Expansion Microscopy - ‘Just add water’
Microscopes are getting beefier and beefier, more complex and expensive, with the sole purpose of being able to see tiny, tiny things just a little bit better. Enter ‘expansion microscopy’, an idea that literally works in the opposite direction to that goal. Instead of ‘zooming in closer’, expansion microscopy aims to ‘blow things up’ in order to see the (once) tiny details (like synapses, or nuclear pores…) on a conventional microscope. Remember those dinosaurs that would expand when you added water as a kid? I sure do…and expansion microscopy works pretty much the same.
Although this technology has been around for a few years, it is just getting started in terms of its ease of use, applicability to different samples (proteins, RNA, DNA, lipids…), and support community. All info on this fascinating technique is available at ExpansionMicroscopy.org.
First described by Edward Boyden and colleagues at the MIT Media Lab in 2015, expansion microscopy is rapidly being applied across fields, species, and disciplines to examine extremely fine structures at the nanoscale (10-20 nm).
Expansion microscopy allows for uniform expansion of a biological sample. Here, we see a brain slice (in panel B) which has been weaved into a polymer mesh with biomolecular anchors. When the polymer is expanded (‘Just add water’), it pulls the biomolecules along with it, maintaining the relative spacing between structures. In (C ) we can see that same brain slice ‘expanded’, revealing tiny pieces of biology previously too small to see (Credit: Chen et al., 2015; Science).
Ed Boyden provided a ‘state of the art’ summary of expansion microscopy to date at a minisymposium today titled “new observations in neuroscience using superresolution microscopy” chaired by Michihiro Igarashi. He gave a quick overview on how they developed the idea that was to become expansion microscopy, through adapting old techniques from the early 1980’s. Next, he discussed the problems of ‘expansion’, the primary one being ’ how can we evenly expand a sample without losing valuable spacial relationships between proteins, DNA, RNA etc…? To overcome this problem they needed to develop biomolecular anchors, which link the molecular target to the polymer mesh. In this way, isometric expansion of the mesh results in the same for the anchored sample.
Using this technique, many researchers have expanded tissues to look at things like synaptic proteins and microtubules at a much finer detail than what was previously possible with conventional confocal microscopes. Others have adapted the technique to work with in situ hybridization, allowing for expansion and quantification of RNA. Dr. Boyden’s lab is also working on expanding non-soft tissues, like bone, and using expansion microscopy in the clinic to diagnose and investigate cancer in unprecedented detail (so called ‘expansion pathology’).
By combining expansion microscopy with RNA visualization (ExFISH) and sequencing (MERFISH), hundreds of transcripts can be examined simultaneously in situ!
Towards the end of the talk, Dr. Boyden highlighted some open questions in the field. These questions focused on a few primary themes:
Can we validate expanded samples below 10-20 nm?
Is expansion ‘pulling’ synapses apart, leading us to false conclusions?
Can we use this technique to probe protein-protein interactions?
Whats the smallest thing we can expand? Can we expand a virus? A DNA origami??
How much can we expand a sample while maintaining all relevant spatial relationships?
To take the last question, Dr. Boyden’s team reasoned, if we can expand something once, why not twice, or thrice?? They put samples through an iterative process allowing for expansion up to 20x the original size!! (shown below)
Iterative Expansion Microscopy allows for sample expansion up to 20x! Panel A shows dendritic spines without expansion, panel B shows the same at 4.5x expansion, and panel C shows dendritic spines at 20x expansion after the iterative process is complete (Credit: Chang et al., 2017; Nature Methods)
A cool side effect of expansion is that it involves filling the sample with water, making it essentially transparent, and useful for long-range circuit mapping at high detail or speeding up techniques like light-sheet microscopy. We are only at the surface of what is possible with this and other super-resolution techniques. I look forward to all the exciting things to come!
See you guys soon! Feel free to follow me on twitter @jborniger and on my website www.jeremyborniger.com
Jeremy C. Borniger, PhD
Department of Psychiatry & Behavioral Sciences
Stanford University SoM