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Super-Beacons: open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy

Pedro M. Pereira, Nils Gustafsson, Mark Marsh, Musa M. Mhlanga, Ricardo Henriques

Preprint posted on February 04, 2020 https://www.biorxiv.org/content/10.1101/2020.01.20.912311v2

Article now published in Traffic at http://dx.doi.org/10.1111/tra.12728

Super-Beacons: making super-resolution live-cell friendly.

Selected by Mariana De Niz

Background

Super-resolution microscopy englobes various different methods that allow imaging at a resolution beyond the limit imposed by diffraction. Over the last few decades, this has allowed imaging at resolutions close to those achieved by electron microscopy, with the advantages of fluorescence and the possibility of live imaging. Localization-based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states – namely photoswitching or emission fluctuations. However, these transitions commonly rely on high-intensity illumination. This imposes two limitations: the requirement of equipment capable of high-intensity illumination, and hindrances to live imaging due to phototoxicity.

In their work, Pereira and colleagues developed open source super-resolution probes named Super-Beacons, which use self-quenching as a mechanism to generate spontaneous, photoswitching-independent of illumination.

 

Key findings and developments

Overall findings and developments

  • Pereira et al designed Super-Beacons, and characterised their photo-switching properties in live-cell compatible imaging conditions (Figure 1).
Figure 1. Super-Beacons photoswitching mechanisms, and structure.

 

Super-Beacons probe design

  • Super-Beacons as photoswitchable probes were designed based on the hypothesis that fluorophore photoswitching can be induced by promoting transient interactions between a fluorophore-quencher pair in a molecule that exhibits two conformational states: ‘closed’-quenched due to fluorophore and quencher contact; and ‘open’-fluorescent where the fluorophore and quencher are not in proximity. It is the transition between closed and open conformations that influences the lifetime of emitting and non-emitting states.
  • A Super-Beacon is a single-stranded DNA (ssDNA) oligo-nucleotide with a fluorophore and a quencher covalently bound to opposing ends. Short, complementary, terminal sequences promote self-hybridization resulting in the stable formation of the hairpin-shaped secondary structure (the ‘closed’ state).
  • The closed state is dominant, but the probe can reversibly transition to a short-lived fluorescent ‘open’ state in a stochastic manner. The rate of transition between states can be modulated by temperature, chemical environment, and choice of oligo-nucleotide sequence.

 

Super-Beacons in vitro characterization

  • The authors characterized Super-Beacons by imaging their photophysical behaviour while immobilized on a coverslip using total internal reflection fluorescence microscopy (TIRFM).
  • They then measured the switching state lifetimes of the Super-Beacons designed for this study. A range of illumination intensities was used, from those compatible with live imaging, to those classically used for STORM. The desirable photoswitching was achieved at 5-fold less illumination than for the control probe.
  • The potential of Super-Beacons as imaging probes was then evaluated, by attaching a biotin modified thymine in the Super-Beacon loop, to a streptavidin-conjugated antibody. This allowed labelling and imaging of beta-tubulin in fixed NIH3T3 cells, with relatively low illumination, and a non-toxic buffer (ie. PBS). This led to the conclusion that the conditions necessary to use Super-Beacons were compatible with live imaging. Moreover, a resolution better than 80nm was achieved upon sample imaging.

 

Super-Beacons and live-cell super-resolution imaging

  • The authors tested the potential of Super-Beacons for live cell SRM imaging, focusing on IFN-induced transmembrane proteins (IFITM-1, -2, and -3).
  • The authors were able to use Super-Beacons to acquire detailed information on the distribution and dynamics of IFITM-1 in live-cell single molecule localization microscopy (SMLM) experiments.
  • Upon comparison of Super-Beacons with classical preparation and imaging using STORM, the authors found negligible visual differences in IFITM1 distribution at the cell surface. Moreover, quantitative analysis using the SQUIRREL algorithm showed that both conditions achieved similar quality.
  • The authors then explored whether the high-fidelity in protein distribution across different illumination intensity regimes was translated into the ability to extract quantitative data; and found no difference between the different illumination regimes.
  • The dynamics of the IFITM1 protein could be visualized and single molecule tracking was performed.
  • Overall, these experiments concluded that Super-Beacons are a promising tool for the analysis of the diffusion of membrane-bound extracellular proteins on the molecular scale, in live cell settings.

 

What I like about this preprint

I like the work of the Henriques lab a lot, in general, in that it has provided extremely useful tools to various fields of research. Moreover, the tools developed- be it software or molecular, have always been in accordance with open science. This work is no exception. Having personally used super-resolution microscopy, I have indeed faced some of the hindrances which Super-Beacons address. I also liked that the manuscript goes well to the point, with the description of the beacons, and the validation experiments performed. The methods section has fine detail, enabling potential users to follow the necessary steps to design and use Super-Beacons. Moreover, the authors openly discuss in detail, the current limitations and possible improvements of Super-Beacons, which allows other scientists wishing to use this tool, to design experiments accordingly. As a side note, the name of the probes is very ingenious.

 

Open questions

*Note: answers to all questions appear at the bottom of this page.

  1. In your work, you explored in detail illumination intensity. Two questions on this: a) why does the ‘on’ ratio change with laser intensity? b) In your discussion you mention the possibility of modifying other parameters such as temperature and chemical environment, to optimize the photoswitching behaviour. Can you expand further on what the possible modifications can be, and whether they are compatible with live imaging without inducing artefacts?
  1. You mention other conjugation options for live cell imaging to overcome some of the current limitations of the Super-Beacons you present in this work. Do you expect any important differences with the conditions you used for your work (eg. the Thiol modification instead of Biotin, or monomeric streptavidin)?
  1. You mention in your work that the on-state lifetime should be similar to the acquisition rate (ie. 10s of milliseconds). From your characterization of Super-Beacons, is there a limitation to the live processes one could visualize, dependent on the dynamic of those processes?
  1. You tested the Super-Beacons in your preprint to study IFN-induced transmembrane proteins. Are Super-Beacons easily introduced into any type of cell/is efficiency high? Since you mention in your work that chemical environments for instance can affect the kinetics of photoswitching, do you envisage there might be differences, based on the sub-cellular structure targeted? Or the conditions tested in vivo?

 

References

  1. Pereira, P.M., Gustafsson, N., Marsh, M., Mhlanga, M.M, Henriques, R., Super-Beacons: open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy, bioRxiv, (2020), doi: 10.1101/2020.01.20.912311

 

Acknowledgements

I am very grateful to Ricardo Henriques for his time, engagement and input. I also thank Mate Palfy for his helpful input.

 

Posted on: 9th March 2020

doi: https://doi.org/10.1242/prelights.17506

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  • Author's response

    Ricardo Henriques shared

    Open questions

    1. In your work, you explored in detail illumination intensity. Two questions on this: a) why does the ‘on’ ratio change with laser intensity? b) In your discussion you mention the possibility of modifying other parameters such as temperature and chemical environment, to optimize the photoswitching behaviour. Can you expand further on what the possible modifications can be, and whether they are compatible with live imaging without inducing artefacts?

    a) The probes have two dominant switching approaches: due to recurring self-hybridization which should be illumination independent; by regular photo-switching photophysics of the fluorophore. When you use low-illumination, the mechanical photoswitching is dominant. However, when you increase illumination then the fluorophores start to be pumped into dark triplet states. This means that the ON-state does have a dependence in the illumination.

    b) For live-cell imaging, you want to use a stable chemical environment and temperature, so the dominant way to tweak photo-switching would be the design of the Super-Beacon structure. For fixed-cells, temperature and chemical environment are an excellent way to tweak photoswitching at the microscopes. This is achieved by changing temperature on an incubator or modifying the chemistry (e.g. salt) on the sample with microfluidics. One thing that might be interesting, but that we have not explored, is that a living cell will have different internal microenvironments that may mean probes will switch differently depending on where they are. Something we can aim to detect in the future.

     

    1. You mention other conjugation options for live cell imaging to overcome some of the current limitations of the Super-Beacons you present in this work. Do you expect any important differences with the conditions you used for your work (eg. the Thiol modification instead of Biotin, or monomeric streptavidin)?

    We spent quite a bit of time playing with the structure of Super-Beacons, giving them overhangs that would bind custom zinc fingers or leucine zipper domains. It would allow us to have strategies for Super-Beacons to bind to protein chimaeras. Unfortunately, we have not succeeded yet. Indeed, there are multiple ways to bind a Super-Beacon to an antibody as you point-out, where biotin-streptavidin is one of the easiest but bulkier ones. We need to find ways to get away from using antibodies and welcome strategies to get there.

     

    1. You mention in your work that the on-state lifetime should be similar to the acquisition rate (ie. 10s of milliseconds). From your characterization of Super-Beacons, is there a limitation to the live processes one could visualize, dependent on the dynamic of those processes?

    It is a hard numbers game. To super-resolve a cellular structure, we need to collect enough blinking events to represent the underlying molecular assembly accurately, in a short enough time so that structural movement is negligible. If it takes a few seconds to collect the data for a structure and during this time it moves 100nm, then our resolution is likely going to be 100nm at best. Unfortunately, this means that we are mostly sensitive to slow-moving structures (e.g.: organelles), and we will miss out on fast events.

     

    1. You tested the Super-Beacons in your preprint to study IFN-induced transmembrane proteins. Are Super-Beacons easily introduced into any type of cell/is efficiency high? Since you mention in your work that chemical environments for instance can affect the kinetics of photoswitching, do you envisage there might be differences, based on the sub-cellular structure targeted? Or the conditions tested in vivo?

    There are several ways to get Super-Beacons inside cells – electroporation, transfection, microinjection. All of them work to some degree. The primary issue that we have faced thus far is that Super-Beacons are very prone to bind unspecifically to unwanted molecular targets. Plus the fact that cells often have an innate immune reaction to finding DNA in their cytoplasm. Effectively this means that there is potential for intracellular live-cell labelling with the Super-Beacons, but there is still work to be done to get them working correctly there. For this we decided to showcase receptors labelled through their extracellular domain in the paper. Once we sort out better ways to label internal structures in living cells, we hope to explore their potential as thermal and chemical sensors.

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