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mounted cells and a hardware autofocus RI mismatch

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Sylvie Le Guyader-2

mounted cells and a hardware autofocus RI mismatch

Hi everyone

I am trying to find some mounting medium with a refractive index close to the one of water (1.33) in order to use our ZDC (Olympus zero drift control, IR-based hardware autofocus) with fixed samples.

Currently we use Dako S3023 and the focus is regularly lost without apparent reason. Andor, who sold us the system, claims that it could be because the IR of the mounting medium doesn’t contrast enough with the one of glass. I asked Dako for the RI of S3023 and they repeatedly stated 1.47.1-5 which is pretty vague but is definitely not 1.33. However I have found here on the confocal server that the RI of Dako S3023 had been measured and would be 1.372.

Does anyone know what the Dako RI is and where I could find a mounting medium that hardens and has an RI close to 1.33? does anyone have any experience with imaging fixed and mounted cells with a hardware autofocus?

Thanks for your help!!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

+46 (0)8 608 9240

Csúcs Gábor

Re: mounted cells and a hardware autofocus RI mismatch

Hallo Sylvie,

Before going more into the details - which objective are you using. The
reliability of the ZDC depends very much on the objective
(irrespectively whether it should "officially" work or not).

Cheers Gabor

>
> Hi everyone
>
> I am trying to find some mounting medium with a refractive index close
> to the one of water (1.33) in order to use our ZDC (Olympus zero drift
> control, IR-based hardware autofocus) with fixed samples.
>
> Currently we use Dako S3023 and the focus is regularly lost without
> apparent reason. Andor, who sold us the system, claims that it could
> be because the IR of the mounting medium doesn’t contrast enough with
> the one of glass. I asked Dako for the RI of S3023 and they repeatedly
> stated 1.47.1-5 which is pretty vague but is definitely not 1.33.
> However I have found here on the confocal server that the RI of Dako
> S3023 had been measured and would be 1.372.
>
> Does anyone know what the Dako RI is and where I could find a mounting
> medium that hardens and has an RI close to 1.33? does anyone have any
> experience with imaging fixed and mounted cells with a hardware autofocus?
>
> Thanks for your help!!
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader
>
> Dept of Biosciences and Nutrition
>
> Karolinska Institutet
>
> Novum
>
> 14157 Huddinge
>
> Sweden
>
> +46 (0)8 608 9240
>

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: +41 44 633 6221
Mobile: +41 79 758 21 58
Fax: +41 44 632 1298
e-mail: [hidden email]

Sylvie Le Guyader-2

Re: mounted cells and a hardware autofocus RI mismatch

Hi Gabor,

Important point! Olympus 60x NA 1.45 TIRF

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157 Huddinge
Sweden
+46 (0)8 608 9240

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: 25 March 2009 14:56
> To: [hidden email]
> Subject: Re: mounted cells and a hardware autofocus RI mismatch
>
> Hallo Sylvie,
>
> Before going more into the details - which objective are you using. The
> reliability of the ZDC depends very much on the objective
> (irrespectively whether it should "officially" work or not).
>
> Cheers Gabor
> >
> > Hi everyone
> >
> > I am trying to find some mounting medium with a refractive index close
> > to the one of water (1.33) in order to use our ZDC (Olympus zero drift
> > control, IR-based hardware autofocus) with fixed samples.
> >
> > Currently we use Dako S3023 and the focus is regularly lost without
> > apparent reason. Andor, who sold us the system, claims that it could
> > be because the IR of the mounting medium doesnt contrast enough with
> > the one of glass. I asked Dako for the RI of S3023 and they repeatedly
> > stated 1.47.1-5 which is pretty vague but is definitely not 1.33.
> > However I have found here on the confocal server that the RI of Dako
> > S3023 had been measured and would be 1.372.
> >
> > Does anyone know what the Dako RI is and where I could find a mounting
> > medium that hardens and has an RI close to 1.33? does anyone have any
> > experience with imaging fixed and mounted cells with a hardware

autofocus?

> >
> > Thanks for your help!!
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader
> >
> > Dept of Biosciences and Nutrition
> >
> > Karolinska Institutet
> >
> > Novum
> >
> > 14157 Huddinge
> >
> > Sweden
> >
> > +46 (0)8 608 9240
> >
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]

Jean-Pierre CLAMME-2

Re: mounted cells and a hardware autofocus RI mismatch

In reply to this post by Sylvie Le Guyader-2

Hello Sylvie

Just curious, why you need the ZDC for fixed samples ? For me using ZDC only make sens for time dependant live imaging.

JP

Confocal Microscopy List <[hidden email]> wrote on 03/25/2009 06:40:31 AM: > Hi everyone
> >
> > I am trying to find some mounting medium with a refractive index > close to the one of water (1.33) in order to use our ZDC (Olympus > zero drift control, IR-based hardware autofocus) with fixed samples.
> >
> > Currently we use Dako S3023 and the focus is regularly lost without > apparent reason. Andor, who sold us the system, claims that it could > be because the IR of the mounting medium doesn’t contrast enough > with the one of glass. I asked Dako for the RI of S3023 and they > repeatedly stated 1.47.1-5 which is pretty vague but is definitely > not 1.33. However I have found here on the confocal server that the > RI of Dako S3023 had been measured and would be 1.372.
> >
> > Does anyone know what the Dako RI is and where I could find a > mounting medium that hardens and has an RI close to 1.33? does > anyone have any experience with imaging fixed and mounted cells with > a hardware autofocus?
> >
> > Thanks for your help!!
> >
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> > Sylvie Le Guyader
> > Dept of Biosciences and Nutrition
> > Karolinska Institutet
> > Novum
> > 14157 Huddinge
> > Sweden
> > +46 (0)8 608 9240
> >

Sylvie Le Guyader-2

Re: mounted cells and a hardware autofocus RI mismatch

Hi again

We are trying to automate imaging with antibody staining on cells treated in many different conditions. Cells are grown and stained on coverslips then mounted. We want to use a pre set number of xy positions (eg 50 positions) that we will use for all coverslips and the z would be found relative to the glass so you can overcome variations in the coverslip thickness this way. Then you trash the data from which you can’t extract anything because there are no cells.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

+46 (0)8 608 9240

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jean-Pierre CLAMME
Sent: 25 March 2009 17:57
To: [hidden email]
Subject: Re: mounted cells and a hardware autofocus RI mismatch

Hello Sylvie

Just curious, why you need the ZDC for fixed samples ? For me using ZDC only make sens for time dependant live imaging.

JP

Confocal Microscopy List<[hidden email]> wrote on 03/25/2009 06:40:31 AM:

> Hi everyone
>
>
>
> I am trying to find some mounting medium with a refractive index
> close to the one of water (1.33) in order to use our ZDC (Olympus
> zero drift control, IR-based hardware autofocus) with fixed samples.
>
>
>
> Currently we use Dako S3023 and the focus is regularly lost without
> apparent reason. Andor, who sold us the system, claims that it could
> be because the IR of the mounting medium doesn’t contrast enough
> with the one of glass. I asked Dako for the RI of S3023 and they
> repeatedly stated 1.47.1-5 which is pretty vague but is definitely
> not 1.33. However I have found here on the confocal server that the
> RI of Dako S3023 had been measured and would be 1.372.
>
>
>
> Does anyone know what the Dako RI is and where I could find a
> mounting medium that hardens and has an RI close to 1.33? does
> anyone have any experience with imaging fixed and mounted cells with
> a hardware autofocus?
>
>
>
> Thanks for your help!!
>
>
>
>
>
> Med vänlig hälsning / Best regards
>
>
>
> Sylvie
>
>
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader
>
> Dept of Biosciences and Nutrition
>
> Karolinska Institutet
>
> Novum
>
> 14157 Huddinge
>
> Sweden
>
> +46 (0)8 608 9240
>
>

Glen MacDonald-2

Re: mounted cells and a hardware autofocus RI mismatch

In reply to this post by Sylvie Le Guyader-2

I've read that Fluoromount G, a hardening mountant from Southern
Biotech, has been measured to an RI of about 1.338.

Regards,
Glen
On Mar 25, 2009, at 6:40 AM, Sylvie Le Guyader wrote:

> 
> Hi everyone
>
>
>
> I am trying to find some mounting medium with a refractive index
> close to the one of water (1.33) in order to use our ZDC (Olympus
> zero drift control, IR-based hardware autofocus) with fixed samples.
>
>
>
> Currently we use Dako S3023 and the focus is regularly lost without
> apparent reason. Andor, who sold us the system, claims that it could
> be because the IR of the mounting medium doesn’t contrast enough
> with the one of glass. I asked Dako for the RI of S3023 and they
> repeatedly stated 1.47.1-5 which is pretty vague but is definitely
> not 1.33. However I have found here on the confocal server that the
> RI of Dako S3023 had been measured and would be 1.372.
>
>
>
> Does anyone know what the Dako RI is and where I could find a
> mounting medium that hardens and has an RI close to 1.33? does
> anyone have any experience with imaging fixed and mounted cells with
> a hardware autofocus?
>
>
>
> Thanks for your help!!
>
>
>
>
>
> Med vänlig hälsning / Best regards
>
>
>
> Sylvie
>
>
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader
>
> Dept of Biosciences and Nutrition
>
> Karolinska Institutet
>
> Novum
>
> 14157 Huddinge
>
> Sweden
>
> +46 (0)8 608 9240
>
>
>

lechristophe

Re: mounted cells and a hardware autofocus RI mismatch

I've tested Fluoromount G as I was looking for hardening mountants for TIRF imaging of fixed cells. Unfortunately, the RI is rising progressively as the Fluoromount hardens, so the same day or next day after mounting I could get some TIRF images (which require a significant difference in RI betwwen the coverslip and the medium, similarily to the hardware autofocus), although of bad quality. But after more than two days it was impossible to get TIRF, because the required angle must have been too high for the very slight RI difference remaining.

So I ended up mounting my cells in PBS( +n-propyl-galate as an anti-fade), so that the RI is 1.33. I struggled to find a good sealant that allowed me to keep my slides (PBS is not viscous like PBS/glycerol and evaporates very rapidly). I tested VALAP, DPX but none were impermeant enough to avoid evaporation after a few days... So if you have a proposition I'd be glad to hear it.

Christophe Leterrier

On Wed, Mar 25, 2009 at 17:37, Glen MacDonald <[hidden email]> wrote:

I've read that Fluoromount G, a hardening mountant from Southern Biotech, has been measured to an RI of about 1.338.

Regards,
Glen
On Mar 25, 2009, at 6:40 AM, Sylvie Le Guyader wrote:



Hi everyone

I am trying to find some mounting medium with a refractive index close to the one of water (1.33) in order to use our ZDC (Olympus zero drift control, IR-based hardware autofocus) with fixed samples.

Currently we use Dako S3023 and the focus is regularly lost without apparent reason. Andor, who sold us the system, claims that it could be because the IR of the mounting medium doesn’t contrast enough with the one of glass. I asked Dako for the RI of S3023 and they repeatedly stated 1.47.1-5 which is pretty vague but is definitely not 1.33. However I have found here on the confocal server that the RI of Dako S3023 had been measured and would be 1.372.

Does anyone know what the Dako RI is and where I could find a mounting medium that hardens and has an RI close to 1.33? does anyone have any experience with imaging fixed and mounted cells with a hardware autofocus?

Thanks for your help!!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

+46 (0)8 608 9240

Guenter Giese

AW: mounted cells and a hardware autofocus RI mismatch

Hi Christophe,

> So I ended up mounting my cells in PBS( +n-propyl-galate as
> an anti-fade), so that the RI is 1.33. I struggled to find a
> good sealant that allowed me to keep my slides (PBS is not
> viscous like PBS/glycerol and evaporates very rapidly). I
> tested VALAP, DPX but none were impermeant enough to avoid
> evaporation after a few days... So if you have a proposition
> I'd be glad to hear it.

Try to store your slides in a humidity chamber on a support located above
the mounting buffer. The mounting buffer (PBS in your case) will provide the
appropriate humidity / vapor pressure to equilibrate the atmosphere /
exchange with your sample.

Please check in advance if your sealant will solubilize in this humid
atmosphere...

Hope this helps

Guenter

>
> Christophe Leterrier
>
>
> On Wed, Mar 25, 2009 at 17:37, Glen MacDonald
> <[hidden email]> wrote:
>
>
> I've read that Fluoromount G, a hardening mountant from
> Southern Biotech, has been measured to an RI of about 1.338.
>
> Regards,
> Glen
> On Mar 25, 2009, at 6:40 AM, Sylvie Le Guyader wrote:
>
>
>
> 
>
> Hi everyone
>
>
>
> I am trying to find some mounting medium with a
> refractive index close to the one of water (1.33) in order to
> use our ZDC (Olympus zero drift control, IR-based hardware
> autofocus) with fixed samples.
>
>
>
> Currently we use Dako S3023 and the focus is
> regularly lost without apparent reason. Andor, who sold us
> the system, claims that it could be because the IR of the
> mounting medium doesnt contrast enough with the one of
> glass. I asked Dako for the RI of S3023 and they repeatedly
> stated 1.47.1-5 which is pretty vague but is definitely not
> 1.33. However I have found here on the confocal server that
> the RI of Dako S3023 had been measured and would be 1.372.
>
>
>
> Does anyone know what the Dako RI is and where
> I could find a mounting medium that hardens and has an RI
> close to 1.33? does anyone have any experience with imaging
> fixed and mounted cells with a hardware autofocus?
>
>
>
> Thanks for your help!!
>
>
>
>
>
> Med vänlig hälsning / Best regards
>
>
>
> Sylvie
>
>
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader
>
> Dept of Biosciences and Nutrition
>
> Karolinska Institutet
>
> Novum
>
> 14157 Huddinge
>
> Sweden
>
> +46 (0)8 608 9240
>
>
>
>
>
>
>

Rosemary.White

Re: AW: mounted cells and a hardware autofocus RI mismatch

How about slathering a thick coating of nail polish on top of the VALAP or
other sealant? We mount plant sections in glycerine jelly and seal with
nail polish - they can last for years without drying out.
cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334

On 26/03/09 9:57 PM, "Guenter Giese" <[hidden email]>
wrote:

> Hi Christophe,
>
>> So I ended up mounting my cells in PBS( +n-propyl-galate as
>> an anti-fade), so that the RI is 1.33. I struggled to find a
>> good sealant that allowed me to keep my slides (PBS is not
>> viscous like PBS/glycerol and evaporates very rapidly). I
>> tested VALAP, DPX but none were impermeant enough to avoid
>> evaporation after a few days... So if you have a proposition
>> I'd be glad to hear it.
>
> Try to store your slides in a humidity chamber on a support located above
> the mounting buffer. The mounting buffer (PBS in your case) will provide the
> appropriate humidity / vapor pressure to equilibrate the atmosphere /
> exchange with your sample.
>
> Please check in advance if your sealant will solubilize in this humid
> atmosphere...
>
> Hope this helps
>
> Guenter
>
>
>
>
>>
>> Christophe Leterrier
>>
>>
>> On Wed, Mar 25, 2009 at 17:37, Glen MacDonald
>> <[hidden email]> wrote:
>>
>>
>> I've read that Fluoromount G, a hardening mountant from
>> Southern Biotech, has been measured to an RI of about 1.338.
>>
>> Regards,
>> Glen
>> On Mar 25, 2009, at 6:40 AM, Sylvie Le Guyader wrote:
>>
>>
>>
>> 
>>
>> Hi everyone
>>
>>
>>
>> I am trying to find some mounting medium with a
>> refractive index close to the one of water (1.33) in order to
>> use our ZDC (Olympus zero drift control, IR-based hardware
>> autofocus) with fixed samples.
>>
>>
>>
>> Currently we use Dako S3023 and the focus is
>> regularly lost without apparent reason. Andor, who sold us
>> the system, claims that it could be because the IR of the
>> mounting medium doesn¹t contrast enough with the one of
>> glass. I asked Dako for the RI of S3023 and they repeatedly
>> stated 1.47.1-5 which is pretty vague but is definitely not
>> 1.33. However I have found here on the confocal server that
>> the RI of Dako S3023 had been measured and would be 1.372.
>>
>>
>>
>> Does anyone know what the Dako RI is and where
>> I could find a mounting medium that hardens and has an RI
>> close to 1.33? does anyone have any experience with imaging
>> fixed and mounted cells with a hardware autofocus?
>>
>>
>>
>> Thanks for your help!!
>>
>>
>>
>>
>>
>> Med vänlig hälsning / Best regards
>>
>>
>>
>> Sylvie
>>
>>
>>
>> @@@@@@@@@@@@@@@@@@@@@@@@
>>
>> Sylvie Le Guyader
>>
>> Dept of Biosciences and Nutrition
>>
>> Karolinska Institutet
>>
>> Novum
>>
>> 14157 Huddinge
>>
>> Sweden
>>
>> +46 (0)8 608 9240
>>
>>
>>
>>
>>
>>
>>

jens rietdorf

Re: mounted cells and a hardware autofocus RI mismatch

In reply to this post by Sylvie Le Guyader-2

Hello Sylvie

polyacrylamid?

Cheers, jens

Sylvie Le Guyader-2 wrote

Hi everyone

I am trying to find some mounting medium with a refractive index close to
the one of water (1.33)

Sylvie Le Guyader

lgelman

Re: mounted cells and a hardware autofocus RI mismatch

In reply to this post by Sylvie Le Guyader-2

Dear Sylvie,

You may want to have a look at this article (see below)… I never tried this mounting medium, but it sounds great. In case you would try it, I would be very grateful to get a feed-back.

Best,

Laurent.

Microsc. Res. Tech. 70:1–9, 2007.

2,20-Thiodiethanol: A New Water Soluble Mounting Medium for High Resolution Optical Microscopy

THORSTEN STAUDT,1,2 MARION C. LANG,1 REBECCA MEDDA,2 JOHANN ENGELHARDT,1 AND STEFAN W. HELL1,2*

ABSTRACT: The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 lm deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,20-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence

quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.

______________________________________

Laurent Gelman, PhD

Friedrich Miescher Institut

Facility for Advanced Imaging and Microscopy

WRO 1066.2.16

Maulbeerstrasse 66

CH-4058 Basel

Tel.: 061 696 43 38 / Cell phone: 079 618 73 69

www.fmi.ch

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: mercredi, 25. mars 2009 14:41
To: [hidden email]
Subject: mounted cells and a hardware autofocus RI mismatch

Hi everyone

I am trying to find some mounting medium with a refractive index close to the one of water (1.33) in order to use our ZDC (Olympus zero drift control, IR-based hardware autofocus) with fixed samples.

Thanks for your help!!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

+46 (0)8 608 9240

Sylvie Le Guyader-2

hardware autofocus reliability

In reply to this post by Csúcs Gábor

Hi list and specifically Gabor

I remembered Gabor's comment from my earlier post on the confocal server
about hardware autofocus so I come and ask for more information.

We have managed to get our ZDC (Olympus hardware autofocus) to work reliably
with our 10x air, 20x oil, 40x oil and 60x oil NA1.35. The 60x oil is a loan
from Olympus after we have had so much trouble getting the ZDC to work
reliably with an Olympus 60x NA1.45 and also with their 60x NA 1.42.
Basically the two highest NA objectives we have do not work reliably. Does
anyone have had a similar experience with hardware autofocus reliability
depending on the objective? Is the problem linked to the variation of lens
quality from one objective to the next or is it related to the NA (ie the
higher the NA, the less reliable the ZDC).

Thanks!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157 Huddinge
Sweden
+46 (0)8 608 9240

autofocus?

Csúcs Gábor

Re: hardware autofocus reliability

Hallo Sylvie,

Unfortunately we have never used/tested an objective with an NA higher
then 1.4.
All the hardware auto-focus solutions (I know of) are reflection based
(IR laser reflection from the air/glass or glass/buffer-sample) surface.
However I don't the realization details. What we have seen with many
systems - that with very low NA - this reflection is too weak so the
auto-focus doesn't work. Nevertheless, high NA should rather help. The
CRIFF system from ASI for example works only with "TIRF objectives".
With lower NA objectives coverslips with reflective coating are
required. What I can imagine is the in the case of the Olympus setup
(when using very high NA objectives) is that the reflected laser beam
doesn't anymore reaches the auto-focus detector. So I'd speculate that
the effect is not objective but NA dependent...

Cheers Gabor

> Hi list and specifically Gabor
>
> I remembered Gabor's comment from my earlier post on the confocal server
> about hardware autofocus so I come and ask for more information.
>
> We have managed to get our ZDC (Olympus hardware autofocus) to work reliably
> with our 10x air, 20x oil, 40x oil and 60x oil NA1.35. The 60x oil is a loan
> from Olympus after we have had so much trouble getting the ZDC to work
> reliably with an Olympus 60x NA1.45 and also with their 60x NA 1.42.
> Basically the two highest NA objectives we have do not work reliably. Does
> anyone have had a similar experience with hardware autofocus reliability
> depending on the objective? Is the problem linked to the variation of lens
> quality from one objective to the next or is it related to the NA (ie the
> higher the NA, the less reliable the ZDC).
>
> Thanks!
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader
> Dept of Biosciences and Nutrition
> Karolinska Institutet
> Novum
> 14157 Huddinge
> Sweden
> +46 (0)8 608 9240
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Gabor Csucs
>> Sent: 25 March 2009 14:56
>> To: [hidden email]
>> Subject: Re: mounted cells and a hardware autofocus RI mismatch
>>
>> Hallo Sylvie,
>>
>> Before going more into the details - which objective are you using. The
>> reliability of the ZDC depends very much on the objective
>> (irrespectively whether it should "officially" work or not).
>>
>> Cheers Gabor
>>
>>> Hi everyone
>>>
>>> I am trying to find some mounting medium with a refractive index close
>>> to the one of water (1.33) in order to use our ZDC (Olympus zero drift
>>> control, IR-based hardware autofocus) with fixed samples.
>>>
>>> Currently we use Dako S3023 and the focus is regularly lost without
>>> apparent reason. Andor, who sold us the system, claims that it could
>>> be because the IR of the mounting medium doesn’t contrast enough with
>>> the one of glass. I asked Dako for the RI of S3023 and they repeatedly
>>> stated 1.47.1-5 which is pretty vague but is definitely not 1.33.
>>> However I have found here on the confocal server that the RI of Dako
>>> S3023 had been measured and would be 1.372.
>>>
>>> Does anyone know what the Dako RI is and where I could find a mounting
>>> medium that hardens and has an RI close to 1.33? does anyone have any
>>> experience with imaging fixed and mounted cells with a hardware
>>>
> autofocus?
>
>>> Thanks for your help!!
>>>
>>> Med vänlig hälsning / Best regards
>>>
>>> Sylvie
>>>
>>> @@@@@@@@@@@@@@@@@@@@@@@@
>>>
>>> Sylvie Le Guyader
>>>
>>> Dept of Biosciences and Nutrition
>>>
>>> Karolinska Institutet
>>>
>>> Novum
>>>
>>> 14157 Huddinge
>>>
>>> Sweden
>>>
>>> +46 (0)8 608 9240
>>>
>>>
>> --
>> Gabor Csucs
>> Light Microscopy Centre, ETH Zurich
>> Schafmattstrasse 18, HPM F16
>> CH-8093, Zurich, Switzerland
>>
>> Web: www.lmc.ethz.ch
>> Phone: +41 44 633 6221
>> Mobile: +41 79 758 21 58
>> Fax: +41 44 632 1298
>> e-mail: [hidden email]
>>