A case for beer – or – what a density gradient medium can do for your microscopy.

Our paper, like so many scientific findings, was brought about by a beer – or more specifically a discussion over a beer.

“I had a beer with David (Drechsel)” Jochen (Rink) said to me after one of our weekly scientific social events at the MPI-CBG. Over their beers they had discussed the challenges we were having imaging planaria and David had suggested we use Iodixanol as a supplement to increase the refractive index of mounting media – a suggestion which proved to be key to our success. While this particular problem and solution were quite straightforward, getting to the point where we could even recognize the problem and thus seek a solution involved a few more people (and a few more beers!) This is the story of how our paper, which describes a simple and straight forward method to correct for spherical aberrations of live tissues thus enabling significantly improved image resolution and quality in deeper tissue layers, came to be.

During my Ph.D. training in cell biology at the University of British Columbia in the laboratory of Jim (Johnson) I became used to thinking of microscopy as a tool rather than a challenge. This changed when I started my postdoctoral work, which focused on imaging cell dynamics during regeneration in planarian flatworms, in Jochen’s lab. Jochen warned me that this would “not be an easy” endeavor and, given how few tools were available for this relatively rare model system, I believed him. Once I started the imaging experiments and realized it was not possible to see any nuclear structure beyond the outermost cell layer however, I started to understand just how difficult the task I had taken on was. I took advantage of the lab’s species collection to obtain and test an unpigmented planarian species and exhausted the resources of our well-equipped light microscopy facility but the epithelium still seemed to act like a black-out curtain. At this point in time I did not have a well-developed understanding of optics in complex tissues however with the help of MPI-CBGs Moritz (Kreysing) and his student Alfonso (Garcia) I began to understand why a lack of pigment does not automatically mean that a tissue becomes optically clear. One of Moritz’ projects had seen a similar “black-out” effect when imaging dense retinal tissue and he had been able to overcome this challenge and image the deeper tissue layers by tuning the refractive index of their mounting media. While this was a bit different from what we were looking to do, as their work focused on fixed tissues, their results did suggest that the problem we were having could be caused by a significant difference in the refractive indexes of the planarian tissue and the aqueous mounting medium we were using at this time, and that if this were the case it might be possible to improve our images by tuning the refractive index of our mounting media. What we needed to do seemed clear however we still faced a challenge – while refractive index adjustment is a core component of state of the art clearing techniques in fixed tissues we had to find a component to tune the refractive index of our mounting medium to that of the sample without harming the live specimen.

I started by trying obvious candidates such as glycerol and sucrose but their high osmolality created a lethal environment for planaria. Halocarbonoils, which we tried next, worked well in fixed planaria but their hydrophobic nature made it impractical for an aquatic model system. After many trials and even more errors, I found myself at an institute social event sharing a beer and discussing these failures with Lennart (Hilbert). Lennart was studying DNA structure by live super-resolution microscopy in Nadine’s (Vastenhouw) lab at the time, and used BSA for refractive index tuning. While BSA was a promising candidate for planaria, the rather low refractive index tuning range and the viscosity and stickiness of saturated BSA solutions limited its usefulness. Lennart also found these limitations frustrating and so, while his approach at that time could not solve my problem, I did find myself a companion in the search for a live compatible refractive index tuning media supplement.

The postdocs had rounds of  beers and discussions week after week but it was not until (of course) the PI got involved that we had our next breakthrough. At the time of Jochen’s forementioned beer with David we were focusing on epithelial cell dynamics because this was really the only cell type we could image in planaria due to the “black-out” effect. David, who was leading the MPI-CBG’s protein expression facility at the time, suggested over that famous beer we should give Iodixanol a try. Like many others he knew Iodixanol as a density gradient medium. While it was widely used for cell or cell organelle isolation, David was also aware though that the stock solution has a rather high refractive index which meant it might be able to meet our particular needs.

After performing some initial tests it became clear that Iodixanol was indeed the reagent we had been looking for. We were able to image past the first cell layer in planaria and significantly improved the live imaging quality in these specimens. Since we were not aware of any compound with similar properties we were eager to test it in other model systems. The diversity of MPI-CBG provided us with access to a number of different model systems and so we tried to improve live imaging in zebrafish embryos. Although this organism is thought to be easy to image, I knew of Lennart’s frustrations thanks to our earlier beers and was happy to share that there might be something which would help.  Since imaging zebrafish was so far already of high quality Iodoxanol supplementation did not lead to the clear “day and night” effect we could observe with planaria. As I am a very self-critical (some would even call it pessimistic) experimentalist I started to doubt the broad applicability of Iodixanol but luckily Lennart has a more optimistic approach to things and just said: “Of course it works, it’s Physics.” – and sure enough it did.

In the end it was the diversity of the institute and the inter-lab interactions which led us to this story. We are happy that together we could establish with Iodixanol a compound which can now compensate for spherical aberrations in vivo and we are eager to hear how it works for the community – ideally over another case of beer!

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