Re: optical clearing of tissue

Posted by Michael Weber-4 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-optical-clearing-of-tissue-tp3783665p3788391.html

Russ,

the objectives are Zeiss W Achroplan (front end covered with ceramics)
with which you can directly dip into water. If they survive treatment
with acids is another question, I am thinking of the glue which holds
the front lens.

Do you use the hydrate and lactic acid/glycerol solutions as one step in
the protocol, or right in your imaging medium?

Michael


Russ Spear schrieb:

> Hi
>
> Could you use some thing like saturated chloral hydrate in water (you'll
> need a drug permit), or a lactic acid/glycerol solution both are water
> miscible. I use these on plant material quite often.  The major problem
> is having to image in water, can you use an objective made for glycerin
> immersion?
>
> Russ
>
> Michael Weber wrote:
>> Dear all,
>>
>> first of all, thanks for the replies off- and online!
>>
>> I should have mentioned a bit more details in my initial post. The
>> embryos get mounted in agarose and investigated with dipping
>> objectives with water as medium, so the RI of the surrounding medium
>> is around 1.33. I expect the RI of the embryo to be higher, so the
>> limiting factor for penetration depth is diffraction between medium
>> and embryo. But, there is nothing I can do about that, right? Clearing
>> with i.e. BABB would not make any sense, if I put the samples back in
>> water-like medium afterwards and do not use oil objectives anyway. So
>> the imaging conditions are already as optimized as possible - is that
>> a valid conclusion, or am I missing something?
>>
>> cheers,
>> Michael
>>
>>
>> Phil Hertzler schrieb:
>>> Mike,
>>>
>>> Methyl salicylate (oil of wintergreen) also works well and smells
>>> better than BABB. :-) Transition through 100% ethanol from aqueous
>>> buffer.  I've stored samples over 15 years in MS without loss of
>>> fluorescence.
>>>
>>> Best regards,
>>>
>>> Phil
>>>
>>> At 01:21 PM 10/7/2009, you wrote:
>>>> Mike
>>>> This is a review that describes our procedure of clearing mammalian and
>>>> insect tissue with BABB. Reprints are available on request
>>>>
>>>> Zucker, R.M.Technical note: Whole insects and Mammalian Embryo Imaging
>>>> with Confocal Microscopy: Morphology and Apoptosis. Cytometry 2006 69A:
>>>> 1143-1152
>>>>
>>>> Best wishes
>>>> bob
>>>>
>>>> Robert M. Zucker, PhD
>>>> U.S. Environmental Protection Agency
>>>> Office of Research and Development
>>>> National Health and Environmental Effects Research Laboratory.
>>>> Toxicology Assessment Division
>>>> Telephone: [phone]   Fax: [phone]
>>>> e-mail: [hidden email]
>>>>
>>>> Mail address: USEPA,ORD,NHEERL,TAD
>>>> Developmental Biology Branch ( MD 67)
>>>> Research Triangle Park, North Carolina, 27711
>>>>
>>>> Shipping address:
>>>> 2525 E.NC Highway 54
>>>> Durham, NC, 27713
>>>>
>>>>
>>>>
>>>>                                                                                                                        
>>>>
>>>>   From:       Michael Weber
>>>> <[hidden email]>                                                                        
>>>>
>>>>                                                                                                                        
>>>>
>>>>   To:        
>>>> [hidden email]                                                                        
>>>>
>>>>                                                                                                                        
>>>>
>>>>   Date:       10/07/2009 11:56
>>>> AM                                                                                      
>>>>
>>>>                                                                                                                        
>>>>
>>>>   Subject:    optical clearing of
>>>> tissue                                                                              
>>>>
>>>>                                                                                                                        
>>>>
>>>>   Sent by:    Confocal Microscopy List
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>>>>                                                                                                                        
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> Dear list,
>>>>
>>>> I am looking for advice on optical "clearing" of fixed tissue before
>>>> staining it and using it for light microscopy. Actually "tissue" is not
>>>> the precise term, since I would like to clear whole fly embryos. This
>>>> process seems to be well established in histology, i.e. using Xylene. I
>>>> also found a commercial product called "Histo-Clear" (National
>>>> Diagnostics), which claims to preserve tissue structures rather well,
>>>> while being less nasty compared to Xylene. Did you guys ever use
>>>> something
>>>> like that? Any input welcome.
>>>>
>>>> cheers,
>>>> Michael
>>>
>>> ------------------------------------------------------------------------
>>> Philip L. Hertzler
>>> Professor
>>> Central Michigan University
>>> Dept. of Biology, Brooks Hall 217
>>> 200 Library Dr.
>>> Mount Pleasant, MI 48859
>>>
>>> Phone: [phone]
>>> Fax: [phone]
>>> Email: [hidden email] or
>>> [hidden email]