I like Russ's system.
My only comment is about the purpose of clearing.
Although cells are mostly water, they do contain protein, lipid and
"cell wall" structures that differ from water in refractive index
(This is why we can see DIC and phase contrast.). Particularly with
plant cells the optical chaos created by these refractive features is
a major limitation on how far into an "intact" tissue one can focus
while still being able to see small features. To avoid this problem,
one can "clear" the specimen by immersing it in liquids (such as
methyl-salicylate, BABB, chloral hydrate or many other liquids)
having RIs of >1.5, which match the RI of the protein/cellwall
structures. ( Structural features composed of lipids or other
materials that may have a lower RI, are often extracted by the
processes that replaces the water with the clearing agent.).
The result is a specimen with an (almost) uniform RI, and therefore
one in which the only contrast visible will be that caused by
absorption or fluorescence. The extent to which the specimen is
"cleared" will depend entirely on the extent to which the RI of the
clearing agent matches that of prominent features in the specimen
(i.e., it will vary with specimen type), but you might want to
remember that liquids that smell may have other biological effects
(on you!).
The remaining problem is that this final clear specimen will usually
have an RI that differs from (is higher than?) the RIs for which
commonly available objectives are corrected (In general the three
options are either 1.519 for oil, 1.448 for glycerol or 1.33 for
water). So even though the cleared specimen looks almost transparent
to the unaided eye, spherical aberration will degrade the microscope
optical performance as one focuses farther and farther into the
specimen.
Assuming that the cleared specimen has an RI higher than that of oil,
one can correct for this over a small range of focus distances, by
using an oil lens and an immersion oil of LOWER RI than that which
one usually uses. How much lower will depend on the thickness of the
layer between the front glass surface of the objective and the
coverslip and on the thickness and RI of the clearing agent between
the other side of the cover slip and the focus plane. It can only
really be determined by trial and error (images of point objects
should "go out of focus" in the same pattern above and below the
plane of focus. ) and because the RI of the oil and the clearing
agent varies with wavelength, the most suitable immersion oil mixture
may be different for different fluorophors.
Alternatively one can use an external SA Corrector such as that sold
by III (No commercial interest!!)
Just another example of how solving one problem can create others.
Jim P.
>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: 919-541-1585 Fax: 919-541-4017
>>>>>>e-mail: [log in to unmask]
>>>>>>
>>>>>>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
>>>>>><[log in to unmask]>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> To:
>>>>>>[log in to unmask]
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Date: 10/07/2009 11:56
>>>>>>AM
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Subject: optical clearing of
>>>>>>tissue
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Sent by: Confocal Microscopy List
>>>>>><[log in to unmask]>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>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: (989) 774-2393
>>>>>Fax: (989) 774-3462
>>>>>Email: [log in to unmask] or
>>>>>[log in to unmask]
--
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Phone: 608-238-3953
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