Leica SP5 Upgrade to gated CW STED?

[Adrian Smith-6]

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Hi all,

We are considering applying for funds to upgrade our SP5 (c. 2008) to
enable CW gated-STED.

Has anyone done this upgrade? How did it go?

One thing in particular that we are concerned about is that we routinely
have the system at 37C for live work (it basically stays at 37 permanently)
but the STED objective is apparently designed for room temperature...

Any comments appreciated,

Thanks,

Adrian Smith
Centenary Institute, Sydney, AU

[Zdenek Svindrych]

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Hi Adrain,

I've been told by Leica people that they support time-gating of their HyD
detectors on SP8 only. But there is no obvious hardware difference between
SP5 and SP8 related to detector gating... Anyway, if you succeed, let me
know. This would be great!

CW-STED has been demonstrated on living cells with various objectives (e.g.
HCX PL APO 100 × /1.40–0.7 Oil, 592nm depletion), so it is possibe. I don't
know what exactly is that special about "special STED 100x/1.4 oil
objective". Some vendors (Nikon) offer pre-selected objectives with best
performance (for extra price).

Also, you probably don't plan to do much STED on living cells, as this is
not really life-friendly technique (but others (like SIM) don't offer the
resolution). I understand your concerns, STED is high tech, end 3D STED is
even more sensitive to abberations. Leica reps should tell you more.

Last bit: living-cell experiments are very time-consuming. STED experiments
even more so (on fixed samples). Try to spare some bucks and get a full
second machine :-).

Good luck with your upgrade!
zdenek svindrych
lf1.cuni.cz


"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,

We are considering applying for funds to upgrade our SP5 (c. 2008) to
enable CW gated-STED.

Has anyone done this upgrade? How did it go?

One thing in particular that we are concerned about is that we routinely
have the system at 37C for live work (it basically stays at 37 permanently)
but the STED objective is apparently designed for room temperature...

Any comments appreciated,

Thanks,

Adrian Smith
Centenary Institute, Sydney, AU"

[Steffen Dietzel]

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Am 28.03.2014 07:50, schrieb Zdenek Svindrych:
> I don't
> know what exactly is that special about "special STED 100x/1.4 oil
> objective".

The chromatic aberration of the STED white 100x is lower than with other
objectives, to make sure that excitation and depletion beam are aligned.
Particularly important if more than one depletion beam is available, as
it is the case now. The chromatic aberration is supposed to be <50 nm
between 450 and 700 nm. All according to Leica, I do not have experience
with it.

Cheers
Steffen


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Steffen Dietzel]

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One thing you might want to keep an eye on is that the Ri of the
immersion oil is temperature sensitive. I am not sure what that means
for STED at 37°. Maybe you can switch oils, to one that has 1.518 at 37.

Steffen


Am 28.03.2014 04:28, schrieb Adrian Smith:

> *****
> 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,
>
> We are considering applying for funds to upgrade our SP5 (c. 2008) to
> enable CW gated-STED.
>
> Has anyone done this upgrade? How did it go?
>
> One thing in particular that we are concerned about is that we routinely
> have the system at 37C for live work (it basically stays at 37 permanently)
> but the STED objective is apparently designed for room temperature...
>
> Any comments appreciated,
>
> Thanks,
>
> Adrian Smith
> Centenary Institute, Sydney, AU
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Alison Dun]

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Hi,

We have a gated-STED on a Leica SP5 using a HyD and it works really well. We have done some CW-STED and the gated-STED is far better. I am not sure about the temperature as we have only done work on fixed samples so far.

Cheers
Ali

Dr. Alison Dun
Technology and Facility Manager
Edinburgh Super Resolution Imaging Consortium (ESRIC)
Heriot-Watt University
Edinburgh
EH14 4AS

Tel: [phone]
@AliDun_esric

Register for our summer school at http://www.esric.org/summer-school.html





On 28 Mar 2014, at 18:06, Steffen Dietzel <[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.
> *****
>
> One thing you might want to keep an eye on is that the Ri of the
> immersion oil is temperature sensitive. I am not sure what that means
> for STED at 37°. Maybe you can switch oils, to one that has 1.518 at 37.
>
> Steffen
>
>
> Am 28.03.2014 04:28, schrieb Adrian Smith:
>> *****
>> 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,
>>
>> We are considering applying for funds to upgrade our SP5 (c. 2008) to
>> enable CW gated-STED.
>>
>> Has anyone done this upgrade? How did it go?
>>
>> One thing in particular that we are concerned about is that we routinely
>> have the system at 37C for live work (it basically stays at 37 permanently)
>> but the STED objective is apparently designed for room temperature...
>>
>> Any comments appreciated,
>>
>> Thanks,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, AU
>>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern