Alexander Rohrbach

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Trapping forces, force constants, and potential depths for dielectric spheres in the presence of spherical aberrations

We present and verify a theoretical model that predicts trapping forces (escape forces), force constants (trap stiffnesses), and trapping potential depths for dielectric spheres with diameters smaller than or equal to the wavelength of the trapping light. Optical forces can be calculated for arbitrary incident light distributions with a two-component approach that determines the gradient and the scattering force separately. We investigate the influence of spherical aberrations that are due to refractive-index mismatch on the maximum trapping force, the force constant, and the potential depth of a trap, which are important for optical tweezer applications. The relationships between the three parameters are explained and studied for different degrees of spherical aberration and various spheres (refractive indices n(s) = 1.39-1.57, radii a = 0.1-0.5 microm, lambda(0) = 1.064 microm). We find that all three parameters decrease when the distance to the coverslip increases. Effects that could make the interpretation of experimental results ambiguous are simulated and explained. Computational results are compared with the experimental data found in the literature. A good coincidence can be established.

Optical trapping of dielectric particles in arbitrary fields

We present a new method to calculate trapping forces of dielectric particles with diameters D < or = lambda in arbitrary electromagnetic, time-invariant fields. The two components of the optical force, the gradient force and the scattering force, are determined separately. Both the arbitrary incident field and the scatterer are represented by plane-wave spectra. The scattering force is determined by means of the momentum transfer in either single- or double-scattering processes. Therefore the second-order Born series is evaluated and solved in the frequency domain by Ewald constructions. Numerical results of our two-force-component approach and an established calculation method are compared and show satisfying agreement. Our procedure is applied to investigate axial trapping by focused waves experiencing effects of aperture illumination and refractive-index mismatch.

Three-dimensional position detection of optically trapped dielectric particles

A theory is presented together with simulation results that describe three-dimensional position detection of a sphere located in a highly focused beam by back-focal plane interferometry. This technique exploits the interference of scattered and unscattered light, which is projected on a quadrant photodiode placed in the back-focal plane of a condenser lens. Due to the Gouy-phase shift inherent in focused beams, it is not only possible to determine the lateral but also the axial position of a spherical particle with nanometer accuracy. In this paper we describe the calculation of arbitrary focused electromagnetic fields, the Gouy phase shift, Mie scattering by focused beams and the resulting position signals using the angular momentum representation. The accuracy and the sensitivity of the detection system are investigated theoretically for various sphere parameters. Both accuracy and sensitivity depend on the incident light distribution as well as on the particle’s properties and position. It is further s...

Trapping and tracking a local probe with a photonic force microscope

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 (PFM). 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 D=λ0/2 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 measur...

Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media

Laser beams that can self-reconstruct their initial beam profile even in the presence of massive phase perturbations are able to propagate deeper into inhomogeneous media. This ability has crucial advantages for light sheet-based microscopy in thick media, such as cell clusters, embryos, skin or brain tissue or plants, as well as scattering synthetic materials. A ring system around the central intensity maximum of a Bessel beam enables its self-reconstruction, but at the same time illuminates out-of-focus regions and deteriorates image contrast. Here we present a detection method that minimizes the negative effect of the ring system. The beams propagation stability along one straight line enables the use of a confocal line principle, resulting in a significant increase in image contrast. The axial resolution could be improved by nearly 100% relative to the standard light-sheet techniques using scanned Gaussian beams, while demonstrating self-reconstruction also for high propagation depths.

A line scanned light-sheet microscope with phase shaped self-reconstructing beams

We recently demonstrated that Microscopy with Self-Reconstructing Beams (MISERB) increases both image quality and penetration depth of illumination beams in strongly scattering media. Based on the concept of line scanned light-sheet microscopy, we present an add-on module to a standard inverted microscope using a scanned beam that is shaped in phase and amplitude by a spatial light modulator. We explain technical details of the setup as well as of the holograms for the creation, positioning and scaling of static light-sheets, Gaussian beams and Bessel beams. The comparison of images from identical sample areas illuminated by different beams allows a precise assessment of the interconnection between beam shape and image quality. The superior propagation ability of Bessel beams through inhomogeneous media is demonstrated by measurements on various scattering media.

Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation.

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5-10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3-5 relative to linear excitation with Gaussian beams. These finding arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the best image resolution, contrast at both a minimum amount of artifacts and signal degradation along the propagation of the beam into the sample.

Three-dimensional tracking of small spheres in focused laser beams: influence of the detection angular aperture

Back-focal-plane interferometry is a method capable of determining the three-dimensional position of a particle with high precision (< 3 nm) at high sampling rates (1 MHz). We investigated theoretically the performance of such a system for dielectric spheres with diameters D = 0.53-3 microm and for metallic spheres with D < or = 300 nm. Good sensitivity and linearity were achieved for a detection angular aperture sin(alpha) of no more than 0.5. A value of sin(alpha) > 0.7 should be used only for dielectric spheres with diameters approximately equal to the laser wavelength. Harmonic optical traps can be calibrated by measurement of the thermal motion of the sphere. We performed Brownian dynamics simulations and subsequent thermal noise analyses to prove that the wrong sin(alpha) incorrectly suggests an increased and nonharmonic axial trapping potential.

Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy

One of main challenges in light-sheet microscopy is to design the light-sheet as extended and thin as possible--extended to cover a large field of view, thin to optimize resolution and contrast. However, a decrease of the beams waist also decreases the illumination beams depth of field. Here, we introduce a new kind of beam that we call sectioned Bessel beam. These beams can be generated by blocking opposite sections of the beams angular spectrum. In combination with confocal-line detection the optical sectioning performance of the light-sheet can be decoupled from the depth of field of the illumination beam. By simulations and experiments we demonstrate that these beams exhibit self-reconstruction capabilities and penetration depths into thick scattering media equal to those of conventional Bessel beams. We applied sectioned Bessel beams to illuminate tumor multicellular spheroids and prove the increase in contrast. Sectioned Bessel beams turn out to be highly advantageous for the investigation of large strongly scattering samples in a light-sheet microscope.