[OpenSPIM] OpenSPIM and optically cleared 3D tissue

Tue Oct 27 07:02:47 CDT 2015

Hi,

I am working on a SPIM microscope for cleared tissue. I plan to put it in  
Wiki at some point, but my boss wants to wait until the paper is written.  
Anyway, I use the approach of a closed chamber and an air objective. My  
chamber is 3D printed with windows made of microscope cover glasses - the  
"cost effective" approach :P. This only work for the non-corrosive media  
though. An alternative is a big glass cuvette. The RI of glass is already  
quite similar to the cleared tissue, so I don't think the windows are a  
big issue. Note that Deisseroth also has a cuvette in his design.

If you find a mounting medium that has the same RI as your sample, and you  
only move the sample while the chamber is stationary (same as in  
OpenSPIM), then you don't have drift, or at least not that much.  However.  
the air objective approach works for small NA, so it's best for small  
magnification - for me 4X/0.13 works nicely. Afterwards the aberrations  
will prevent you from achieving the resolution that you would have in air  
(there is a paper about it, Silvestri "Correcting spherical aberrations in  
confocal light sheet microscopy: A theoretical study"). It's a good way to  
start while collecting funds for a proper objective ;)

For mounting medium, the easiest and cheapest solution is to go the oil  
way, like in the CUBIC papers (Susaki et al, see in Methods/Imaging). The  
stuff that is used for confocal microscopy - FocusClear, sRIMS, Reagent 2,  
you name it - is usually too expensive, too unstable, or both (i.e.  
Reagent 2 for CUBIC tends to crystallize if your lab's air-conditioning is  
set too low, sRIMS also does something very weird).

If you have more specific questions, go ahead and ask. Greetings,
Monika

W dniu .10.2015 o 19:16 Michael Weber <weber at mpi-cbg.de> pisze:

> Hi Jamie,
>
> if you are using hazardous clearing media and don't have a dedicated  
> dipping objective available, one idea would be to use long-working  
> distance air >objectives and a smaller, closed glass chamber. The main  
> issue I would expect from such a combination would be a drift of your  
> detection focal plane >when moving the sample through the light sheet,  
> because the light has to travel through layers of different refractive  
> indices, and you change the >"thickness" of those layers. But this drift  
> should be linear and could be corrected by adapting the distance between  
> light sheet and detection >objective while acquiring a z-stack. So you  
> would either need to motorize the mirror for translating the light  
> sheet, or your detection objective, and >then implement the drift  
> correction in the software. Might be worth a try, unless I overlooked  
> something important here.
>
> Cheers,
> Michael
>
> On Oct 24, 2015, at 8:27 AM, Jamie Flynn <Jamie.Flynn at uon.edu.au> wrote:
>
>> Hi Pete,
>>
>> Thanks for the link. Some very impressive images from the mesolens.  
>> You¹re
>> right that pickings are slim for commercially available lenses. Leica  
>> and
>> Olympus have some objectives that are suitable for various types of
>> cleared tissue, but come with hefty price tags. Apparently Zeiss have
>> something in the works as well (not that it¹ll be cheap!). The RI range  
>> of
>> imaging media for different clearing techniques is obviously one of the
>> bigger challenges (e.g. 1.45 for Focusclear and 1.56 for DBE). Will look
>> into a custom design as you suggest.
>>
>> Cheers
>> Jamie
>>
>>
>>
>> On 23/10/2015 7:16 pm, "Peter Gabriel Pitrone" <pitrone at mpi-cbg.de>  
>> wrote:
>>
>>> with the exeption of the "Meso-lens" from Prof. Brad Amos... But I'm  
>>> sure
>>> it uses glue, and you probably can not afford it.
>>>
>>> http://www2.mrc-lmb.cam.ac.uk/research/technology-transfer/mesolens-micros
>>> copy/
>>>
>>>
>>>
>>> ----- Original Message -----
>>> From: "Peter Gabriel Pitrone" <pitrone at mpi-cbg.de>
>>> To: "Jamie Flynn" <Jamie.Flynn at uon.edu.au>
>>> Cc: openspim at openspim.org
>>> Sent: Friday, October 23, 2015 10:14:06 AM
>>> Subject: Re: [OpenSPIM] OpenSPIM and optically cleared 3D tissue
>>>
>>> Hey Jamie,
>>>
>>> I would have someone design the lenses (and therefor chamber as well)  
>>> for
>>> you made in a way the is fluid/liquid proof WITHOUT using glue, also
>>> keeping in mind the absolute largest (NA in terms of detection lens,  
>>> and)
>>> working distance possible so you could put in bigger tissues. I doubt
>>> that there is a objective on the market that would meet your  
>>> requirements.
>>>
>>> Regards,
>>> Pete
>>>
>>> ----- Original Message -----
>>> From: "Jamie Flynn" <Jamie.Flynn at uon.edu.au>
>>> To: openspim at openspim.org
>>> Sent: Friday, October 23, 2015 1:58:29 AM
>>> Subject: [OpenSPIM] OpenSPIM and optically cleared 3D tissue
>>>
>>> Hi everyone,
>>> We¹re building a dual illumination OpenSPIM at the Hunter Medical
>>> Research Institute in Newcastle, Australia. Our ai m is to have the  
>>> scope
>>> and analysis pipeline used as a core facility for lab groups to image  
>>> 3D
>>> tumour cell cultures, developing embryos and most importantly for us,
>>> optically cleared 3D tissue samples (e.g. CLARITY, CUBIC, 3DISCO, Scale
>>> etc.). Imaging cleared 3D samples would be an interesting avenue for
>>> OpenSPIM and it¹d be great to hear about any hardware modifications or
>>> mounting protocols others may have used to do it.
>>>
>>> If anyone wants to try clearing out their own tissue samples, my
>>> colleagues and I put together a handbook that describes the reagents,
>>> equipment and detailed protocols for ŒPassive CLARITY¹. We compiled the
>>> protocols outlined in Tomer et al. Nat. Prot. 9(7) 2014 and Yang et al.
>>> Cell (158) 2014 and added in our own refinements (after much trial and
>>> error!). It¹s a very simple technique and has worked really well in our
>>> hands. Here¹s the link. Download it to your desktop in case the link  
>>> gets
>>> broken:
>>> https://drive.google.com/open?id=0BzIZDgZFR2srNFk2dHFqT1UycFk
>>>
>>> All the best--------------------
>>> Jamie Flynn (PhD)University of Newcastle
>>> Hunter Medical Research Institute
>>> Room 3613 level 3 east
>>> 1 Kookaburra Circuit, New Lambton Heights
>>> NSW 2305, Australia.
>>> pH: +612 40420468Mobile: +61 416003787
>>> Email: jamie.flynn at uon.edu.au
>
>>>> _____________
>
> Michael Weber
> Postdoc, Huisken lab
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108, 01307 Dresden
> Tel. 0049 351/2102837
>
> http://www.mpi-cbg.de/huisken
>

-- 
Dr Monika Pawłowska
Nencki Institute
02-093 Warsaw
Pasteura 3
Poland
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