Scattering of illumination light from a laser is a severe problem especially when imaging in thick media. Although this effect occurs in nearly every imaging process, it can be well perceived and ...analyzed in configurations where the optical axes for illumination and detection are perpendicular to each other. In this paper I present a theoretical perspective of how to extend the point-spread function arithmetic from ideal imaging to realistic imaging including ghost images. These ghost images are generated by scattered light and are low-correlated with the ideal image. Numerical simulations of the propagation of four different types of illumination beams through a cluster of spheres illustrate the effects of inhomogeneous object illumination. Clear differences between a conventional plane-wave illumination, a static light-sheet, and a laterally scanned Gaussian beam, but also relative to a scanned Bessel beam, can be observed.
The first quantitative agreement between measured and calculated stiffnesses of optically trapped particles in the subwavelength regime is presented. It is shown for all three dimensions that the ...measured extent of harmonic optical trapping potentials for dielectric spheres comes very close to the theoretically predicted extent, provided all known instrumental parameters are considered. The recently predicted strong asymmetry of the trapping potential due to the electric field's linear polarization has been verified in all three directions. This effect vanishes for spheres with diameters d approximately lambda, which exhibit the strongest trap stiffnesses.
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 beam's 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.
In modern 3D microscopy, holding and orienting arbitrary biological objects with optical forces instead of using coverslips and gel cylinders is still a vision. Although optical trapping forces are ...strong enough and related photodamage is acceptable, the precise (re-) orientation of large specimen with multiple optical traps is difficult, since they grab blindly at the object and often slip off. Here, we present an approach to localize and track regions with increased refractive index using several holographic optical traps with a single camera in an off-focus position. We estimate the 3D grabbing positions around several trapping foci in parallel through analysis of the beam deformations, which are continuously measured by defocused camera images of cellular structures inside cell clusters. Although non-blind optical trapping is still a vision, this is an important step towards fully computer-controlled orientation and feature-optimized laser scanning of sub-mm sized biological specimen for future 3D light microscopy.
Optical tweezers are a flexible manipulation tool used to grab micro-objects at a specific point, but a controlled manipulation of objects with more complex or changing shapes is hardly possible. ...Here, we demonstrate, by time-sharing optical forces, that it is possible to adapt the shape of the trapping potential to the shape of an elongated helical bacterium. In contrast to most other trapped objects, this structure can continuously change its helical shape (and therefore its mechanical energy), making trapping it much more difficult than trapping tiny non-living objects. The shape deformations of the only 200-nm-thin bacterium (Spiroplasma) are measured space-resolved at 800 Hz by exploiting local phase differences in coherently scattered trapping light. By localizing each slope of the bacterium we generate high-contrast, super-resolution movies in three dimensions, without any object staining. This approach will help in investigating the nanomechanics of single wall-less bacteria while reacting to external stimuli on a broad temporal bandwidth.
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.
We investigate binding and plasmonic coupling between optically trapped 80 nm silver spheres using a combination of spectroscopic sensing and 3D interferometric laser particle tracking on a 1 μs time ...scale. We demonstrate that nanoparticle coupling can be either spontaneous or induced by another particle through confinement of diffusion. We reveal ultrafast entries and exits of nanoparticles inside the optical trap, fast particle rearrangements before binding, and dimer formation allowing new insights into nanoparticle self-assembly.
Phagocytic particle uptake is crucial for the fate of both living cells and pathogens. Invading particles have to overcome fluctuating lipid membranes as the first physical barrier. However, the ...energy and the role of the fluctuation-based particle-membrane interactions during particle uptake are not understood. We tackle this problem by indenting the membrane of differently composed Giant Unilamellar Vesicles (GUVs) with optically trapped particles until particle uptake. By continuous 1 MHz tracking and autocorrelating the particle's positions within 30µs delays for different indentations, the fluctuations' amplitude, the damping, the mean forces, and the energy profiles were obtained. Remarkably, the uptake energy into a GUV becomes predictable since it increases for smaller fluctuation amplitudes and longer relaxation time. Our observations could be explained by a mathematical model based on continuous suppression of fluctuation modes. Hence, the reduced particle uptake energy for protein-ligand interactions LecA-Gb3 or Biotin-Streptavidin results also from pronounced, low-friction membrane fluctuations.
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.
Background The evolution of the IgE response to the numerous allergen molecules of Dermatophagoides pteronyssinus is still unknown. Objectives We sought to characterize the evolutionary patterns of ...the IgE response to 12 molecules of D pteronyssinus from birth to adulthood and to investigate their determinants and clinical relevance. Methods We investigated the clinical data and sera of 722 participants in the German Multicenter Allergy Study, a birth cohort started in 1990. Diagnoses of current allergic rhinitis (AR) related to mite allergy and asthma were based on yearly interviews at the ages of 1 to 13 years and 20 years. IgE to the extract and 12 molecules of D pteronyssinus were tested by means of ImmunoCAP and microarray technology, respectively, in sera collected at ages 1, 2, 3, 5, 6, 7, 10, 13, and 20 years. Exposure to mites at age 6 and 18 months was assessed by measuring Der p 1 weight/weight concentration in house dust. Results One hundred ninety-one (26.5%) of 722 participants ever had IgE to D pteronyssinus extract (≥0.35 kUA /L). At age 20 years, their IgE recognized most frequently Der p 2, Der p 1, and Der p 23 (group A molecules; prevalence, >40%), followed by Der p 5, Der p 7, Der p 4, and Der p 21 (group B molecules; prevalence, 15% to 30%) and Der p 11, Der p 18, clone 16, Der p 14, and Der p 15 (group C molecules; prevalence, <10%). IgE sensitization started almost invariably with group A molecules and expanded sequentially first to group B and finally to group C molecules. Early IgE sensitization onset, parental hay fever, and higher exposure to mites were associated with a broader polymolecular IgE sensitization pattern. Participants reaching the broadest IgE sensitization stage (ie, ABC) had significantly higher risk of mite-related AR and asthma than unsensitized participants. IgE to Der p 1 or Der p 23 at age 5 years or less predicted asthma at school age. Conclusions Parental hay fever and early exposure to D pteronyssinus allergens promote IgE polysensitization to several D pteronyssinus molecules, which in turn predicts current mite-related AR and current/future asthma. These results might inspire predictive algorithms and prevention strategies against the progression of IgE sensitization to mites toward AR and asthma.
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