PA-GFP

[Antonio Jose Pereira]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi,

We're using 405nm laser to activate PA-GFP tubulin.

We're witnessing a behaviour which was unexpected: we see the much-wanted sudden signal increase in the subsequent GFP channel acquisitions (488 excitation) but the issue is that the signal keeps increasing, sometimes to as much as 130%, in a timescale of the order of a few minutes after the 405 pulse.

After this couple of minutes, either the increase is not significant anymore or it is potentially masked by contributions of a decay resulting from residual turnover (although we're doing taxol stabilization) or photo-bleaching.

We essentially ruled out some residual contribution of the 488 imaging laser to photo-conversion (however, this was performed in the absence of an initial 405 pulse).

Any ideas what this might mean?
Is it any meaningful to think of a population of pa-gfp molecules which undergoes a delayed conversion to the right conformation ...?
Has anyone come across this phenomenon of an increase in signal when trying to calculate photobleaching using Taxol-stabilized microtubules?

I appreciate your input.

Antonio

António José Pereira - Chromosome Instability and Dynamics lab
i3S / IBMC , Universidade do Porto, [phone] (ext 6011)

[Knecht, David]

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Hi- This is tubulin so you are activating both microtubules and free dimer.  Is the initial activation highly localized or more diffuse?  Is the increase over time highly localized or diffuse?  Are you activating in a small spot or over a large area?  One possibility I am thinking of is that you are activating free protein in planes above and below your imaging plane which then localizes into your imaging plane.  Dave

Dr. David Knecht
Professor
Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

> On Sep 30, 2016, at 10:06 AM, Antonio Jose Pereira <[hidden email]> wrote:
>
> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi,
>
> We're using 405nm laser to activate PA-GFP tubulin.
>
> We're witnessing a behaviour which was unexpected: we see the much-wanted sudden signal increase in the subsequent GFP channel acquisitions (488 excitation) but the issue is that the signal keeps increasing, sometimes to as much as 130%, in a timescale of the order of a few minutes after the 405 pulse.
>
> After this couple of minutes, either the increase is not significant anymore or it is potentially masked by contributions of a decay resulting from residual turnover (although we're doing taxol stabilization) or photo-bleaching.
>
> We essentially ruled out some residual contribution of the 488 imaging laser to photo-conversion (however, this was performed in the absence of an initial 405 pulse).
>
> Any ideas what this might mean?
> Is it any meaningful to think of a population of pa-gfp molecules which undergoes a delayed conversion to the right conformation ...?
> Has anyone come across this phenomenon of an increase in signal when trying to calculate photobleaching using Taxol-stabilized microtubules?
>
> I appreciate your input.
>
> Antonio
>
> António José Pereira - Chromosome Instability and Dynamics lab
> i3S / IBMC , Universidade do Porto, [phone] (ext 6011)
>
>
>

[jerie]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Antonio,

is really no 405nm light reaching the sample after switching 'off' the laser? Is a shutter or AOTF used to block any residual emission from the source?

Cheers, jens.

Jens Rietdorf
Visiting Scientist
Fundação Oswaldo Cruz, Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Rio de Janeiro,  Brazil.

Am 30.09.2016 11:06 schrieb "Antonio Jose Pereira" <[hidden email]>:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi,

We're using 405nm laser to activate PA-GFP tubulin.

We're witnessing a behaviour which was unexpected: we see the much-wanted sudden signal increase in the subsequent GFP channel acquisitions (488 excitation) but the issue is that the signal keeps increasing, sometimes to as much as 130%, in a timescale of the order of a few minutes after the 405 pulse.

After this couple of minutes, either the increase is not significant anymore or it is potentially masked by contributions of a decay resulting from residual turnover (although we're doing taxol stabilization) or photo-bleaching.

We essentially ruled out some residual contribution of the 488 imaging laser to photo-conversion (however, this was performed in the absence of an initial 405 pulse).

Any ideas what this might mean?
Is it any meaningful to think of a population of pa-gfp molecules which undergoes a delayed conversion to the right conformation ...?
Has anyone come across this phenomenon of an increase in signal when trying to calculate photobleaching using Taxol-stabilized microtubules?

I appreciate your input.

Antonio

António José Pereira - Chromosome Instability and Dynamics lab
i3S / IBMC , Universidade do Porto, [phone] (ext 6011)