Characterization of diffusing sub-10 nm nano-objects using single anti-resonant element optical fibers.

Accurate characterization of diffusing nanoscale species is increasingly important for revealing processes at the nanoscale, with fiber-assisted nanoparticle-tracking-analysis representing a new and promising approach in this field. In this work, we uncover the potential of this approach for the characterization of very small nanoparticles (<20 nm) through experimental studies, statistical analysis and the employment of a sophisticated fiber and chip design. The central results is the characterization of diffusing nanoparticles as small as 9 nm with record-high precision, corresponding to the smallest diameter yet determined for an individual nanoparticle with nanoparticle-tracking-analysis using elastic light scattering alone. Here, the detectable scattering cross-section is limited only by the background scattering of the ultrapure water, thus reaching the fundamental limit of Nanoparticle-Tracking-Analysis in general. The obtained results outperform other realizations and allow access to previously difficult to address application fields such as understanding nanoparticle growth or control of pharmaceuticals.

Locally Structured On-Chip Optofluidic Hollow-Core Light Cages for Single Nanoparticle Tracking.

Nanoparticle tracking analysis (NTA) is a widely used methodology to investigate nanoscale systems at the single species level. Here, we introduce the locally structured on-chip optofluidic hollow-core light cage, as a novel platform for waveguide-assisted NTA. This hollow waveguide guides light by the antiresonant effect in a sparse array of dielectric strands and includes a local modification to realize aberration-free tracking of individual nano-objects, defining a novel on-chip solution with properties specifically tailored for NTA. The key features of our system are (i) well-controlled nano-object illumination through the waveguide mode, (ii) diffraction-limited and aberration-free imaging at the observation site, and (iii) a high level of integration, achieved by on-chip interfacing to fibers. The present study covers all aspects relevant for NTA including design, simulation, implementation via 3D nanoprinting, and optical characterization. The capabilities of the approach to precisely characterize practically relevant nanosystems have been demonstrated by measuring the solvency-induced collapse of a nanoparticle system which includes polymer brush-based shells that react to changes in the liquid environment. Our study unlocks the advantages of the light cage approach in the context of NTA, suggesting its application in various areas such as bioanalytics, life science, environmental science, or nanoscale material science in general.

Colorimetric Biosensors Based on Polymer/Gold Hybrid Nanoparticles: Topological Effects of the Polymer Coating.

Gold nanoparticles decorated with analyte recognition units can form the basis of colorimetric (bio)sensors. The presentation of those recognition units may play a critical role in determining sensor sensitivity. Herein, we use a model system to investigate the effect of the architecture of a polymeric linker that connects gold nanoparticles with the recognition units. Our results show that the number of the latter that can be adsorbed during the assembly of the colorimetric sensors depends on the linker topology. We also show that this may lead to substantial differences in colorimetric sensor performance, particularly in situations in which the interactions with the analyte are comparably weak. Finally, we discuss design principles for efficient colorimetric sensor materials based on our findings.

Nanoparticle Tracking in Single-Antiresonant-Element Fiber for High-Precision Size Distribution Analysis of Mono- and Polydisperse Samples.

Accurate determination of the size distribution of nanoparticle ensembles remains a challenge in nanotechnology-related applications due to the limitations of established methods. Here, a microstructured fiber-assisted nanoparticle tracking analysis (FaNTA) realization is introduced that breaks existing limitations through the recording of exceptionally long trajectories of rapidly diffusing polydisperse nanoparticles, resulting in excellent sizing precision and unprecedented separation capabilities of bimodal nanoparticle mixtures. An effective-single-mode antiresonant-element fiber allows to efficiently confine nanoparticles in a light-guiding microchannel and individually track them over more than 1000 frames, while aberration-free imaging is experimentally confirmed by cross-correlation analysis. Unique features of the approach are (i) the highly precise determination of the size distribution of monodisperse nanoparticle ensembles (only 7% coefficient of variation) and (ii) the accurate characterization of individual components in a bimodal mixture with very close mean diameters, both experimentally demonstrated for polymer nanospheres. The outstanding performance of the FaNTA realization can be quantified by introducing a new model for the bimodal separation index. Since FaNTA is applicable to all types of nano-objects down to sub-20 nm diameters, the method will improve the precision standard of mono- and polydisperse nanoparticle samples such as nano-plastics or extracellular vesicles.

Three-dimensional tracking of nanoparticles by dual-color position retrieval in a double-core microstructured optical fiber.

Elastic light scattering-based three-dimensional (3D) tracking of objects at the nanoscale level is essential for unlocking the dynamics of individual species or interactions in fields such as biology or surface chemistry. In this work, we introduce the concept of dual-color 3D tracking in a double-core microstructured optical fiber that for the first time allows for full 3D reconstruction of the trajectory of a diffusing nanoparticle in a water-filled fiber-integrated microchannel. The use of two single-mode cores provides two opposite decaying evanescent fields of different wavelengths within the microchannel, bypassing spatial domains of ambiguous correlation between the scattered intensity and position. The novelty of the fiber design is the use of two slightly different single-mode cores, preventing modal crosstalk and thus allowing for longitudinally invariant dual-color illumination across the entire field of view. To demonstrate the capabilities of the scheme, a single gold nanosphere (80 nm) diffusing in the water-filled microchannel was tracked for a large number of images (about 32 000) at a high frame rate (1.389 kHz) over a long time (23 s), with the determined hydrodynamic diameters matching expectations. The presented 3D tracking approach yields unique opportunities to unlock processes at the nanoscale level and is highly relevant for a multitude of fields, particularly within the context of understanding sophisticated interaction of diffusing species with functionalized surfaces within the context of bioanalytics, nanoscale materials science, surface chemistry or life science.

Ultrahigh numerical aperture meta-fibre for flexible optical trapping.

Strong focusing on diffraction-limited spots is essential for many photonic applications and is particularly relevant for optical trapping; however, all currently used approaches fail to simultaneously provide flexible transportation of light, straightforward implementation, compatibility with waveguide circuitry, and strong focusing. Here, we demonstrate the design and 3D nanoprinting of an ultrahigh numerical aperture meta-fibre for highly flexible optical trapping. Taking into account the peculiarities of the fibre environment, we implemented an ultrathin meta-lens on the facet of a modified single-mode optical fibre via direct laser writing, leading to a diffraction-limited focal spot with a record-high numerical aperture of up to NA ≈ 0.9. The unique capabilities of this flexible, cost-effective, bio- and fibre-circuitry-compatible meta-fibre device were demonstrated by optically trapping microbeads and bacteria for the first time with only one single-mode fibre in combination with diffractive optics. Our study highlights the relevance of the unexplored but exciting field of meta-fibre optics to a multitude of fields, such as bioanalytics, quantum technology and life sciences.

Tracking and Analyzing the Brownian Motion of Nano-objects Inside Hollow Core Fibers.

Tracking and analyzing the individual diffusion of nanoscale objects such as proteins and viruses is an important methodology in life science. Here, we show a sensor that combines the efficiency of light line illumination with the advantages of fluidic confinement. Tracking of freely diffusing nano-objects inside water-filled hollow core fibers with core diameters of tens of micrometers using elastically scattered light from the core mode allows retrieving information about the Brownian motion and the size of each particle of the investigated ensemble individually using standard tracking algorithms and the mean squared displacement analysis. Specifically, we successfully measure the diameter of every gold nanosphere in an ensemble that consists of several hundreds of 40 nm particles, with an individual precision below 17% (±8 nm). In addition, we confirm the relevance of our approach with respect to bioanalytics by analyzing 70 nm λ-phages. Overall these features, together with the strongly reduced demand for memory space, principally allows us to record thousands of frames and to achieve high frame rates for high precision tracking of nanoscale objects.

Three dimensional spatiotemporal nano-scale position retrieval of the confined diffusion of nano-objects inside optofluidic microstructured fibers.

Understanding the dynamics of single nano-scale species at high spatiotemporal resolution is of utmost importance within fields such as bioanalytics or microrheology. Here we introduce the concept of axial position retrieval via scattered light at evanescent fields inside a corralled geometry using optofluidic microstructured optical fibers allowing to unlock information about diffusing nano-scale objects in all three spatial dimensions at kHz acquisition rate for several seconds. Our method yields the lateral positions by localizing the particle in a wide-field microscopy image. In addition, the axial position is retrieved via the scattered light intensity of the particle, as a result of the homogenized evanescent fields inside a microchannel running parallel to an optical core. This method yields spatial localization accuracies <3 nm along the transverse and <21 nm along the retrieved directions. Due to its unique properties such as three dimensional tracking, straightforward operation, mechanical flexibility, strong confinement, fast and efficient data recording, long observation times, low background scattering, and compatibility with microscopy and fiber circuitry, our concept represents a new paradigm in light-based nanoscale detection techniques, extending the capabilities of the field of nanoparticle tracking analysis and potentially allowing for the observation of so far inaccessible processes at the nanoscale level.

Thermal illumination limits in 3D Raman microscopy: A comparison of different sample illumination strategies to obtain maximum imaging speed.

Confocal Raman microscopy is a powerful tool for material science and biomedical research. However, the low Raman scattering cross-section limits the working speed, which reduces the applicability for large and sensitive samples. Here, we discuss the fundamental physical limits of Raman spectroscopy with respect to signal-to-noise, sample load and how to achieve maximal imaging speed. For this, we develop a simple model to describe arbitrary far field light microscopes and their thermal influence on the sample. This model is used to compare the practical applicability of point- and line-confocal microscopes as well as wide-field-, light sheet- and light line illumination, for the measurement of 3D biological samples. The parallelization degree of the illumination can positively affect the imaging speed as long as it is not limited by thermal sample heating. In case of heat build-up inside the sample, the advantages of parallelization can be lost due to the required attenuation of excitation and the working speed can drop below that of a sequential method. We show that for point like illumination, the exposure time is thermally not as critical for the sample as the irradiance, while for volume like illumination, the exposure time and irradiance result in the same thermal effect. The results of our theoretical study are experimentally confirmed and suggest new concepts of Raman microscopy, thus extending its applicability. The developed model can be applied to Raman imaging as well as to other modes (e.g. two- or three- photon imaging, STED, PALM/STORM, MINFLUX) where thermal effects impose a practical limit due to the high irradiance required.

cellSTORM-Cost-effective super-resolution on a cellphone using dSTORM.

High optical resolution in microscopy usually goes along with costly hardware components, such as lenses, mechanical setups and cameras. Several studies proved that Single Molecular Localization Microscopy can be made affordable, relying on off-the-shelf optical components and industry grade CMOS cameras. Recent technological advantages have yielded consumer-grade camera devices with surprisingly good performance. The camera sensors of smartphones have benefited of this development. Combined with computing power smartphones provide a fantastic opportunity for "imaging on a budget". Here we show that a consumer cellphone is capable of optical super-resolution imaging by (direct) Stochastic Optical Reconstruction Microscopy (dSTORM), achieving optical resolution better than 80 nm. In addition to the use of standard reconstruction algorithms, we used a trained image-to-image generative adversarial network (GAN) to reconstruct video sequences under conditions where traditional algorithms provide sub-optimal localization performance directly on the smartphone. We believe that "cellSTORM" paves the way to make super-resolution microscopy not only affordable but available due to the ubiquity of cellphone cameras.

Automated distinction of shearing and distortion artefacts in structured illumination microscopy.

Any motion during an image acquisition leads to an artefact in the final image. Structured illumination microscopy (SIM) combines several raw images into one high-resolution image and is thus particularly prone to these motion artefacts. Their unpredictable shape cannot easily be distinguished from real high-resolution content. We previously implemented a motion detection specifically for SIM, which had two shortcomings which are solved here. First, the brightness dependency of the motion signal is removed. Second, the empirical threshold of the calculated motion signal was not a threshold at a maximum allowed artefact. Here we investigate which artefacts are still acceptable and which linear movement creates them. Thus, the motion signal is linked with the maximal strength of the expected artefact. A signal-to-noise analysis including classification successfully distinguishes between artefact-free imaging, shearing and distortion artefacts in biological specimens. A shearing, as in wide-field microscopy, is the dominant reconstruction artefact, while distortions arise not until surprisingly fast movements.

Motion artefact detection in structured illumination microscopy for live cell imaging.

The reconstruction process of structured illumination microscopy (SIM) creates substantial artefacts if the specimen has moved during the acquisition. This reduces the applicability of SIM for live cell imaging, because these artefacts cannot always be recognized as such in the final image. A movement is not necessarily visible in the raw data, due to the varying excitation patterns and the photon noise. We present a method to detect motion by extracting and comparing two independent 3D wide-field images out of the standard SIM raw data without needing additional images. Their difference reveals moving objects overlaid with noise, which are distinguished by a probability theory-based analysis. Our algorithm tags motion-artefacts in the final high-resolution image for the first time, preventing the end-user from misinterpreting the data. We show and explain different types of artefacts and demonstrate our algorithm on a living cell.

fastSIM: a practical implementation of fast structured illumination microscopy.

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm2, free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator.

We describe a two-beam interference structured illumination fluorescence microscope. The novelty of the presented system lies in its simplicity. A programmable spatial light modulator (ferroelectric LCoS) in an intermediate image plane enables precise and rapid control of the excitation pattern in the specimen. The contrast of the projected light pattern is strongly influenced by the polarization state of the light entering the high NA objective. To achieve high contrast, we use a segmented polarizer. Furthermore, a mask with six holes blocks unwanted components in the spatial frequency spectrum of the illumination grating. Both these passive components serve their purpose in a simpler and almost as efficient way as active components. We demonstrate a lateral resolution of 114.2 ± 9.5 nm at a frame rate of 7.6 fps per reconstructed 2D slice.