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The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. However, optical aberrations and fixed fluorophore dipoles can lead to non-isotropic and distorted PSFs, thereby complicating and biasing conventional fitting approaches. In addition, some researchers deliberately modify the PSF by introducing specific phase shifts in order to provide improved sensitivity, e.g., for localizing molecules in 3D, or for determining the dipole orientation. For devising an experimental approach, but also for interpreting obtained data it would be helpful to have a simple visualization tool which calculates the expected PSF for the experiment in mind. To address this need, we have developed a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. It incorporates a wide range of microscope and fluorophore parameters, enabling an accurate representation of various imaging conditions. Further, our app directly provides the Cramer-Rao bound for assessing the best achievable localization precision under given conditions. In addition to facilitating the simulation of PSFs of isotropic emitters, our application provides simulations of fixed dipole orientations as encountered, e.g., in cryogenic single-molecule localization microscopy applications. Moreover, it supports the incorporation of optical aberrations and phase manipulations for PSF engineering, as well as the simulation of crowded environments with overlapping molecules. Importantly, our software allows for the fitting of custom aberrations directly from experimental data, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.

Single molecule localization microscopy relies on the precise quantification of the position of single dye emitters in a sample. This precision is improved by the number of photons that can be detected from each molecule. It is therefore recommendable to increase illumination times for the recording process. Particularly recording at cryogenic temperatures dramatically reduces photobleaching and thereby allows a massive increase in illumination times to several seconds. As a downside, microscope instabilities may well introduce jitter during such long illuminations, deteriorating the localization precision. In this paper, we theoretically demonstrate that a parallel recording of fiducial marker beads together with a novel fitting approach accounting for the full drift trajectory allows for largely eliminating drift effects for drift magnitudes of several hundred nanometers per frame., Comment: 12 pages, 7 figures

Single molecule localization microscopy has the potential to resolve structural details of biological samples at the nanometer length scale. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of ~1nm, thereby reaching the corresponding Cram\'{e}r Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.

Single molecule localization microscopy is a recently developed superresolution imaging technique to visualize structural properties of single cells. The basic principle consists in chemically attaching fluorescent dyes to the molecules, which after excitation with a strong laser may emit light. To achieve superresolution, signals of individual fluorophores are separated in time. In this paper we follow the physical and chemical literature and derive mathematical models describing the propagation of light emitted from dyes in single molecule localization microscopy experiments via Maxwell's equations. This forms the basis of formulating inverse problems related to single molecule localization microscopy. We also show that the current status of reconstruction methods is a simplification of more general inverse problems for Maxwell's equations as discussed here., Comment: 38 pages, 10 figures

Absorption microscopy is a powerful technique, enabling the detection of single non- fluorescent molecules at room temperature. So far, the molecular absorption has been probed optically via the attenuation of a probing laser. The sensitivity of optical probing is not only restricted by background scattering, but it is fundamentally limited by laser shot noise. Here, we present nanomechanical photothermal microscopy, which overcomes the scattering and shot noise limit by detecting the sample absorption directly with a temperature sensitive substrate. We use nanomechanical silicon nitride drums, whose resonant frequency detunes with local heating. Individual Au nanoparticles with diameters from 10 nm - 200 nm and single molecules (Atto 633) are scanned with a 305 {\mu}W heating laser with a peak irradiance of 330 {\mu}W/{\mu}m2. Using stress-optimized drums, we achieve a sensitivity of 45 fW/sqrt(Hz), which results in a signal-to-noise ratio of >60 for a single molecule. Our method has important consequences for a wide range of applications, such as imaging, absorption analysis and spectrochemical analysis of non-fluorescent samples.