cross excitation of Alexa647 wirh 488nm wavelength

[Pawel Pasierbek]

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Dear All,

Not strictly confocal question, but maybe somebody could have a hint.

One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
(Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
we use a single filter cube with a triple filter for 488, 561, 633
excitation and respective emission (Zeiss HE77).

Using control slide with Alexa647 we get cross excitation with the 488nm
laser. The emission is definitely in the Alexa647 range. I have also looked
at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
even 405nm light.

Has anybody observed such a phenomenon, or something happened to our
Alexa647??? (it is 6 months old, stored   at -80oC).

I will be grateful for your input.

All the best from Vienna,
Pawel


-----------------------------------------------------
Pawel Pasierbek
Microscopy Specialist
BIOOPTICS

IMBA - Instute of Molecular Biotechnology
IMP - Institute of Molecular Pathology

Dr. Bohr-Gasse 7
A-1030 Vienna
Austria

http://cores.imp.ac.at/biooptics/

www.imba.oeaw.ac.at/
www.imp.ac.at

[mcammer]

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Back in 2002 we had this problem.  I was told by one of the chemists at one of the companies providing dyes that this had something to do with either partial synthesis of the Cy5 or cleavage of the Cy5 due to excessive light.  Also, this was discussed briefly in the messages around this one:
http://lists.umn.edu/cgi-bin/wa?A2=ind0209&L=confocalmicroscopy&P=165
-Michael C.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pawel Pasierbek
Sent: Tuesday, February 15, 2011 10:01 AM
To: [hidden email]
Subject: cross excitation of Alexa647 wirh 488nm wavelength

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Dear All,

Not strictly confocal question, but maybe somebody could have a hint.

One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
(Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
we use a single filter cube with a triple filter for 488, 561, 633
excitation and respective emission (Zeiss HE77).

Using control slide with Alexa647 we get cross excitation with the 488nm
laser. The emission is definitely in the Alexa647 range. I have also looked
at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
even 405nm light.

Has anybody observed such a phenomenon, or something happened to our
Alexa647??? (it is 6 months old, stored   at -80oC).

I will be grateful for your input.

All the best from Vienna,
Pawel


-----------------------------------------------------
Pawel Pasierbek
Microscopy Specialist
BIOOPTICS

IMBA - Instute of Molecular Biotechnology
IMP - Institute of Molecular Pathology

Dr. Bohr-Gasse 7
A-1030 Vienna
Austria

http://cores.imp.ac.at/biooptics/

www.imba.oeaw.ac.at/
www.imp.ac.at

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[yuansheng sun]

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Hi Pawel,

We used the combination of Alexa488-Alexa555-Alexa647 for a
three-color FRET work.  Only little excitation of Alexa647 at the
Argon 488 laser line was observed.  The ratio of the Alexa647-alone
intensity excited by the Argon 488 nm versus that excited by the HeNe
633 nm is about 2%.  Of course, this depends on the powers of the two
lasers.  How much of the Alexa647 is excited by the 488 nm in your
imaging conditions?

Sheng


Yuansheng Sun
Research Scientist
Keck Center for Cellular Imaging
University of Virginia



On Tue, Feb 15, 2011 at 10:01 AM, Pawel Pasierbek <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> Not strictly confocal question, but maybe somebody could have a hint.
>
> One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
> (Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
> we use a single filter cube with a triple filter for 488, 561, 633
> excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the 488nm
> laser. The emission is definitely in the Alexa647 range. I have also looked
> at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
> even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).
>
> I will be grateful for your input.
>
> All the best from Vienna,
> Pawel
>
>
> -----------------------------------------------------
> Pawel Pasierbek
> Microscopy Specialist
> BIOOPTICS
>
> IMBA - Instute of Molecular Biotechnology
> IMP - Institute of Molecular Pathology
>
> Dr. Bohr-Gasse 7
> A-1030 Vienna
> Austria
>
> http://cores.imp.ac.at/biooptics/
>
> www.imba.oeaw.ac.at/
> www.imp.ac.at
>

[Watkins, Simon C]

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The problem is that TIRF is a very high signal to noise technique, so even the smallest excitation shows up in the final image.  Because of this its really essential to use an emission filter wheel when using  a multipass cube (which is essential for ensuring correct registration etc).
S

Simon C. Watkins Ph.D, FRC Path
Professor and Vice Chair Cell Biology and Physiology
Professor Immunology Director Center for Biologic Imaging
BSTS 225
University of Pittsburgh
3500 Terrace St
Pittsburgh PA 15261
412-352-2277
www.cbi.pitt.edu

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of yuansheng sun
Sent: Tuesday, February 15, 2011 11:39 AM
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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Hi Pawel,

We used the combination of Alexa488-Alexa555-Alexa647 for a
three-color FRET work.  Only little excitation of Alexa647 at the
Argon 488 laser line was observed.  The ratio of the Alexa647-alone
intensity excited by the Argon 488 nm versus that excited by the HeNe
633 nm is about 2%.  Of course, this depends on the powers of the two
lasers.  How much of the Alexa647 is excited by the 488 nm in your
imaging conditions?

Sheng


Yuansheng Sun
Research Scientist
Keck Center for Cellular Imaging
University of Virginia



On Tue, Feb 15, 2011 at 10:01 AM, Pawel Pasierbek <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> Not strictly confocal question, but maybe somebody could have a hint.
>
> One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
> (Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
> we use a single filter cube with a triple filter for 488, 561, 633
> excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the 488nm
> laser. The emission is definitely in the Alexa647 range. I have also looked
> at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
> even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).
>
> I will be grateful for your input.
>
> All the best from Vienna,
> Pawel
>
>
> -----------------------------------------------------
> Pawel Pasierbek
> Microscopy Specialist
> BIOOPTICS
>
> IMBA - Instute of Molecular Biotechnology
> IMP - Institute of Molecular Pathology
>
> Dr. Bohr-Gasse 7
> A-1030 Vienna
> Austria
>
> http://cores.imp.ac.at/biooptics/
>
> www.imba.oeaw.ac.at/
> www.imp.ac.at
>

[Martin Wessendorf-2]

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

On 2/15/2011 9:01 AM, Pawel Pasierbek wrote:

> One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
> (Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
> we use a single filter cube with a triple filter for 488, 561, 633
> excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the 488nm
> laser. The emission is definitely in the Alexa647 range. I have also looked
> at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
> even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).

This sounds simply like "bleed-through" if you're using a triple filter.
  The Alexa 647 will be excitable by shorter wavelengths, albeit
inefficiently.

I know of no way around the problem except blocking the emission of the
Alexa 647.  Could you split your output to two cameras with two
different emission filters?  (--You would also probably need to be able
to switch off GFP excitation when imaging the Alexa.)

Good luck!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Ignatius, Mike-2]

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*****

From our resident expert Dr. Iain Johnson.

"The AF647 dye itself is very stable in water, although the maleimide portion will hydrolyze rapidly to unreactive maleaimic acids in water - but dye will be fine.  - I have my own data on this (measured the fluorescence quantum yield from the same aqueous stock solution on two occcasions separated by ~12 months. QYs identical within experimental error).  Longer wavelegth carbocyanines (AF750 etc) are a different matter.  There the dye definitely falls apart over a period of months storage in aqueous at 4C.

Using a multiband filter you give up spectral isolation in return for speed.  There is really no way around this. But since it appears that they only have the 488 laser on when they do this experiment, the blame can't be entirely laid at the door of the filter.  Although the excitation spectrum of AF647 looks almost at baseline at 488 nm, it never goes completely to zero on the blue side of the 0-0 band (this is true for pretty much all polyatomic fluorophores).  The extinction of AF647 is about 0.3% of max at 488 nm.  This is equates to about 750 cm-1 M-1 - quite sufficient to give a detectable signal when you are using laser excitation."

As communicated by Mike Ignatius, Molecular Probes/Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, February 15, 2011 9:22 AM
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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

On 2/15/2011 9:01 AM, Pawel Pasierbek wrote:

> One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
> (Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
> we use a single filter cube with a triple filter for 488, 561, 633
> excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the 488nm
> laser. The emission is definitely in the Alexa647 range. I have also looked
> at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
> even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).

This sounds simply like "bleed-through" if you're using a triple filter.
  The Alexa 647 will be excitable by shorter wavelengths, albeit
inefficiently.

I know of no way around the problem except blocking the emission of the
Alexa 647.  Could you split your output to two cameras with two
different emission filters?  (--You would also probably need to be able
to switch off GFP excitation when imaging the Alexa.)

Good luck!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Jennifer Clarke]

Dear Pawel and list

I can confirm from our experience that a 488nm laser line does definitely cause some excitation of Alexa647.
 
We routinely do FCS with APD detectors using combinations of peptides conjugated to either Alexa488 or Alexa647 and fusion proteins tagged with either mEGFP or mKate
and when we've run controls with Alexa647 being the ONLY dye present and only the 488nm laser line, even at exceedingly low power (what we use to image Alexa488 or mEGFP with APDs), we definitely do see emission from the Alexa647 (although of course fairly minimal compared to excitation with 633nm laser).
I'm sure on any system 488nm excitation at sufficient level to excite most common green dyes would also give some cross-excitation of Alexa647 during simultaneous scanning.

Kind regards
Jen

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ignatius, Mike
Sent: Wednesday, 16 February 2011 06:03
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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

From our resident expert Dr. Iain Johnson.

"The AF647 dye itself is very stable in water, although the maleimide portion will hydrolyze rapidly to unreactive maleaimic acids in water - but dye will be fine.  - I have my own data on this (measured the fluorescence quantum yield from the same aqueous stock solution on two occcasions separated by ~12 months. QYs identical within experimental error).  Longer wavelegth carbocyanines (AF750 etc) are a different matter.  There the dye definitely falls apart over a period of months storage in aqueous at 4C.

Using a multiband filter you give up spectral isolation in return for speed.  There is really no way around this. But since it appears that they only have the 488 laser on when they do this experiment, the blame can't be entirely laid at the door of the filter.  Although the excitation spectrum of AF647 looks almost at baseline at 488 nm, it never goes completely to zero on the blue side of the 0-0 band (this is true for pretty much all polyatomic fluorophores).  The extinction of AF647 is about 0.3% of max at 488 nm.  This is equates to about 750 cm-1 M-1 - quite sufficient to give a detectable signal when you are using laser excitation."

As communicated by Mike Ignatius, Molecular Probes/Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, February 15, 2011 9:22 AM
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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

On 2/15/2011 9:01 AM, Pawel Pasierbek wrote:

> One of our users is doing 2 color TIRF experiment, using GFP and
> Alexa647 (Alexa Fluor 647 C5 maleimid). Because the time resolution is
> very critical, we use a single filter cube with a triple filter for
> 488, 561, 633 excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the
> 488nm laser. The emission is definitely in the Alexa647 range. I have
> also looked at the sample with Confocal (LSM710) and can excite Alexa
> 647 with 488nm and even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).

This sounds simply like "bleed-through" if you're using a triple filter.
  The Alexa 647 will be excitable by shorter wavelengths, albeit inefficiently.

I know of no way around the problem except blocking the emission of the Alexa 647.  Could you split your output to two cameras with two different emission filters?  (--You would also probably need to be able to switch off GFP excitation when imaging the Alexa.)

Good luck!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Hu Xian]

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Dear List,

We also confirm that this phenomenon.

Alexa647 is the far-red dye we use for dSTORM experiment, for Alexa647 to "blink" other than adding the redox cocktail describe in Sauer's paper, 488 illumination works well too.
Actually no of the other non-specific line seems to be able to trigger the "blinking" of Alexa647 under such circumstances, so it is rather specific.


Thanks.

Regards,
Edna



________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Jen Clarke [[hidden email]]
Sent: Wednesday, February 16, 2011 7:40 AM
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

Dear Pawel and list

I can confirm from our experience that a 488nm laser line does definitely cause some excitation of Alexa647.

We routinely do FCS with APD detectors using combinations of peptides conjugated to either Alexa488 or Alexa647 and fusion proteins tagged with either mEGFP or mKate
and when we've run controls with Alexa647 being the ONLY dye present and only the 488nm laser line, even at exceedingly low power (what we use to image Alexa488 or mEGFP with APDs), we definitely do see emission from the Alexa647 (although of course fairly minimal compared to excitation with 633nm laser).
I'm sure on any system 488nm excitation at sufficient level to excite most common green dyes would also give some cross-excitation of Alexa647 during simultaneous scanning.

Kind regards
Jen

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ignatius, Mike
Sent: Wednesday, 16 February 2011 06:03
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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

From our resident expert Dr. Iain Johnson.

"The AF647 dye itself is very stable in water, although the maleimide portion will hydrolyze rapidly to unreactive maleaimic acids in water - but dye will be fine.  - I have my own data on this (measured the fluorescence quantum yield from the same aqueous stock solution on two occcasions separated by ~12 months. QYs identical within experimental error).  Longer wavelegth carbocyanines (AF750 etc) are a different matter.  There the dye definitely falls apart over a period of months storage in aqueous at 4C.

Using a multiband filter you give up spectral isolation in return for speed.  There is really no way around this. But since it appears that they only have the 488 laser on when they do this experiment, the blame can't be entirely laid at the door of the filter.  Although the excitation spectrum of AF647 looks almost at baseline at 488 nm, it never goes completely to zero on the blue side of the 0-0 band (this is true for pretty much all polyatomic fluorophores).  The extinction of AF647 is about 0.3% of max at 488 nm.  This is equates to about 750 cm-1 M-1 - quite sufficient to give a detectable signal when you are using laser excitation."

As communicated by Mike Ignatius, Molecular Probes/Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, February 15, 2011 9:22 AM
To: [hidden email]
Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength

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

On 2/15/2011 9:01 AM, Pawel Pasierbek wrote:

> One of our users is doing 2 color TIRF experiment, using GFP and
> Alexa647 (Alexa Fluor 647 C5 maleimid). Because the time resolution is
> very critical, we use a single filter cube with a triple filter for
> 488, 561, 633 excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the
> 488nm laser. The emission is definitely in the Alexa647 range. I have
> also looked at the sample with Confocal (LSM710) and can excite Alexa
> 647 with 488nm and even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored   at -80oC).

This sounds simply like "bleed-through" if you're using a triple filter.
  The Alexa 647 will be excitable by shorter wavelengths, albeit inefficiently.

I know of no way around the problem except blocking the emission of the Alexa 647.  Could you split your output to two cameras with two different emission filters?  (--You would also probably need to be able to switch off GFP excitation when imaging the Alexa.)

Good luck!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Terri Bruce]

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

Sent from my iPad

On Feb 15, 2011, at 12:23 PM, "Watkins, Simon C" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The problem is that TIRF is a very high signal to noise technique, so even the smallest excitation shows up in the final image.  Because of this its really essential to use an emission filter wheel when using  a multipass cube (which is essential for ensuring correct registration etc).
> S
>
> Simon C. Watkins Ph.D, FRC Path
> Professor and Vice Chair Cell Biology and Physiology
> Professor Immunology Director Center for Biologic Imaging
> BSTS 225
> University of Pittsburgh
> 3500 Terrace St
> Pittsburgh PA 15261
> 412-352-2277
> www.cbi.pitt.edu
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of yuansheng sun
> Sent: Tuesday, February 15, 2011 11:39 AM
> To: [hidden email]
> Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Pawel,
>
> We used the combination of Alexa488-Alexa555-Alexa647 for a
> three-color FRET work.  Only little excitation of Alexa647 at the
> Argon 488 laser line was observed.  The ratio of the Alexa647-alone
> intensity excited by the Argon 488 nm versus that excited by the HeNe
> 633 nm is about 2%.  Of course, this depends on the powers of the two
> lasers.  How much of the Alexa647 is excited by the 488 nm in your
> imaging conditions?
>
> Sheng
>
>
> Yuansheng Sun
> Research Scientist
> Keck Center for Cellular Imaging
> University of Virginia
>
>
>
> On Tue, Feb 15, 2011 at 10:01 AM, Pawel Pasierbek <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>>
>> Not strictly confocal question, but maybe somebody could have a hint.
>>
>> One of our users is doing 2 color TIRF experiment, using GFP and Alexa647
>> (Alexa Fluor 647 C5 maleimid). Because the time resolution is very critical,
>> we use a single filter cube with a triple filter for 488, 561, 633
>> excitation and respective emission (Zeiss HE77).
>>
>> Using control slide with Alexa647 we get cross excitation with the 488nm
>> laser. The emission is definitely in the Alexa647 range. I have also looked
>> at the sample with Confocal (LSM710) and can excite Alexa 647 with 488nm and
>> even 405nm light.
>>
>> Has anybody observed such a phenomenon, or something happened to our
>> Alexa647??? (it is 6 months old, stored   at -80oC).
>>
>> I will be grateful for your input.
>>
>> All the best from Vienna,
>> Pawel
>>
>>
>> -----------------------------------------------------
>> Pawel Pasierbek
>> Microscopy Specialist
>> BIOOPTICS
>>
>> IMBA - Instute of Molecular Biotechnology
>> IMP - Institute of Molecular Pathology
>>
>> Dr. Bohr-Gasse 7
>> A-1030 Vienna
>> Austria
>>
>> http://cores.imp.ac.at/biooptics/
>>
>> www.imba.oeaw.ac.at/
>> www.imp.ac.at
>>

[Pawel Pasierbek]

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

Dear All,

Thank you for your input. The excitation of the Alexa647 wee see with quite
high 488nm laser power (we need to use it due to a very! low signal from the
GFP).

Yes, we will try hardware solutions - filter wheel, dual view and 2 cameras
system...

From what I have learnt so far, one can really nicely excite the Alexa647
with shorter wavelengths... unfortunately.

I will let you know what kind of a solution we found.

All the best,
Pawel