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As Ryan has alluded, the most likely answer is that Alexa 568 has some residual excitation at 405 nm (without looking at the spectra I'd guess that this would be on the order of 5%) - because A568 is a much brighter dye than A405 even 5% excitation might give you comparable signal strengths. The best solution would be to use individual band pass filters for *detection* (due to A568 having a finite crossection at 405, changing excitation filters is unlikely to get you very far). Linear unmixing might work if that's not possible, although you generally want to get the crosstalk below ~ 10% before this becomes a reliable option.
cheers,
David
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From: "Ryan, Kevin" <[log in to unmask]>
To: [log in to unmask]
Sent: Tuesday, 6 November 2012 10:13 AM
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:[log in to unmask]] On Behalf Of Ke Peng
Sent: Monday, November 05, 2012 3:42 PM
To: [log in to unmask]
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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