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- ItemDirect supercritical angle localization microscopy for nanometer 3D superresolution([London] : Nature Publishing Group UK, 2021) Dasgupta, Anindita; Deschamps, Joran; Matti, Ulf; Hübner, Uwe; Becker, Jan; Strauss, Sebastian; Jungmann, Ralf; Heintzmann, Rainer; Ries, Jonas3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.
- ItemA versatile and customizable low-cost 3D-printed open standard for microscopic imaging([London] : Nature Publishing Group UK, 2020) Diederich, Benedict; Lachmann, René; Carlstedt, Swen; Marsikova, Barbora; Wang, Haoran; Uwurukundo, Xavier; Mosig, Alexander S.; Heintzmann, RainerModern microscopes used for biological imaging often present themselves as black boxes whose precise operating principle remains unknown, and whose optical resolution and price seem to be in inverse proportion to each other. With UC2 (You. See. Too.) we present a low-cost, 3D-printed, open-source, modular microscopy toolbox and demonstrate its versatility by realizing a complete microscope development cycle from concept to experimental phase. The self-contained incubator-enclosed brightfield microscope monitors monocyte to macrophage cell differentiation for seven days at cellular resolution level (e.g. 2 μm). Furthermore, by including very few additional components, the geometry is transferred into a 400 Euro light sheet fluorescence microscope for volumetric observations of a transgenic Zebrafish expressing green fluorescent protein (GFP). With this, we aim to establish an open standard in optics to facilitate interfacing with various complementary platforms. By making the content and comprehensive documentation publicly available, the systems presented here lend themselves to easy and straightforward replications, modifications, and extensions.
- ItemCorrecting systematic errors by hybrid 2D correlation loss functions in nonlinear inverse modelling(San Francisco, California, US : PLOS, 2023) Mayerhöfer, Thomas G.; Noda, Isao; Pahlow, Susanne; Heintzmann, Rainer; Popp, JürgenRecently a new family of loss functions called smart error sums has been suggested. These loss functions account for correlations within experimental data and force modeled data to obey these correlations. As a result, multiplicative systematic errors of experimental data can be revealed and corrected. The smart error sums are based on 2D correlation analysis which is a comparably recent methodology for analyzing spectroscopic data that has found broad application. In this contribution we mathematically generalize and break down this methodology and the smart error sums to uncover the mathematic roots and simplify it to craft a general tool beyond spectroscopic modelling. This reduction also allows a simplified discussion about limits and prospects of this new method including one of its potential future uses as a sophisticated loss function in deep learning. To support its deployment, the work includes computer code to allow reproduction of the basic results.
- ItemIMAGE-IN: Interactive web-based multidimensional 3D visualizer for multi-modal microscopy images(San Francisco, California, US : PLOS, 2022) Gupta, Yubraj; Costa, Carlos; Pinho, Eduardo; A. Bastião Silva, Luís; Heintzmann, RainerAdvances in microscopy hardware and storage capabilities lead to increasingly larger multidimensional datasets. The multiple dimensions are commonly associated with space, time, and color channels. Since “seeing is believing”, it is important to have easy access to user-friendly visualization software. Here we present IMAGE-IN, an interactive web-based multidimensional (N-D) viewer designed specifically for confocal laser scanning microscopy (CLSM) and focused ion beam scanning electron microscopy (FIB-SEM) data, with the goal of assisting biologists in their visualization and analysis tasks and promoting digital work-flows. This new visualization platform includes intuitive multidimensional opacity fine-tuning, shading on/off, multiple blending modes for volume viewers, and the ability to handle multichannel volumetric data in volume and surface views. The software accepts a sequence of image files or stacked 3D images as input and offers a variety of viewing options ranging from 3D volume/surface rendering to multiplanar reconstruction approaches. We evaluate the performance by comparing the loading and rendering timings of a heterogeneous dataset of multichannel CLSM and FIB-SEM images on two devices with installed graphic cards, as well as comparing rendered image quality between ClearVolume (the ImageJ open-source desktop viewer), Napari (the Python desktop viewer), Imaris (the closed-source desktop viewer), and our proposed IMAGE-IN web viewer.
- ItemOptical Sectioning and High Resolution in Single-Slice Structured Illumination Microscopy by Thick Slice Blind-SIM Reconstruction(San Francisco, California, US : PLOS, 2015) Jost, Aurélie; Tolstik, Elen; Feldmann, Polina; Wicker, Kai; Sentenac, Anne; Heintzmann, Rainer; Degtyar, Vadim E.The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out-of-focus fluorescence and diffraction limited resolution. In this work, we show that a single slice of Structured Illumination Microscopy (two or three beam SIM) data can be processed to provide an image exhibiting tight sectioning and high transverse resolution. Our reconstruction algorithm is adapted from the blind-SIM technique which requires very little knowledge of the illumination patterns. It is thus able to deal with illumination distortions induced by the sample or illumination optics. We named this new algorithm thick slice blind-SIM because it models a three-dimensional sample even though only a single two-dimensional plane of focus was measured.
- ItemThe role of risk communication in public health interventions. An analysis of risk communication for a community quarantine in Germany to curb the SARS-CoV-2 pandemic(San Francisco, California, US : PLOS, 2021) Scholz, Juliane; Wetzker, Wibke; Licht, Annika; Heintzmann, Rainer; Scherag, André; Weis, Sebastian; Pletz, Mathias; Betsch, Cornelia; Bauer, Michael; Dickmann, Petra; Frey, RosemaryBackground: Separating ill or possibly infectious people from their healthy community is one of the core principles of non-pharmaceutical interventions. However, there is scarce evidence on how to successfully implement quarantine orders. We investigated a community quarantine for an entire village in Germany (Neustadt am Rennsteig, March 2020) with the aim of better understanding the successful implementation of quarantine measures. Methods: This cross-sectional survey was conducted in Neustadt am Rennsteig six weeks after the end of a 14-day mandatory community quarantine. The sample size consisted of 562 adults (64% of the community), and the response rate was 295 adults, or 52% (33% of the community). Findings: National television was reported as the most important channel of information. Contact with local authorities was very limited, and partners or spouses played a more important role in sharing information. Generally, the self-reported information level was judged to be good (211/289 [73.0%]). The majority of participants (212/289 [73.4%]) approved of the quarantine, and the reported compliance was 217/289 (75.1%). A self-reported higher level of concern as well as a higher level of information correlated positively with both a greater acceptance of quarantine and self-reported compliant behaviour. Interpretation: The community quarantine presented a rare opportunity to investigate a public health intervention for an entire community. In order to improve the implementation of public health interventions, public health risk communication activities should be intensified to increase both the information level (potentially leading to better compliance with community quarantine) and the communication level (to facilitate rapport and trust between public health authorities and their communities). © 2021 Scholz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- ItemsimpleISM—A straight forward guide to upgrade from confocal to ISM(San Francisco, California, US : PLOS, 2022) Goswami, Monalisa; Lachmann, René; Kretschmer, Robert; Heintzmann, RainerResolution in a confocal laser scanning microscopes (CLSM) can be improved if the pinhole is closed. But closing the pinhole will deteriorate the signal to noise ratio (SNR). A simple technique to improve the SNR while keeping the resolution same by upgrading the system to an image scanning microscope. In this paper, we explain in detail, based on an Olympus Fluoview 300 system, how a scanning microscope can be upgraded into an image scanning microscope (ISM) using a simple camera-based detector and an Arduino Due providing a galvo driving and camera synchronization signals. We could confirm a resolution improvement as well as superconcentration and made the interesting observation of a reduced influence of laser fluctuations.
- ItemA Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum(San Francisco, California, US : PLOS, 2015) Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg; Harder, TilmannDiatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.
- ItemNonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength(San Francisco, California, US : PLOS, 2016) Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, RainerStructured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.
- ItemBessel beam CARS of axially structured samples([London] : Macmillan Publishers Limited, part of Springer Nature, 2015) Heuke, Sandro; Zheng, Juanjuan; Akimov, Denis; Heintzmann, Rainer; Schmitt, Michael; Popp, JürgenWe report about a Bessel beam CARS approach for axial profiling of multi-layer structures. This study presents an experimental implementation for the generation of CARS by Bessel beam excitation using only passive optical elements. Furthermore, an analytical expression is provided describing the generated anti-Stokes field by a homogeneous sample. Based on the concept of coherent transfer functions, the underling resolving power of axially structured geometries is investigated. It is found that through the non-linearity of the CARS process in combination with the folded illumination geometry continuous phase-matching is achieved starting from homogeneous samples up to spatial sample frequencies at twice of the pumping electric field wave. The experimental and analytical findings are modeled by the implementation of the Debye Integral and scalar Green function approach. Finally, the goal of reconstructing an axially layered sample is demonstrated on the basis of the numerically simulated modulus and phase of the anti-Stokes far-field radiation pattern.