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Comment by Zee2 | Hacker News Reader

For some reason, what actually stands out to me in this paper is the method in which they verified the rotational motion. They used single-molecule fluorescence and optically tracked the circular trajectories that the single molecule traced out while spinning. That's the most impressive part, in my opinion... I didn't know we could even resolve fluorescing particles on that scale, much less track their trajectories over time.

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Single molecule fluroescence has been around for a while; when I was in grad school, students in another lab were doing this. They'd label motor proteins (which use energy to move in a specific direction) to visualize them on a surface and calculate their velocity.

The particle can be much smaller than the resolution, as long as it's really bright, it will just sort of "smear out" over multiple adjacent pixels and it's possible, with some arcane trickery, to then localize to a sub-pixel.

See the section titled "Fluorescence microscopy data analysis". Basically, when you have a single molecule fluorescing you "just" need to do some math to figure out the center of the samples over time. See https://www.microscope.healthcare.nikon.com/products/super-r... for an overview

thanks for sharing.

since you sound like an expert, do you know if this technique works for live imaging of RNA molecules < 200 nucleotides?

or would tagging such a small molecule potentially alter biological processes and contaminate results?

[edited to clarify live-imaging requirement]

FISH works for both DNA and RNA. When articles have pretty colors lighting up the inside of a cell, it's likely FISH.

First google hit is a 2020 summary of RNA-FISH, "Technical review and guide to RNA fluorescence in situ hybridization":

  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085896/

thanks for the link. i saw this already; it's very useful.

to clarify, the question was meant for live imaging and the risk of altering biological processes for target RNA molecules < 200 nucleotides.

I am not exactly an expert, I just happened to do a deep dive into microscopy techniques a couple years ago :-)

The general term for these types of techniques (e.g., ones that let you image things below the "diffraction limit", which is roughly half the wavelength of light being used to image, see [1]), is super resolution microscopy[2]. There are a few other types you might find interesting.

1: https://en.wikipedia.org/wiki/Diffraction-limited_system 2: https://en.wikipedia.org/wiki/Super-resolution_microscopy

thanks for sharing, will check these out!

based on your understanding, do you think it's possible to do live imaging of RNA molecules < 200 nucleotides -- without altering biological processes?

super resolution microscopy references DNA imaging but doesn't delve into contamination risk, which is the critical bit.

the first link didn't mention DNA/RNA applications at all.

do you mind sharing the other types you recommend investigating?

https://www.nature.com/articles/s41586-019-1397-7

this paper came out a few years ago using super resolution fluorescence and dna origami to track unwinding of dna by single helicase enzymes! its not an easy technique but it is doable with the right equipment (the 2014 Nobel Prize in chemistry was for super resolution microscopy)

That's absolutely fascinating! The use of single-molecule fluorescence to verify rotational motion is indeed impressive. I'm curious, how do you think this groundbreaking technique could potentially impact future research in the field?

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