Posted by
Ke Peng on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Problem-with-penetrating-signals-in-the-2nd-channel-tp7579271p7579274.html
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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
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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
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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.
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From: Confocal Microscopy List [mailto:
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Sent: Monday, November 05, 2012 3:42 PM
To:
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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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