Making Hidden Cell Particle Interactions Visible by Thermal Noise Frequency Decomposition.

Thermal noise drives cellular structures, bacteria, and viruses on different temporal and spatial scales. Their weak interactions with their environment can change on subsecond scales. However, particle interactions can be hidden or invisible-even when measured with thermal noise sensitivity, leading to misconceptions about their binding behavior. Here, it is demonstrated how invisible particle interactions at the cell periphery become visible by MHz interferometric thermal noise tracking and frequency decomposition at a spectral update rate of only 0.5 s. The particle fluctuations are analyzed in radial and lateral directions by a viscoelastic modulus G(ω,tex ) over the experiment time tex , revealing a surprisingly similar, frequency dependent response for different cell types. This response behavior can be explained by a mathematical model for molecular scale elasticity and damping. The method to reveal hidden interactions is tested at two examples: the stiffening of macrophage filopodia tips within 2 s with particle contact invisible by the fluctuation width. Second, the extent and stiffness of the soft cell glycocalyx is measured, which can be sensed by a particle only on microsecond-timescales, but which remains invisible on time-average. This concept study shows how to uncover hidden cellular interactions, if particle motions are measured at high-speed.

100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales.

Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions.

Deep-ROCS: from speckle patterns to superior-resolved images by deep learning in rotating coherent scattering microscopy.

Rotating coherent scattering (ROCS) microscopy is a label-free imaging technique that overcomes the optical diffraction limit by adding up the scattered laser light from a sample obliquely illuminated from different angles. Although ROCS imaging achieves 150 nm spatial and 10 ms temporal resolution, simply summing different speckle patterns may cause loss of sample information. In this paper we present Deep-ROCS, a neural network-based technique that generates a superior-resolved image by efficient numerical combination of a set of differently illuminated images. We show that Deep-ROCS can reconstruct super-resolved images more accurately than conventional ROCS microscopy, retrieving high-frequency information from a small number (6) of speckle images. We demonstrate the performance of Deep-ROCS experimentally on 200 nm beads and by computer simulations, where we show its potential for even more complex structures such as a filament network.

Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy.

Cells change their shape within seconds, cellular protrusions even on subsecond timescales enabling various responses to stimuli of approaching bacteria, viruses or pharmaceutical drugs. Typical response patterns are governed by a complex reorganization of the actin cortex, where single filaments and molecules act on even faster timescales. These dynamics have remained mostly invisible due to a superposition of slow and fast motions, but also due to a lack of adequate imaging technology. Whereas fluorescence techniques require too long integration times, novel coherent techniques such as ROCS microscopy can achieve sufficiently high spatiotemporal resolution. ROCS uses rotating back-scattered laser light from cellular structures and generates a consistently high image contrast at 150 nm resolution and frame rates of 100 Hz-without fluorescence or bleaching. Here, we present an extension of ROCS microscopy that exploits the principles of dynamic light scattering for precise localization, visualization and quantification of the cytoskeleton activity of mouse macrophages. The locally observed structural reorganization processes, encoded by dynamic speckle patterns, occur upon distinct mechanical stimuli, such as soft contacts with optically trapped beads. We find that a substantial amount of the near-membrane cytoskeleton activity takes place on millisecond timescales, which is much faster than reported ever before.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy.

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle's hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 µm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 µs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions.