Super-resolution imaging: when biophysics meets nanophotonics

Abstract

Probing light–matter interaction at the nanometer scale is one of the most fascinating topics of modern optics. Its importance is underlined by the large span of fields in which such accurate knowledge of light–matter interaction is needed, namely nanophotonics, quantum electrodynamics, atomic physics, biosensing, quantum computing and many more. Increasing innovations in the field of microscopy in the last decade have pushed the ability of observing such phenomena across multiple length scales, from micrometers to nanometers. In bioimaging, the advent of super-resolution single-molecule localization microscopy (SMLM) has opened a completely new perspective for the study and understanding of molecular mechanisms, with unprecedented resolution, which take place inside the cell. Since then, the field of SMLM has been continuously improving, shifting from an initial drive for pushing technological limitations to the acquisition of new knowledge. Interestingly, such developments have become also of great interest for the study of light–matter interaction in nanostructured materials, either dielectric, metallic, or hybrid metallic-dielectric. The purpose of this review is to summarize the recent advances in the field of nanophotonics that have leveraged SMLM, and conversely to show how some concepts commonly used in nanophotonics can benefit the development of new microscopy techniques for biophysics. To this aim, we will first introduce the basic concepts of SMLM and the observables that can be measured. Then, we will link them with their corresponding physical quantities of interest in biophysics and nanophotonics and we will describe state-of-the-art experiments that apply SMLM to nanophotonics. The problem of localization artifacts due to the interaction of the fluorescent emitter with a resonant medium and possible solutions will be also discussed. Then, we will show how the interaction of fluorescent emitters with plasmonic structures can be successfully employed in biology for cell profiling and membrane organization studies. We present an outlook on emerging research directions enabled by the synergy of localization microscopy and nanophotonics.

Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis

“…One of its most successful and widely applied variants is single-molecule localization microscopy (SMLM), such as photoactivatable localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), direct STORM (dSTORM), points accumulation for imaging in nanoscale topography (PAINT), or its more popular variant DNA-PAINT. , Recently, SMLM was extended to the realm of fluorescence lifetime imaging microscopy (FLIM). Conventional (non-super-resolution) FLIM by itself has found multiple applications across many research fields ranging from material sciences to biology and medicine. , A particularly attractive FLIM application is lifetime-based multiplexed imaging of cells, where lifetime information combined with spectral separation allows for the simultaneous imaging of different targets inside a cell. , Recent technical developments allowed for combining FLIM with SMLM to realize fluorescence lifetime SMLM (FL-SMLM). − …”

Localising two sub-diffraction emitters in 3D using quantum correlation microscopy

“…The quest for super-resolution imaging and localisation of fluorophores is one of the most important challenges for optical microscopy [1][2][3][4][5], a convenient imaging modality for both functional and live-cell imaging. However, the length scale for visible light restricts imaging to of order 500 nm because of the diffraction limit.…”

Event-based vision sensor enables fast and dense single-molecule localization microscopy

“…For a recent review of the field of SMLM, refer to [14,15] While retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging of immunolabelled samples [16,17,18,19], spatial analysis of protein clusters [20,21] or protein 1 dynamics in the cell [22], single-molecule microscopy is also used in a much broader range of applications exploiting other information carried by the point-spread function (PSF). A non exhaustive list of them includes accessing the spectrum of the dyes for multicolor imaging [23,24], retrieving the emitter's orientation via polarization measurements [25], or even probing the local environment of the molecules through modifications of the fluorescence intensity [26,27], or the fluorescent state lifetime [28,29,30,31].…”

“…For recent reviews of the field of SMLM, refer to [11,12] Retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging [13,14,15,16], spatial analysis of protein clusters [17,18] or protein dynamics in the cell [19]. Still, the point-spread function (PSF) carries significant additional information, giving access to the spectrum of the dyes for multicolor imaging [20,21], the emitter's orientation [22], the local environment of the molecules through modifications of the fluorescence intensity [23,24], and fluorescent state lifetime [25,26,27,28].…”