the results here:
> http://microscopynotes.com/880/opals/
>
> 2.  If anyone has successfully done spectral unmixing  with these probes
> (six Opals plus dapi) using a Zeiss 880, we would be very interested in
> tips on how to do this.
> We have lasers at 405, 440 (or 445), 458, 488, 514, 561, 594, 633 nm and a
> detector we could use for emission from 690 to 750 nm.  Therefore, we
> expect this is really a problem of spectral unmixing of five probes and
> adding in the dapi and Opal 690 as separate sequential detections.
>
> Cheers--
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270  [hidden email]<mailto:
> [hidden email]>  http://nyulmc.org/micros
> http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the
> intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
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>