Zeiss objective with CUBIC clearing reagent

[Gordon, Peter]

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Hi all,

I was just wondering if anyone has tried dipping the 20x water dipping Zeiss objective directly into CUBIC clearing Reagent 1. I currently image cleared tissues through a coverslide but avoiding this would be most helpful.

Peter

[Moulding, Dale]

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Hi Peter,

I'd double check with your Zeiss rep if I was you, but every time I have asked the answer has always been yes for all aqueous based clearing reagents (scale, clarity, opticlear etc). As long as they aren’t solvent based it is fine.

However, when I've done this with opticlear the images were terrible dipped directly into opticlear (RI 1.47 I think), and much much better using a coverslip over the sample, with water between objective and coverslip. I guess it is the depth of the wrong RI the objective looks through. 2.5mm of RI 1.47 gives terrible spherical aberration, but looking through water to the coverslip minimises the distance you are looking through the wrong RI.

Cheers

Dale

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gordon, Peter
Sent: 04 October 2018 14:54
To: [hidden email]
Subject: Zeiss objective with CUBIC clearing reagent

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Hi all,

I was just wondering if anyone has tried dipping the 20x water dipping Zeiss objective directly into CUBIC clearing Reagent 1. I currently image cleared tissues through a coverslide but avoiding this would be most helpful.

Peter

[Mark Scott-2]

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Hi Peter,

Speak to your local Zeiss rep, they have a company Powerpoint detailing the “OK to use” and “Banned” clearing methods for use with their lenses.  

Basically, I was told any of the aqueous methods such as Scale, CUBIC, Clarity, FocusClear etc are absolutely fine with their lenses.   There are some question marks over FRUIT and things like BABB/3DISCO are no go areas.

While you can use CUBIC with their lenses (although again, worth double checking with the Zeiss rep where you are), the biggest issue will be the R.I. mis-match.  I think you will find the images you get will be terrible quality and imaging through a coverslip may actually be better anyway.  When I tried CUBIC even with Zeiss’ clearing objective on the Z.1 and matched the R.I. to around 1.47 it was still prone to blurry images.

Thanks
Mark

_______________________________________
 
Mark Scott
Senior Microscopist
Institute of Molecular Bioscience
Level 6 North, 306 Carmody Road (Building 80)
The University of Queensland
Brisbane, Queensland, 4072, Australia
https://imb.uq.edu.au/microscopy
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On 5/10/18, 12:17 am, "Confocal Microscopy List on behalf of Gordon, Peter" <[hidden email] on behalf of [hidden email]> wrote:

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    Hi all,
   
    I was just wondering if anyone has tried dipping the 20x water dipping Zeiss objective directly into CUBIC clearing Reagent 1. I currently image cleared tissues through a coverslide but avoiding this would be most helpful.
   
    Peter
   
   

[Gordon, Peter]

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Thanks for replying Dale and Mark,

I'll do a comparison experiment this week and see how it turns out. Glad I moved away from BABB, this wouldn't have even been an option before.

Peter

Dr Peter Gordon BSc Hons
Division of Cancer Studies
Room 2.32 New Hunt's House
King's College London
02078486174

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Moulding, Dale
Sent: 04 October 2018 16:06
To: [hidden email]
Subject: Re: Zeiss objective with CUBIC clearing reagent

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Hi Peter,

I'd double check with your Zeiss rep if I was you, but every time I have asked the answer has always been yes for all aqueous based clearing reagents (scale, clarity, opticlear etc). As long as they aren’t solvent based it is fine.

However, when I've done this with opticlear the images were terrible dipped directly into opticlear (RI 1.47 I think), and much much better using a coverslip over the sample, with water between objective and coverslip. I guess it is the depth of the wrong RI the objective looks through. 2.5mm of RI 1.47 gives terrible spherical aberration, but looking through water to the coverslip minimises the distance you are looking through the wrong RI.

Cheers

Dale

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gordon, Peter
Sent: 04 October 2018 14:54
To: [hidden email]
Subject: Zeiss objective with CUBIC clearing reagent

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Hi all,

I was just wondering if anyone has tried dipping the 20x water dipping Zeiss objective directly into CUBIC clearing Reagent 1. I currently image cleared tissues through a coverslide but avoiding this would be most helpful.

Peter