A guide to small fluorescent probes for single-molecule biophysics

Abstract

The explosive growth of single-molecule techniques is transforming our understanding of biology, helping to develop new physics inspired by emergent biological processes, and leading to emerging areas of nanotechnology. Key biological and chemical processes can now be probed with new levels of detail, one molecule at a time, from the nanoscopic dynamics of nature's molecular machines to an ever-expanding range of exciting applications across multiple length and time scales. Their common feature is an ability to render the underlying distribution of molecular properties that ensemble averaging masks and to reveal new insights into complex systems containing spatial and temporal heterogeneity. Small fluorescent probes are among the most adaptable and versatile for single-molecule sensing applications because they provide high signal-to-noise ratios combined with excellent specificity of labeling when chemically attached to target biomolecules or embedded within a host material. In this review, we examine recent advances in probe designs, their utility, and applications and provide a practical guide to their use, focusing on the single-molecule detection of nucleic acids, proteins, carbohydrates, and membrane dynamics. We also present key challenges that must be overcome to perform successful single-molecule experiments, including probe conjugation strategies, identify tradeoffs and limitations for each probe design, showcase emerging applications, and discuss exciting future directions for the community.

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…UAAs have found a wide range of utility in biological science. − However, in order for a UAA to be useful as a spectroscopic and/or imaging reporter of proteins, it must meet several requirements: (1) it should be able to produce a detectable, distinguishable, and interpretable signal (e.g., a vibrational or fluorescence signal) with desired spectroscopic properties; (2) it should be a simple derivative of one of the canonical amino acids; (3) it should be chemically stable; (4) it should not significantly perturb the structural and functional properties of the protein in question; and (5) it should be able to be incorporated into proteins via either a chemical or biological method.…”

Boosting the toolbox for live imaging of translation

“…However, no such optimization has been made for the SunTag system in the context of a developing organism, although photo-physical properties of molecules can be variable between different systems (i.e. in vitro experiments, cell culture, tissues, organisms...) 24,36,37 .…”

“…Since then, several new FPs have been and continue to be discovered and engineered 22,23 . The constant race for better FPs generally implies the optimization for fast folding, pH resistance as well as improvement of photo-physic properties 24 .…”

“…However, no such optimization has been made for the SunTag system in the context of a developing organism, although photo-physical properties of molecules can be variable between different systems (i.e. in vitro experiments, cell culture, tissues, organisms…) 24,36,37 . We tested five different GFP variants for their brightness, photobleaching and propensity to aggregation in fixed and live embryos.…”

Extended BODIPYs as Red–NIR Laser Radiation Sources with Emission from 610 nm to 750 nm

“…This technique demands molecular probes working in the so-called biological or therapeutic window (beyond 650 nm), owing to the deep-tissue penetration of this long-wavelength radiation (2–5 cm) [ 7 , 8 , 9 , 10 ]. These fluorophores should be chemically robust and photostable in order to display long lasting fluorescent images, and readily available for post-functionalization to allow targetable labelling [ 11 ]. Another aspect of these red–NIR luminophores is the development of low-cost excitation sources in optical fibers applied in telecommunications (telecom range beyond 800 nm) owing to the lower interference and scattering of this long-wavelength radiation with its surroundings, allowing the light to travel longer distances [ 12 , 13 ].…”