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Barlow, Andrew on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Problem-with-penetrating-signals-in-the-2nd-channel-tp7579271p7579280.html
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Hi Aromis,
If you're using an UltraView that is controlled by Volocity, then you also have the option of correcting cross talk using spectral unmixing.
If you need any help with this option, please don't hesitate to contact or team of technical support specialists, just follow the link in the help menu for their contact details.
Regards,
Andy
Andrew Barlow PhD | Market Development Leader, CI & A
PerkinElmer | For the Better
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-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Tong Yan
Sent: 06 November 2012 00:45
To:
[hidden email]
Subject: Re: Problem with 'penetrating' signals in the 2nd channel
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Hi,
We have encountered same problems on our PE spinning disc before not only on Alexa 568. We add in one single emission band (460/W70) for blue signal to solve the problem.
Regards,
TONG Yan
NUS Centre for BioImaging Sciences
Department of Biological Sciences
National University of Singapore
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Ryan, Kevin
Sent: Tuesday, November 06, 2012 5:13 AM
To:
[hidden email]
Subject: Re: Problem with 'penetrating' signals in the 2nd channel
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This sounds like a cross-talk issue. Check your excitation filters - since the emission filter you're using has a dual-band acceptance with broad bandpasses, only your excitation filter determines what fluoresces. See if the 405 excitation filter you're using overlaps the Alexa 568 excitation curve at all. I expect that it does - and it doesn't take much for visible crosstalk.
Options:
- Use a different set of excitation filters to minimize crosstalk.
- Correct your data afterwards: determine the percentage of the Alexa 568 crosstalk using an Alexa 568 only calibration sample (note any exposure time - I suggest calibrating with equal times), then post-process to subtract the Alexa 568 image(s) times that scaling from the Alexa 405 image(s).
405_image est. = 405_image - [ 568_image * (405 time)/(568 time) * 568_crosstalk_scalar ]
Kevin Ryan
Media Cybernetics, Inc.
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Ke Peng
Sent: Monday, November 05, 2012 3:42 PM
To:
[hidden email]
Subject: Problem with 'penetrating' signals in the 2nd channel
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Dear Everyone:
I'm doing multi-color sequential imaging of a protein complex and have met a confusing situation:
I used Alexa 568 labeled 2nd Ab to probe one protein and I found that the signal from the Alexa 568 could be detected in the 405 channel, the channel that is supposed to detect dyes like Alexa 405. We are using the Pelkin Elmer spinning disc microscope and the emission filter used is filter 3 (445nm, 615nm). This filter is used for emission discrimination for both Alexa 405 and Alexa 568. The sequence I used in the imaging was 1: Alexa 568; 2: eGFP; 3: Alexa 405. The excitation and emission wavelengths of these dyes are well separated and should work well in multicolor imaging.
I was wondering whether this is due to the fact that the large amount of Alexa 568 excited in the first step kept emitting and photons were captured again through the filter (445nm, 615nm) while signals were being detected for the Alexa 405 in the 3rd step. It does not sound very likely as the fluorophore should 'finish' emitting within the range of nanoseconds when excitation is removed and there is an eGFP detection (lasts about 300ms) step in between the two detection. But I have no other explanation at the moment. Does anyone of you have similar experience and/or have a clue how this happened?
Thanks a lot for your help in advance.
Best regards,
Aromis
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