Leica STED and 592 Notch Filter Issue

[Mark Scott-2]

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Hi All,

When using our 592nm STED laser on our SP8 system, we are noticing an issue with our notch filter not going into place as expected.  When the STED laser is set to 0% with the shutter open the STED laser is producing excitation with the 592nm laser of our sample as the notch filter is not going into position.  When the 592nm laser is set to even 0.1% power the notch filter goes into position automatically and we do not get the excitation.

Has anyone with a Leica STED 3X system seen this issue with their own system or willing to try and replicate the problem for me?

I am trying to ascertain if the issue is a failure on our system or a bug/oversight in the system as a whole where we need to ensure the shutter is closed even when the laser is on 0% power.  

It is also worth noting that manually selecting the correct notch filter for this 592nm STED laser does not solve the problem when the laser is on 0%, it is showing it being selected but we are still getting excitation.

Thanks

_______________________________________

Mark Scott
Senior Microscopist
Institute of Molecular Bioscience
Level 6 North, 306 Carmody Road (Building 80)
The University of Queensland
Brisbane, Queensland, 4072, Australia
http://imb.uq.edu.au/microscopy
[hidden email]

[Steffen Dietzel]

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Hi Mark,

I am not sure I completely understand your issue, but I assume you have
a dye such as A594 in your sample and you see some excitation of it by
the 592-depletion laser although this one is on zero percent (but with
open shutter).

In any case, since the notch filter is before the detector (after the
AOBS), it will in no case prevent the generation of fluorescence by the
STED beam, but only that reflected (!) light from the STED laser reaches
the detector. If the fluorescence has a large enough Stokes shift, it
should not be hindered by the notch filter.

We did have an issue with a leaky AOTF with the STED 592 laser when the
592 was used and then tuned down to zero. (not when a different STED
laser was last used). Which might not be so surprising since a very
small leakage of a several 100 mW laser is still a lot. We have
determined this with the 592, I assumed that it was the same with 660
and 775, but we did not test (due to the lack of suitable dyes for this
excitation at the time.) I hear the problem was solved with some
software update, we didn't get around to check. The easy way to avoid
the issue was to close the shutter in a sequential setting that does not
use the STED laser. So that's what we did (and still do) and once we
were aware of the problem, it wasn't one any more.

Have fun with your STED, so far we have best results with a combo of
near red (A594 or Aberior Star 580) and far red (Atto 647N, Aberior Star
635P (P is important) or SIR-dyes), both depleted with 775. It was
pretty exciting a couple of times comparing the results to confocal :-)

Steffen


Am 07.08.2018 um 06:02 schrieb Mark Scott:

> *****
> 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 All,
>
> When using our 592nm STED laser on our SP8 system, we are noticing an issue with our notch filter not going into place as expected.  When the STED laser is set to 0% with the shutter open the STED laser is producing excitation with the 592nm laser of our sample as the notch filter is not going into position.  When the 592nm laser is set to even 0.1% power the notch filter goes into position automatically and we do not get the excitation.
>
> Has anyone with a Leica STED 3X system seen this issue with their own system or willing to try and replicate the problem for me?
>
> I am trying to ascertain if the issue is a failure on our system or a bug/oversight in the system as a whole where we need to ensure the shutter is closed even when the laser is on 0% power.
>
> It is also worth noting that manually selecting the correct notch filter for this 592nm STED laser does not solve the problem when the laser is on 0%, it is showing it being selected but we are still getting excitation.
>
> Thanks
>
> _______________________________________
>
> Mark Scott
> Senior Microscopist
> Institute of Molecular Bioscience
> Level 6 North, 306 Carmody Road (Building 80)
> The University of Queensland
> Brisbane, Queensland, 4072, Australia
> http://imb.uq.edu.au/microscopy
> [hidden email]
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Mark Scott-2]

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Thanks Steffen,

It looks like it is something like the leaky AOTF exciting the 594 dye that you mentioned.  Engineer visit already booked so will hopefully sort the problem easily.

We’re definitely getting some great results and had some 3-colour 3D STED images using nothing but Alexa dyes off there the other day which looked great. Will keep having a try of some of the others you suggested as well.

Cheers
Mark

On 8/8/18, 8:57 pm, "Confocal Microscopy List on behalf of Steffen Dietzel" <[hidden email] on behalf of [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 Mark,
   
    I am not sure I completely understand your issue, but I assume you have
    a dye such as A594 in your sample and you see some excitation of it by
    the 592-depletion laser although this one is on zero percent (but with
    open shutter).
   
    In any case, since the notch filter is before the detector (after the
    AOBS), it will in no case prevent the generation of fluorescence by the
    STED beam, but only that reflected (!) light from the STED laser reaches
    the detector. If the fluorescence has a large enough Stokes shift, it
    should not be hindered by the notch filter.
   
    We did have an issue with a leaky AOTF with the STED 592 laser when the
    592 was used and then tuned down to zero. (not when a different STED
    laser was last used). Which might not be so surprising since a very
    small leakage of a several 100 mW laser is still a lot. We have
    determined this with the 592, I assumed that it was the same with 660
    and 775, but we did not test (due to the lack of suitable dyes for this
    excitation at the time.) I hear the problem was solved with some
    software update, we didn't get around to check. The easy way to avoid
    the issue was to close the shutter in a sequential setting that does not
    use the STED laser. So that's what we did (and still do) and once we
    were aware of the problem, it wasn't one any more.
   
    Have fun with your STED, so far we have best results with a combo of
    near red (A594 or Aberior Star 580) and far red (Atto 647N, Aberior Star
    635P (P is important) or SIR-dyes), both depleted with 775. It was
    pretty exciting a couple of times comparing the results to confocal :-)
   
    Steffen
   
   
    Am 07.08.2018 um 06:02 schrieb Mark Scott:
    > *****
    > 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 All,
    >
    > When using our 592nm STED laser on our SP8 system, we are noticing an issue with our notch filter not going into place as expected.  When the STED laser is set to 0% with the shutter open the STED laser is producing excitation with the 592nm laser of our sample as the notch filter is not going into position.  When the 592nm laser is set to even 0.1% power the notch filter goes into position automatically and we do not get the excitation.
    >
    > Has anyone with a Leica STED 3X system seen this issue with their own system or willing to try and replicate the problem for me?
    >
    > I am trying to ascertain if the issue is a failure on our system or a bug/oversight in the system as a whole where we need to ensure the shutter is closed even when the laser is on 0% power.
    >
    > It is also worth noting that manually selecting the correct notch filter for this 592nm STED laser does not solve the problem when the laser is on 0%, it is showing it being selected but we are still getting excitation.
    >
    > Thanks
    >
    > _______________________________________
    >
    > Mark Scott
    > Senior Microscopist
    > Institute of Molecular Bioscience
    > Level 6 North, 306 Carmody Road (Building 80)
    > The University of Queensland
    > Brisbane, Queensland, 4072, Australia
    > http://imb.uq.edu.au/microscopy
    > [hidden email]
    >
    --
    ------------------------------------------------------------
    Steffen Dietzel, PD Dr. rer. nat
    Ludwig-Maximilians-Universität München
    Biomedical Center (BMC)
    Head of the Core Facility Bioimaging
   
    Großhaderner Straße 9
    D-82152 Planegg-Martinsried
    Germany
   
    http://www.bioimaging.bmc.med.uni-muenchen.de