Covid has hit every one of us, and all in different ways. During the first national lockdown (in the UK we’re now on number 3), my daily life changed a lot. The University had shut down, so I was no longer going in to do research, and the nursery had shut down so the little man was kept home. And the wife was doing online learning for her studies, so I had a lot of time on my hands, most of which was spent trying to tire out and otherwise distract a toddler.
One of the things I did to stay motivated in science was to sign up to interesting webinars. This week I’ll be talking about one that I found particularly interesting hosted by the people at Inscopix (who make head-mounted miniscopes; www.inscopix.com): Ed Boyden talking about some of the tools he’s developed.
Ed Boyden, along with Karl Deisseroth, literally invented the field of optogenetics1, so this was bound to be an interesting listen. In fact, Inscopix archived the presentation, so I would urge anyone reading this to go check it out2. He went into a number of his recent advances for interrogating neuronal circuits, including optimising genetically-encoded voltage sensors and soma-targeted GCaMP.
The development that I found most interesting was expansion microscopy, a method for “nanoscale microscopy over extended scales”3. Not the most obvious description for a technique that lets you improve your microscope resolution by expanding the sample from the inside. Essentially, you infuse your sample with a swellable acrylamide polymer, anchor to cellular proteins as a scaffold, polymerise then add water to expand equally in all directions at once.
This enables you to drastically improve your imaging resolution without having to use ludicrously advanced (and expensive and difficult) microscopes (Figure 1 shows successive zooms of the same sample pre- and post-expansion). They managed to improve the imaging resolution to the point of distinguishing pre- and post-synapses in a brain section; this is otherwise extremely challenging to do using light microscopy, due to the diffraction limit of light causing blurriness at such limited distances.
However, this was not enough for Ed Boyden, so he decided to take expansion microscopy a step further with iterative expansion. He described this as “like PCR, but for expanding samples”. Essentially you do iterative rounds of polymer infusions and swelling to produce massive increases in sample volume and separation between particles. You can see the progression of improved resolution of “Brainbow” neuronal circuitry imaging in Figure 2.
Once again, this was not enough for Ed Boyden, who has since developed a technique for sequencing RNA in situ on a slice, leading to the precise mapping of RNA sequences across a section5. However, this leads me to my point for presenting this methodology (beyond my interest in cool new techniques), which is to emphasise the need to only use methods or technology for relevant applications. By which, I mean that it is all too easy to get lured in by some fancy new tech, and then spend a lot of time and money getting them working in your own research (and I promise you, it will take you a lot longer than you think, it always looks a lot easier and cleaner in the pioneering papers than it will be to do it yourself).
In fact, my first thought upon seeing these methods was to think of how I might use it in my own research. However, this level of resolution is just not necessary for me at all; there have only been a few occasions over the past decade that I actually needed any microscopy more advanced than “normal” epifluorescence, and that could be achieved with pretty basic confocal imaging.
I’ve come to realise that it is always better to stick with what you know (obviously doing iterative improvements as and when needed), and only progress to more advanced techniques when there is a definitive need for it, and have a plan for the work you plan to do with it and how it will improve your research impact. And assume it will take twice as long to get it running well compared to what you think.
Finally, always make sure you find a friend who can show you how to do a new technique before diving in for yourself; if you don’t know anyone personally, there are plenty of ways to find someone who can help, such as at conferences or workshops, or even just email people who’ve published in your field (academics tend to be happy to help, even someone who is technically a rival).
If you seek help, you will not only save yourself potentially huge amounts of cash on suboptimal equipment, but also huge amounts of wasted time and resources learning and optimising the technique. This is a lesson I wish I had learned years ago; I would have saved a lot of time and effort, so I hope you will too.
1. Boyden et al., Nature Neuroscience 8, 1263-1268 (2005) Millisecond timescale, genetically targeted optical control of neural activity.
3. Chen et al., Science 346(6221), 543 (2015) Expansion Microscopy.
4. Chang et al., Nature Methods 14, 593-599 (2017) Iterative expansion microscopy.
5. Alon et al., BioRxiv (2020) Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems.