COLM for rapid and high-resolution imaging of intact brains and other tissues

raju

Hi Folks,

I would like to introduce COLM (CLARITY Optimized Light-sheet Microscope) for high-speed imaging of large clarified samples at high resolution.

At the least three major innovations were needed over the light sheet microscopy idea to make COLM a reality. The details are here:
http://www.nature.com/nprot/journal/v9/n7/full/nprot.2014.123.html
And, additional details will be added here:
http://clarityresourcecenter.com/COLM.html

COLM specs in brief:
* An entire mouse brain can be imaged in 3-4 hours at half a micron resolution, and in about a day at quarter of micron resolution (spine level details).
* A mouse brain is about 3 TB of raw data at half a micron resolution, and about 10 TB at quarter micron. Therefore, COLM does real-time data compression (4-5 fold loss-less compression).
* In current version, a tissue of 1.4 cm x 1.4 cm x 4.5 cm size can be imaged at quarter of micron resolution.
* Current version can image up to 4 colors in one sample. 6 LASER engine is suggested in the current design.
* A very advanced, intuitive and easy-to-use control software (more or less a push-button system).

A conscious effort was made to develop COLM with off-the-shelf parts. Very minimum custom manufacturing needed. Current cost is estimated at less than 400K (excluding control software, which we will provide free-of-cost), and will become cheaper. I would estimate a system assembly time of a few days, once all parts are available.

Although I will be very happy to help spread the technology, I do realize that many labs may not be equipped for custom-built COLM. Therefore, currently exploring commercialization options to make it easily accessible and even more easier to use, provided there is enough interest.

As far as I know there is no other system that can image as large a sample at as high-resolution in as quick time as COLM.

Thanks,

Raju Tomer

nicolasrenier

Thanks Raju for this development. I also found light-sheet microscopy to be excellent for this kind of large field of view imaging. I'm not tech-savvy, but I'll be interested to delve into the details of your microscopy optimisations. We use a commercial light-sheet microscope (from LaVision), which gives us already great results, but your technique might make it even better.

Just a concern, it seems that you have aberrant reflections in some panels in your paper that leads to duplicated neurons at high mag (see panels 8b). Do you think this is an issue with COLM, and that it could be easily fixed? I also noticed in traditional light-sheet that if the focus point of the light sheet is misaligned with the center of the lens, it can create those optical artifacts.

raju

Thanks Nicolas. La Vision backbone could potentially be adapted for COLM, though not easily. Not sure what kind of resolution you get out of it (CUBIC paper folks used La vision system, and honestly I was not much impressed by the resolution of the images). I think the most important advance of COLM is the ability to image "entire brain" at "near dendritic spine" resolution in quick time. And that needed three major innovations outlined in Figure 4. In my opinion, all three are absolutely needed to even take a well focused high-resolution stack, going from dorsal all the way to ventral, let alone imaging the whole brain.

I see your point about Figure 8b. I also noticed it and thought that it could have been a stitching artifact with neighboring tiles not completely aligning on the edges (there is no possibility of this to be an optical aberration, because of Figure 4b innovation). But then if you follow vertically in the image, you can rule it out. In fact in other images similarly clustered neurons are also present. It seems to be a feature of PV neurons (I see similar arrangements in gabaergic labelled neurons as well: a tandem repetition of 4/5 very similar looking cells in a row in lot many places in the cortex. This to me (a relatively new neuroscientist) sounds like a novel observation made possible by unbiased whole brain imaging capability).

Lemme know if you are interested in looking at ways to adapt La Vision system for COLM for your personal use. Or if La vision folks are interested in contacting us / stanford otl ...

nicolasrenier

Thanks for your comments Raju!

I am also puzzled by the pictures in the Cubic paper, but from my experience with the LaVision system, I feel it has more to do with the clearing than the actual microscope.

I will definitely mention your imaging strategy to the LaVision folks, but it might be easier to build a system from scratch (or wait for commercial options!) than trying to retrofit the current microscope. I feel the NA of the macro zoom objective and light sheet generating lenses might not be adequate.

I will stick with "traditional" light sheet for now, because I feel it is good enough for what I'm doing, but I'll definitely get back in touch if I have precise questions to implement your advanced light sheet system.

raju

great, thanks Nicolas. Please feel free for any comments/questions.

Gabriel_Arisi

Dear @raju,

I would like to adapt openspim configurations. Instead of using a rotating chamber I
want to use a moving xy motors to obtain a z image stack.

Do you think that is the best approach? Congrats on the COLM publication!

Best regards