[OpenSPIM] OpenSPIM and optically cleared 3D tissue

Tue Oct 27 08:41:57 CDT 2015

They wouldn't, PLEASE don't even think about it...

They'd be eaten alive!

----- Original Message -----
From: "Jamie Flynn" <Jamie.Flynn at uon.edu.au>
To: "Michael Weber" <weber at mpi-cbg.de>
Cc: openspim at openspim.org
Sent: Tuesday, October 27, 2015 2:10:49 PM
Subject: Re: [OpenSPIM] OpenSPIM and optically cleared 3D tissue

Hi Michael, 

Nice idea. Especially motorising the corner mirrors as it would also make an ‘autofocus’ function during each z-step possible. Not just for the air/immersion fluid mismatch, but for RI inconsistencies throughout cleared samples as was done in ‘CLARITY optimised light sheet microsc opy’ (COLM – Tomer et al., Nat Protoc 9(7), 2014). 

Luckily most of the immersion fluids for CLARITY are fairly innocuous, so the dipping objectives should be fine. Although I’m not sure how the dipping objectives would fare with the organic solvents used in techniques like BABB or 3DISCO… 

Cheers 
Jamie 

From: < openspim-bounces at openspim.org > on behalf of Michael Weber < weber at mpi-cbg.de > 
Date: Monday, 26 October 2015 5:16 am 
To: " openspim at openspim.org " < openspim at openspim.org > 
Subject: Re: [OpenSPIM] OpenSPIM and optically cleared 3D tissue 

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 40420468 
Mobile: +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 

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