Problem with 'penetrating' signals in the 2nd channel

[Ke Peng]

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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

[Kevin Ryan]

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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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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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[David Baddeley]

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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


________________________________
 From: "Ryan, Kevin" <[hidden email]>
To: [hidden email]
Sent: Tuesday, 6 November 2012 10:13 AM
Subject: Re: Problem with 'penetrating' signals in the 2nd channel
 
*****
To join, leave or search the confocal microscopy listserv, go to:
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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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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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[Ke Peng]

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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:[hidden email]]
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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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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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Cybernetics and their affiliates and is intended for the exclusive and
confidential use of the intended recipient. Any use, dissemination,
printing, or copying of this transmission and its attachment(s) is strictly
prohibited. If you are not the intended recipient, please do not read,
print, copy, distribute or take action in reliance upon this message.  If
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###########

[Tim Feinstein-2]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:[hidden email]]
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> ############################################################################
> ##########
> CONFIDENTIALITY NOTICE:
> This email transmission and its attachments contain confidential and
> proprietary information of Princeton Instruments, Acton Research, Media
> Cybernetics and their affiliates and is intended for the exclusive and
> confidential use of the intended recipient. Any use, dissemination,
> printing, or copying of this transmission and its attachment(s) is strictly
> prohibited. If you are not the intended recipient, please do not read,
> print, copy, distribute or take action in reliance upon this message.  If
> you have received this in error, please notify the sender immediately by
> telephone or return email and promptly delete all copies of the original
> transmission and its attachments from your computer system.
> ############################################################################
> ###########

[Ke Peng]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:[hidden email]]
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> ######################################################################
> ######
> ##########
> CONFIDENTIALITY NOTICE:
> This email transmission and its attachments contain confidential and
> proprietary information of Princeton Instruments, Acton Research,
> Media Cybernetics and their affiliates and is intended for the
> exclusive and confidential use of the intended recipient. Any use,
> dissemination, printing, or copying of this transmission and its
> attachment(s) is strictly prohibited. If you are not the intended
> recipient, please do not read, print, copy, distribute or take action
> in reliance upon this message.  If you have received this in error,
> please notify the sender immediately by telephone or return email and
> promptly delete all copies of the original transmission and its

attachments from your computer system.
> ######################################################################
> ######
> ###########

[dbstongy]

*****
To join, leave or search the confocal microscopy listserv, go to:
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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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
######################################################################################
CONFIDENTIALITY NOTICE:
This email transmission and its attachments contain confidential and proprietary information of Princeton Instruments, Acton Research, Media Cybernetics and their affiliates and is intended for the exclusive and confidential use of the intended recipient. Any use, dissemination, printing, or copying of this transmission and its attachment(s) is strictly prohibited. If you are not the intended recipient, please do not read, print, copy, distribute or take action in reliance upon this message.  If you have received this in error, please notify the sender immediately by telephone or return email and promptly delete all copies of the original transmission and its attachments from your computer system.
#######################################################################################

[Barlow, Andrew]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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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