Article
Biochemical Research Methods
Soeren Doose
Summary: Single-molecule localization microscopy has become an important tool in biomedical research. The LOCAN Python library provides well-defined data structures and analysis methods for analyzing localization data in a script or computable notebook.
Article
Chemistry, Physical
Cecilia Zaza, German Chiarelli, Ludovit P. Zweifel, Mauricio Pilo-Pais, Evangelos Sisamakis, Fabio Barachati, Fernando D. Stefani, Guillermo P. Acuna
Summary: Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Article
Chemistry, Multidisciplinary
Antonio Garcia, Shannon J. Saluga, David J. Dibble, Pia A. Lopez, Nozomi Saito, Suzanne A. Blum
Summary: This study for the first time imaged the selectivity of individual molecular catalysts for two different reactions in real time at the single-catalyst level, revealing that catalyst selectivity may be unexpectedly spatially and time-variable in ruthenium-catalyzed polymerization.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2021)
Article
Chemistry, Multidisciplinary
Aleksandra K. Adamczyk, Teun A. P. M. Huijben, Miguel Sison, Andrea Di Luca, Stefano Vanni, Sophie Brasselet, Kim I. Mortensen, Fernando D. Stefani, Mauricio Pilo-Pais, Guillermo P. Acuna
Summary: This study demonstrates a controlled orientation of single molecules in DNA origami by linking them to oligonucleotide strands and leaving unpaired bases. The number of unpaired bases affects the stretching and orientation of the fluorophore linkers. These findings expand the application of DNA origami in the fabrication of nanodevices involving orientation-dependent molecular interactions.
Article
Biophysics
Daniel F. Nino, Joshua N. Milstein
Summary: In recent years, significant advances have been made in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events becomes more difficult, affecting the accuracy of measurements. The study provides a working definition of the dynamic range for quantitative SMLM and explores the factors affecting it.
BIOPHYSICAL JOURNAL
(2021)
Article
Multidisciplinary Sciences
Sanghyeon Park, Yonghyeon Jo, Minsu Kang, Jin Hee Hong, Sangyoon Ko, Suhyun Kim, Sangjun Park, Hae Chul Park, Sang-Hee Shim, Wonshik Choi
Summary: The authors applied label-free wavefront sensing adaptive optics to single-molecule localization microscopy (SMLM) for deep-tissue super-resolution imaging. The proposed system allows for the measurement and correction of complex tissue aberrations, enabling SMLM to resolve sub-diffraction morphologies in deep tissues.
NATURE COMMUNICATIONS
(2023)
Article
Chemistry, Multidisciplinary
Yaya Hao, Mingqiang Li, Qian Zhang, Jiye Shi, Jiang Li, Qian Li, Chunhai Fan, Fei Wang
Summary: This study developed a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. The research has important implications for expanding the functionality of CRISPR molecular machines and developing integrated molecular circuits and high-throughput nucleic acid detection.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2022)
Article
Chemistry, Multidisciplinary
Stefan Krause, Evelyn Ploetz, Johann Bohlen, Patrick Schuler, Renukka Yaadav, Florian Selbach, Florian Steiner, Izabela Kaminska, Philip Tinnefeld
Summary: Researchers screened 10 preparation methods for graphene-on-glass and presented an optimized protocol for single-molecule and super-resolution imaging; they utilized DNA origami technology to detect graphene quality, showing discrepancies between conventional techniques such as light microscopy, atomic force microscopy, and Raman spectroscopy for graphene characterization.
Article
Biotechnology & Applied Microbiology
Andrew E. S. Barentine, Yu Lin, Edward M. Courvan, Phylicia Kidd, Miao Liu, Leonhard Balduf, Timy Phan, Felix Rivera-Molina, Michael R. Grace, Zach Marin, Mark Lessard, Juliana Rios Chen, Siyuan Wang, Karla M. Neugebauer, Joerg Bewersdorf, David Baddeley
Summary: This study presents an integrated acquisition and analysis platform for single-molecule localization microscopy, which enables the imaging and analysis of 10,000 cells per day. The platform utilizes microscopy-specific data compression, distributed storage, and distributed analysis to increase throughput and allows for automated analysis initiation from the microscope. The platform also supports imaging of multiple cells in multi-well sample formats.
NATURE BIOTECHNOLOGY
(2023)
Article
Chemistry, Physical
Narima Eerqing, Hsin-Yu Wu, Sivaraman Subramanian, Serge Vincent, Frank Vollmer
Summary: This study developed an integrated optical setup that combines optoplasmonic and DNA-PAINT techniques for detecting oligonucleotides. The experimental results of both techniques were compared and complementary insights into single molecule processes were provided. Hybridisation events were observed in the same sample cell using fluorescence and optoplasmonic sensor signals, revealing the accumulation of irreversible hybridisation events in optoplasmonic sensing and the novel physicochemical mechanisms that stabilize DNA hybridisation on optically-excited plasmonic nanoparticles.
NANOSCALE HORIZONS
(2023)
Article
Chemistry, Multidisciplinary
Maria Sanz-Paz, Fangjia Zhu, Nicolas Bruder, Karol Kolataj, Antonio I. Fernandez-Dominguez, Guillermo P. Acuna
Summary: By modifying the local density of photonic states, optical nanoantennas can alter the emission spectrum of a dye. In this study, DNA origami was used to precisely position an individual dye around a gold nanorod and observe how it affects the emission spectrum. The spectral overlap between the nanorod resonance and different vibrational levels of the dye's excitonic ground state can lead to strong suppression or enhancement of transitions.
Article
Chemistry, Multidisciplinary
Renjie Niu, Chunyuan Song, Fei Gao, Weina Fang, Xinyu Jiang, Shaokang Ren, Dan Zhu, Shao Su, Jie Chao, Shufen Chen, Chunhai Fan, Lianhui Wang
Summary: This study introduces a DNA origami based nanoprinting strategy for transferring DNA strands to the surface of gold nanocubes, generating stereo-controlled gold nanostructures with specific geometry and composition for stronger surface enhanced Raman scattering signal amplification.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2021)
Article
Nanoscience & Nanotechnology
Abhinav Banerjee, Micky Anand, Simanta Kalita, Mahipal Ganji
Summary: DNA-PAINT super-resolution imaging is used to measure the free energy of dinucleotide base stacking at the single-molecule level. Results show that addition of a single dinucleotide base stacking can greatly stabilize the DNA duplex nanostructure. These findings are important for designing functional DNA nanostructures and predicting the local DNA structure.
NATURE NANOTECHNOLOGY
(2023)
Article
Chemistry, Multidisciplinary
Fatih N. Gur, Susanne Kempter, Florian Schueder, Christoph Sikeler, Maximilian J. Urban, Ralf Jungmann, Philipp C. Nickels, Tim Liedl
Summary: The study demonstrates a DNA origami switch that utilizes the transition from double-stranded DNA to single-stranded DNA to drive a reversible conformational change. By using the unique properties of DNA, the switch exerts a contractive force produced by the extended single-stranded DNA to achieve large-scale motion. The operation of this switch is shown through various microscopy techniques, illustrating the intricate forces at play in nanoscale physical systems operating in fluids.
ADVANCED MATERIALS
(2021)
Article
Chemistry, Multidisciplinary
Kristina Huebner, Himanshu Joshi, Aleksei Aksimentiev, Fernando D. Stefani, Philip Tinnefeld, Guillermo P. Acuna
Summary: The study introduces a new technique to determine the orientation of single fluorophores in DNA origami structures, providing insights into the relationship between the fluorophores, environment, and structure. Results indicate that the orientation of fluorophores is influenced more by the specific molecular environment than the type of fluorophore.
Review
Microbiology
Andrew A. Bridges, Jojo A. Prentice, Ned S. Wingreen, Bonnie L. Bassler
Summary: This article discusses the network features and mechanisms of bacterial collective behaviors, including quorum sensing and second-messenger cyclic dimeric GMP (c-di-GMP) signal transduction. The article emphasizes ligand specificity and sensitivity, signal integration mechanisms, single-input sensory systems versus coincidence detectors, and tuning of input-output dynamics via feedback regulation. It summarizes how different features of signal transduction systems allow groups of bacteria to successfully interpret and collectively react to dynamically changing environments.
ANNUAL REVIEW OF MICROBIOLOGY
(2022)
Article
Biology
Carola Sparn, Eleni Dimou, Annalena Meyer, Roberto Saleppico, Sabine Wegehingel, Matthias Gerstner, Severina Klaus, Helge Ewers, Walter Nickel
Summary: The study reveals that Glypican-1 (GPC1) is the primary factor driving the unconventional secretion of Fibroblast growth factor 2 (FGF2), while its role in FGF2 signaling into cells is dispensable. Furthermore, the research provides insights into the structural basis of GPC1-dependent FGF2 secretion. These findings have significant implications for the key role of GPC1 in tumor progression.
Article
Cell Biology
Fabio Lolicato, Roberto Saleppico, Alessandra Griffo, Annalena Meyer, Federica Scollo, Bianca Pokrandt, Hans-Michael Muller, Helge Ewers, Hendrik Hahl, Jean-Baptiste Fleury, Ralf Seemann, Martin Hof, Britta Brugger, Karin Jacobs, Ilpo Vattulainen, Walter Nickel
Summary: This study found that cholesterol can promote the recruitment and translocation of FGF2 in cells, as well as enhance its binding to PI(4,5)P2. Through molecular dynamics simulations and experiments, it was revealed that cholesterol modulates the binding of FGF2 to PI(4,5)P2 by increasing the visibility of PI(4,5)P2 on the membrane surface, inducing the clustering of PI(4,5)P2 molecules, and increasing membrane tension.
JOURNAL OF CELL BIOLOGY
(2022)
Article
Cell Biology
Konstanty Cieslinski, Wu Yu-Le, Lisa Nechyporenko, Sarah Janice Hoerner, Duccio Conti, Michal Skruzny, Jonas Ries
Summary: In this study, the authors used single-molecule localization microscopy to visualize individual kinetochore complexes in budding yeast, and proposed a quantitative model of the budding yeast kinetochore based on their comprehensive dataset.
JOURNAL OF CELL BIOLOGY
(2023)
Article
Cell Biology
Markus Mund, Aline Tschanz, Yu-Le Wu, Felix Frey, Johanna L. Mehl, Marko Kaksonen, Ori Avinoam, Ulrich S. Schwarz, Jonas Ries
Summary: The study reveals the three-dimensional shapes of clathrin coats during endocytosis and introduces a novel Cooperative Curvature Model that accurately describes the changes in shapes and dynamics. The findings provide insights into the mechanism of clathrin coat remodeling during endocytosis and offer a potential general model for clathrin coat remodeling on the plasma membrane.
JOURNAL OF CELL BIOLOGY
(2023)
Article
Biochemical Research Methods
Yu-Le Wu, Philipp Hoess, Aline Tschanz, Ulf Matti, Markus Mund, Jonas Ries
Summary: Quantitative data analysis is crucial for extracting biological insights from the coordinates of single fluorophores in a single-molecule localization microscopy (SMLM) workflow. LocMoFit, an open-source framework, is introduced to fit an arbitrary model to localization coordinates, extract meaningful parameters, and select suitable models. It enables the analysis of complex, heterogeneous, and dynamic structures, and has been demonstrated in assembling multi-protein distribution maps, calculating single-particle averages, and performing time-resolved reconstruction of dynamic processes from static snapshots. Extensive simulation and visualization routines validate the robustness of LocMoFit, and tutorials are provided to enhance users' extraction of information from SMLM data.
Letter
Biochemical Research Methods
Klaus C. Gwosch, Francisco Balzarotti, Jasmin K. Pape, Philipp Hoess, Jan Ellenberg, Jonas Ries, Ulf Matti, Roman Schmidt, Steffen J. Sahl, Stefan W. Hell
Article
Optics
Jan Becker, Takahiro Deguchi, Alexander Juegler, Ronny Foerster, Uwe Huebner, Jonas Ries, Rainer Heintzmann
Summary: This paper proposes a method to improve the signal-to-noise ratio (SNR) of an imaging system by splitting the pupil and simultaneously acquiring and recombining two sub-images. This method achieves better SNR for transmitting high spatial frequency information without capturing more photons.
Article
Chemistry, Multidisciplinary
Ria Thielhorn, Isabelle Heing-Becker, Nadja Huempfer, Jakob Rentsch, Rainer Haag, Kai Licha, Helge Ewers
Summary: Expansion microscopy (ExM) is a technique that enlarges a hydrogel-embedded facsimile of a biological sample to improve resolution below the diffraction limit. However, gel formation and digestion cause a loss in label. In this study, a small molecule agent combining targeting, fluorescent labeling, and gel linkage was developed to overcome this issue, resulting in improved fluorescence signal retention and the resolution of nuclear pores. Mechanistic insight into dye retention in ExM was also provided.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2023)
Editorial Material
Cell Biology
Serge Mostowy, Aurelie Bertin, Helge Ewers
Article
Biochemical Research Methods
Shuang Fu, Wei Shi, Tingdan Luo, Yingchuan He, Lulu Zhou, Jie Yang, Zhichao Yang, Jiadong Liu, Xiaotian Liu, Zhiyong Guo, Chengyu Yang, Chao Liu, Zhen-li Huang, Jonas Ries, Mingjie Zhang, Peng Xi, Dayong Jin, Yiming Li
Summary: We propose a deep-learning method for precise localization of spatially variant point emitters, covering the full chip of a modern sCMOS camera. By using a graphic processing unit-based vectorial point spread function fitter, we accurately model the spatially variant PSF of a high numerical aperture objective in the entire field of view. Combined with deformable mirror-based optimal PSF engineering, we demonstrate high-accuracy three-dimensional single-molecule localization microscopy over a large volume, enabling imaging of mitochondria and nuclear pore complexes in entire cells without hardware scanning, with a 100-fold increase in throughput compared to existing techniques.
Article
Multidisciplinary Sciences
Michael C. A. Dyhr, Mohsen Sadeghi, Ralitsa Moynova, Carolin Knappe, Burcu Kepsutlu Cakmak, Stephan Werner, Gerd Schneider, James McNally, Frank Noe, Helge Ewers
Summary: Cryo-soft X-ray tomography (cryo-SXT) is a powerful method for investigating cell ultrastructure, offering high resolution and contrast without the need for labeling. A new automated 3D segmentation pipeline based on semi-supervised deep learning enables high-throughput analysis of tomographic data with limited manual annotations and variations in conditions.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2023)
Article
Multidisciplinary Sciences
Takahiro Deguchi, Malina K. Iwanski, Eva-Maria Schentarra, Christopher Heidebrecht, Lisa Schmidt, Jennifer Heck, Tobias Weihs, Sebastian Schnorrenberg, Philipp Hoess, Sheng Liu, Veronika Chevyreva, Kyung-Min Noh, Lukas C. Kapitein, Jonas Ries
Summary: We developed a live-cell tracking method with nanometer spatial and millisecond temporal resolution using MINFLUX super-resolution technique. This method allowed us to resolve the precise stepping motion of motor protein kinesin-1 on microtubules in living cells. Tracking of motors on fixed cell microtubules also revealed the architecture of the microtubule cytoskeleton with protofilament resolution.
Article
Engineering, Electrical & Electronic
Joran Deschamps, Christian Kieser, Philipp Hoess, Takahiro Deguchi, Jonas Ries
Summary: Modern microscopy increasingly relies on microscope automation for improved throughput, reproducibility, and observation of rare events. Arduino microcontrollers are widely used for automation due to their low cost and accessibility, while field programmable gate arrays (FPGA) are capable of high-speed and parallel signal processing. However, configuring FPGA remains complex. In this work, an affordable FPGA called MicroFPGA with an open-source and user-friendly programming language is used to create a versatile microscope control platform.
Article
Biochemical Research Methods
Yu-Le Wu, Philipp Hoess, Aline Tschanz, Ulf Matti, Markus Mund, Jonas Ries
Summary: This paper presents an open-source framework called LocMoFit for fitting arbitrary models to localization coordinates. The framework has significant potential for analyzing complex, heterogeneous, and dynamic structures, and can be used for assembling protein distribution maps, calculating single-particle averages, and performing time-resolved analysis of highly dynamic processes.
