http://confocal-microscopy-list.275.s1.nabble.com/Problem-with-penetrating-signals-in-the-2nd-channel-tp7579271p7579275.html
This is a classic case of cross-talk. 405nm light excites a lot of things, which is one reason why it makes a great wavelength for across-the-board photobleaching. Most older PE scopes do not have a motorized emission filter wheel, which is what you need here. Your best bet for this particular experiment is either to install an aftermarket filter wheel in front of the CCD or else use something like Alexa488 instead of the 405nm dye.
Dept. of Pharmacology & Chemical Biology
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> Thanks a lot for your reply. In our system excitation are provided by laser
> lines: 405 (Diode) and 561 (DPSS). So maybe the excitation filter is not
> relevant in this case?
>
> Aromis
>
> -----邮件原件-----
> 发件人: Confocal Microscopy List [mailto:
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> 代表 Ryan, Kevin
> 发送时间: Montag, 5. November 2012 22:13
> 收件人:
[hidden email]
> 主题: 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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