Re: optical clearing of tissue

Posted by Russell Spear on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-optical-clearing-of-tissue-tp3783665p3788668.html

Hi Micheal

I just checked the refractive index of pure glycerol 1.47, lactic
acid/glycerol (1:3) 1.44, and sat. chloral hydrate 1.60.

I normally infiltrate my specimens through a graded series of water /
clearing agent steps (how fast and how large the steps are depends on
the size and how delicate the material is).  Plant material normally
takes a long time because I have large specimens.

I have not damaged a objective by using glycerol alone or by mounting
lactic acid/glycerol cleared material in pure glycerol for immersion
imaging but my Leica 20X and 40X NA. 1.15 & 1.2 are glycerol immersion
objectives.

When I image chloral cleared material I mount the specimen in chloral
hydrate and coverslip it, then use standard immersion oil between the
coverslip and a standard 60X or 100X objective.

You should check with your microscope rep. about how suitable your
objectives are for glycerol immersion work.

Russ

Michael Weber wrote:

> 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
>>>>> <[hidden email]>                                              
>>>>>
>>>>>                                                                                                                        
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> 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]