Posted by
Ke Peng on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Problem-with-penetrating-signals-in-the-2nd-channel-tp7579271p7579276.html
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Hello All:
With your input I kept searching for the excitation/emission spectra of
Alexa568, I found a more complete documentation
(
http://depts.washington.edu/keck/DV-RT/Filtersets%20Live-Cell.html ) and I
guess here is the reason why (just like some of you said):
Alexa568 is excited at 405 with the efficiency of about 5% (exactly as David
said, which was not shown on the website of Invitrogen) -> the residual
signal will get through the dual-band emission filter (445nm, 615nm) -> the
signal was recorded as derived from Alexa 405 -> crosstalk
Thank you all for clarifying this situation!
An obvious solution might be to separate the 445 and 615 emission filter but
I don't know whether this will be really feasible.
I'm doing quadrupole imaging using Alexa 405, eGFP, Alexa568 and Alexa 647.
If you have experience and/or better ideas of choosing fluorophores please
share a generous hint.
Thanks again.
Cheers,
Ke
-----邮件原件-----
发件人: Confocal Microscopy List [mailto:
[hidden email]]
代表 Tim Feinstein
发送时间: Montag, 5. November 2012 23:15
收件人:
[hidden email]
主题: Re: Problem with 'penetrating' signals in the 2nd channel
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Hi Ke,
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.
cheers,
TF
Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology University of Pittsburgh, School of
Medicine BST W1301, 200 Lothrop St.
Pittsburgh, PA 15261
On Nov 5, 2012, at 4:54 PM, Ke Peng wrote:
> *****
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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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