Progress and perspectives in single-molecule optical spectroscopy

Abstract

We review some of the progress of single-molecule optical experiments in the past 20 years and propose some perspectives for the coming years. We particularly focus on methodological advances in fluorescence, super-resolution, photothermal contrast and interferometric scattering, and briefly discuss a few of the applications. These advances have enabled the exploration of new emitters, quantum optics, the chemistry and biology of complex heterogeneous systems, nanoparticles and plasmonics, detection and study of non-fluorescing and non-absorbing nano-objects. We conclude by proposing some ideas for future experiments. The field will move towards more and better signals of a broader variety of objects, and towards a sharper view of the surprising complexity of the nanoscale world of single (bio-)molecules, nanoparticles and their nano-environments.

Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan

“…This is achieved because of the combination of (i) nanophotonic UV antenna to enhance the signal, (ii) detailed analysis to reduce the background intensity, and (iii) chemical photostabilizing agents to avoid fluorescence saturation. Our results provide guidelines on how to extend plasmonics into the UV regime − and further develop label-free single molecule spectroscopy. ,,− Earlier works using UV aluminum nanophotonics were restricted to proteins containing a large number of Trp residues such as β-galactosidase (156 Trps) , and streptavidin (24 Trps) . Here we improve the sensitivity by more than 1 order of magnitude, down to the single tryptophan level.…”

“…1−3 This property opens fascinating opportunities to investigate proteins in their native state without introducing any external fluorescent label. 4,5 Avoiding the fluorescence labeling not only simplifies the protein preparation and purification steps but also importantly rules out all the necessary controls to ensure the fluorescent label does not affect the protein. 6−11 Tryptophan (Trp), the brightest of the three aromatic amino acids, has a 12% quantum yield in water and an absorption cross section 20× lower than typical fluorescent dyes.…”

“…Proteins containing aromatic amino acids (tryptophan, tyrosine, and phenylalanine) are naturally fluorescent when excited in the deep ultraviolet (UV). − This property opens fascinating opportunities to investigate proteins in their native state without introducing any external fluorescent label. , Avoiding the fluorescence labeling not only simplifies the protein preparation and purification steps but also importantly rules out all the necessary controls to ensure the fluorescent label does not affect the protein. − …”

“…Altogether, the data presented in Figure demonstrate that a protein bearing a single Trp residue can be detected using UV-FCS. We envision that the methods developed here to optimize the UV autofluorescence signal to background ratio will be useful to a wide range of future studies on label-free single protein spectroscopy, ,,− as well as the advancement of plasmonics into the UV range. ,,, UV-FCS can provide information about local concentration, diffusion properties, and autofluorescence brightness per molecule to shine new light on protein interaction dynamics with ligands or other molecular partners. − While in scattering microscopy the interference signal scales with the third power of the nanoparticle diameter, ,,, UV-FCS is less sensitive to the protein size, relying more on its tryptophan content. The TNase proteins detected here have a molecular weight less than 20 kDa, opening the possibility to detect label-free proteins with molecular weights in the single-digit kDa range.…”

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements

“…Since its debut in the early 1990s, single-emitter fluorescence detection has found applications in many fields, ranging from biophysics to quantum optics [1][2][3][4][5]. Dynamic and sensitive measurements under fluidic conditions, however, remain challenging because diffusion restricts the observation time and the detected photon counts, hampering the investigation of both slow and fast phenomena.…”