The thermal position fluctuations of a single micron-sized sphere immersed in a fluid were recorded by optical trapping interferometry with nanometer spatial and microsecond temporal resolution. We find, in accord with the theory of Brownian motion including hydrodynamic memory effects, that the transition from ballistic to diffusive motion is delayed to significantly longer times than predicted by the standard Langevin equation. This delay is a consequence of the inertia of the fluid. On the shortest time scales investigated, the sphere's inertia has a small, but measurable, effect.
An improved type of scanning probe microscope system able to measure soft interactions between an optically trapped probe and local environment is presented. Such a system that traps and tracks thermally fluctuating probes to measure local interactions is called a photonic force microscope ͑PFM͒. The instrument can be used to study two-dimensional and three-dimensional surface forces, molecular binding forces, entropic and viscoelastic forces of single molecules, and small variations in particle flow, local diffusion, and viscosities. We introduce and characterize a PFM, and demonstrate its outstanding stability and very low noise. The probe's position can be measured within a precision of 0.2-0.5 nm in three dimensions at a 1 MHz sampling rate. The trapping system facilitates stable trapping of latex spheres with diameter Dϭ 0 /2 at laser powers as low as 0.6 mW in the focal plane. The ratio between the trapping stiffness and laser power was able to be optimized for various trapping conditions. The measured trap stiffnesses coincide well with the calculated stiffnesses obtained from electromagnetic theory. The design and the features of the novel PFM setup are discussed. The optical and thermodynamical principles as well as signal analysis are explained. Applications for three-dimensional, hard-clipping interaction potentials are shown. The technique discussed in this article and the results presented should be of great interest also to people working in the fields of classical optical tweezing, particle tracking, interferometry, surface inspection, nanotechnology, and scanning probe microscopy.
Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.
The force field of optical tweezers is commonly assumed to be conservative, neglecting the complex action of the scattering force. Using a novel method that extracts local forces from trajectories of an optically trapped particle, we measure the three-dimensional force field experienced by a Rayleigh particle with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is nonconservative with the nonconservative component increasing radially away from the optical axis, in agreement with the Gaussian beam model of the optical trap.
The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data.
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