Relocating Single Molecules in Super-Resolved Fluorescence Lifetime Images near a Plasmonic Nanostructure

Abstract

Single-molecule localization microscopy is a powerful technique with vast potential to study lightmatter interactions at the nanoscale. Nanostructured environments can modify the fluorescence emission of single molecules and the induced decay-rate modification can be retrieved to map the local density of optical states (LDOS). However, the modification of the emitter's point spread function (PSF) can lead to its mislocalization, setting a major limitation to the reliability of this approach. In this paper, we address this by simultaneously mapping the position and decay rate of single-molecules and by sorting events by their decay rate and PSF size. With the help of numerical simulations, we are able to infer the dipole orientation and to retrieve the real position of mislocalized emitters. We have applied our approach of single-molecule fluorescence lifetime imaging microscopy (smFLIM) to study the LDOS modification of a silver nanowire over a field of view of ~10 µm 2 with a single-molecule localization precision of ~15 nm. This is possible thanks to the combined use of an EMCCD camera and an array of single-photon avalanche diodes, enabling multiplexed and super-resolved fluorescence lifetime imaging.

Imaging and Localization of Single Emitters near Plasmonic Particles of Different Size, Shape, and Material

“…Furthermore, by fixing the orientation of the emitter, not only the effect of emitter location but also orientation could be studied to reveal polarization-dependent properties that are usually lost due to orientation averaging. Ideally, multimodal imaging may enable the collection of emission intensity, PSF shape, 17 fluorescence lifetime, 29 emission polarization, 42 and single-molecule spectra 43 to obtain a complete picture of particle–emitter coupling. Such experiments will shed light on the effect of a 3D photonic environment on optical imaging and will extend the realm of super-resolution microscopy to nano- and micrometer-sized colloids.…”

Super-resolution imaging: when biophysics meets nanophotonics

“…FDTD simulations support the observations. Further developments of this technique allowed to increase the field-of-view from 1 μm 2 to 10 μm 2 , thanks to the use of a 8 × 1 SPAD array [133]. More details about this experiment will be given in Section 4.4.…”

“…effects such as LDOS changes, PSF changes, or radiation pattern changes, from a weakness into a strength. Blanquer et al [133] showed that it is possible to correlate lifetime information (smFLIM measurements) and real space imaging for "relocating" molecule localizations to their correct position. In essence, the correlation of lifetime changes with the width of the observed PSF allows to pinpoint more accurately the dipole position and orientation of fluorophores near a nanophotonic structure of interest.…”

“…Up to date, only few technologies are available for wide-field FLIM with single-molecule sensitivity. Among them are timegated SPAD arrays [133,188], electron multiplying microchannel plates (MCP) [189,190], electro-optical modulators [191], or gated optical image intensifiers [192]. One of the most promising technologies for fluorescence-lifetime wide-field SMLM so far is the commercially available lifetime camera LINCam (Photonscore), see Figure 11a.…”

Partial Cloaking of a Gold Particle by a Single Molecule

“…The yellow circle and the magenta cross in Fig. 2(b) mark the lateral positions of the GNP and M0, respectively, but it should be borne in mind that the results can contain systematic errors due to the redirection of the molecular emission by the GNP [40][41][42].…”