Ries Lab

Superresolution Microscopy for Structural Cell Biology

We develop superresolution microscopy technologies to visualize the structure and dynamics of molecular machines in cells on the nanoscale. We use these techniques to investigate the dynamic structural organization of the machinery that drives clathrin-mediated endocytosis.

Research in the Ries Lab

Previous and current research

Like nanoscopic machines, protein assemblies carry out a multitude of cellular processes. Understanding their function and mechanics requires knowing their in situ structural organisation and dynamics, which are barely accessible to classical structural biology techniques (EM, NMR, crystallography). Superresolution microscopy, and specifically Single-Molecule Localization Microscopy (SMLM), is an ideal tool to study these assemblies in their natural cellular environment and to understand their modus operandi.

In our group, we push the limits of superresolution microscopy by developing optical, biological and computational methods:

To enable quantitative measurements, we developed novel reference samples , taking advantage of the well-defined symmetry and stoichiometry of the nuclear pore complex (NPC). These standards allow quantifying the resolution of microscopes, labeling efficiencies, and the precise copy numbers of proteins in complexes.

To achieve highest 3D resolution, we are developing new analysis tools and the new microscope technologies Supercritical Angle Localization Microscopy and 4Pi-SMLM.

High-throughput superresolution microscopy and a comprehensive analysis software enable the acquisition of large datasets and their interpretation with powerful statistics.

The main biological question that drives technology development in my group is clathrin-mediated endocytosis. We achieved a first breakthrough by applying our high-throughput superresolution microscopy to determine the nanoscale distribution of 23 endocytosis proteins in over 100’000 yeast endocytic structures. As the superresolution images contained timing markers, we could computationally reconstruct the dynamic molecular architecture of a forming endocytic vesicle from this massive data set. This allowed us to discover how actin generates and transfers the force to pull in a membrane vesicle.

Future projects and goals

Our research vision is to develop the microscopy technologies that will allow us to visualize the structure and the dynamics of molecular machines in living cells on the nanoscale. This will add key technology to enable the emerging field of in situ structural biology, and, maybe most importantly, make the dimension of time accessible to structural analysis. By visualizing the conformational and compositional changes macromolecular complexes undergo during their functional cycle, we will be able to see molecular machines in action and obtain unprecedented insights into the mechanisms of the core machinery of life.

To fulfill this vision, we will further develop our advanced microscopes for maximal 3D resolution and multi-color on native samples, and develop computational tools to reconstruct dynamic protein assemblies from thousands of snapshot images. We will establish the novel MINFLUX superresolution technology to directly image dynamic conformational changes of protein assemblies in living cells with nanometer spatial and millisecond temporal resolution. To highlight specific protein complexes in defined functional states inside electron densities we will build seamless workflows for correlative superresolution microscopy and electron tomography. We will drive the development of these technologies by investigating the dynamic structural organization of the endocytic machinery in yeast and mammals.

We will continue to make these technologies available to our biological collaborators and as open-source to the community to investigate structure – function relationships of other cellular machines.


Ries Lab 2018

Group Members

Jonas Ries
Group Leader
Takahiro Deguchi
Takahiro Deguchi
Angelica Maria Estrada Pacheco
Angelica Maria Estrada Pacheco
Research Technician
Philipp Hoess
Philipp Hoess
PhD Student
Sheng Liu
Sheng Liu
Ulf Matti
Ulf Matti
Research Technician
Aline Tschanz
Aline Tschanz
PhD Student
Jervis Vermal Thevathasan
Jervis Vermal Thevathasan
Yu-Le Wu
Yu-Le Wu
PhD Student
Tomas Noordzij
Tomas Noordzij
Master Student

Associated PostDocs

Rafael Martins Galupa
Markus Mund

Open positions

We are always looking for motivated Master students and interns with a background in physics, programming or biology. Please contact Jonas directly.

If you want to join as a PhD student, please note that admission is exclusively via the EMBL PhD program.




SMAP (Superresolution Microscopy Analysis Platform) is a comprehensive software framework for single-molecule localization microscopy that can be used for fitting of raw data and subsequent analysis.
It is published in Nature Methods.
The most up-to-date source code can be obtained from GitHub.
You can download a compiled version for Windows and Mac, an example dataset and check the documentation.
The additional external software necessary to run SMAP can be downloaded from the respective websites: Bioformats MATLAB Toolbox (bfmatlab.zip) and Micro-Manager 1.4.22


fit3Dcspline is a GPU based 3D single molecule fitter for arbitrary, experimental point spread functions (PSF).
It was published in Nature Methods and is part of SMAP (see above).
Data obtained from the standalone version can be visualized using Felix Woitzel's Pointcloud-Loader.

Excitation Intensities

dSTORM example data sets (Diekmann et al., 2020, Nature Methods).
SMLM (dSTORM) example data showing the effect of different excitation intensities (unprocessed TIF-stacks and SMAP-fitted localizations files).

Direct Supercritical Angle Localization microscopy (dSALM)

Preprint on bioRxiv
Example data and step-by-step guide for analysing dSALM data



The GitHub repository RiesPieces contains small and large internal projects from the lab that we hope can benefit other research groups. More specifically, you can find 3D designs for microscope parts, our recipe for focus stabilization, electronics pieces and many more.


In the GitHub repository LaserEngine you can find the design (machined parts and the optical path) of a custom laser box that is operated with cheap laser diodes. Furthermore, it contains the description of the necessary electronics and how to build the agitation module to scramble the modes of the multimode illumination. You can find more information in the corresponding paper.

Easier Micro-manager User interface (EMU)

EMU is a Micro-manager plugin that controls the device properties of the microscope via an accessible GUI. It is distributed with Micro-Manager 2.0-gamma (nightly build) or can be installed from its source on GitHub. There and in the publication you can find more information on its functionalities and how to adapt it to your microscope setup.

Nup96 cell lines

In these cell lines, the nuclear pore component Nup96 is homozygously labeled with mEGFP, SNAP-tag, HaloTag, or the photoconvertible fluorescent protein mMaple. They can be used as 3D resolution standards for calibration and quality control, to quantify absolute labeling efficiencies and as precise reference standards for molecular counting. You can find more information in the respective paper. The cell lines are available from the cell line repository CLS.



EMU: reconfigurable graphical user interfaces for Micro-Manager
Deschamps J, Ries J.
BMC Bioinformatics doi: 10.1186/s12859-020-03727-8
Corresponding GitHub repository
Previously on bioRxiv doi: 10.1101/2020.03.18.997494
Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation
Aramesh M, Mergenthal S, Issler M, Weber F, Plochberger B, Qin X, Liska R, Duda G, Huppa J, Ries J, Schuetz G, Klotzsch E.
How good are my data? Reference standards in superresolution microscopy
Mund M, Ries J.
Molecular Biology of the Cell doi: 10.1091/mbc.E19-04-0189
Quantitative Data Analysis in Single-Molecule Localization Microscopy
Wu Y, Tschanz A, Krupnik L, Ries J.
Trends in Cell Biology doi: 10.1016/j.tcb.2020.07.005
SMAP - A Modular Superresolution Microscopy Analysis Platform for SMLM Data
Ries J.
Nature Methods doi: 10.1038/s41592-020-0938-1
Previously on bioRxiv doi: 10.1101/2020.07.24.219188
More information and links to the software can be found above
Teaching deep neural networks to localize single molecules for super-resolution microscopy
Speiser A, Müller LR, Matti U, Obara CJ, Legant WR, Ries J, Macke JH, Turaga SC.
arXiv: 1907.00770
Optimizing imaging speed and excitation intensity for single-molecule localization microscopy
Diekmann R, Kahnwald M, Schoenit A, Deschamps J, Matti U, Ries J.
Nature Methods doi: 10.1038/s41592-020-0918-5
A link to the data can be found above
Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution
Dasgupta A, Deschamps J, Matti U, Hübner U, Becker J, Strauss S, Jungman R, Heintzmann R, Ries J.
Accurate 4Pi single-molecule localization using an experimental PSF model
Li Y, Buglakova E, Zhang Y, Thevathasan JV, Bewersdorf J, Ries J.
Optics Letters doi: 10.1364/OL.397754
Previously on bioRxiv doi: 10.1101/2020.03.18.997163
Identification of novel synaptonemal complex components inC. elegans
Hurlock ME, Čavka I, Kursel LE, Haversat J, Wooten M, Nizami Z, Turniansky R, Hoess P, Ries J, Gall JG, Rog O, Köhler S, Kim Y.
Journal of Cell Biology doi: 10.1083/jcb.201910043
Nanoscale pattern extraction from relative positions of sparse 3D localisations
Curd A, Leng J, Hughes R, Cleasby A, Rogers B, Trinh C, Baird M, Takagi Y, Tiede C, Sieben C, Manley S, Schlichthaerle T, Jungmann R, Ries J, Shroff H, Peckham M.
MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells
Gwosch K, Pape JK, Balzarotti F, Hoess P, Ellenberg J, Ries J, Hell SW.
Nature Methods doi: 10.1038/s41592-019-0688-0
Previously on bioRxiv doi: 10.1101/734251
Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging
Zhang Y, Schroeder LK, Lessard MD, Kidd P, Chung J, Song Y, Benedetti L, Li Y, Ries J, Grimm JB, Lavis LD, De Camilli P, Rothman JE, Baddeley D & Bewersdorf J.
Nature Methods doi: 10.1038/s41592-019-0676-4
Previously on bioRxiv doi: 10.1101/613174
A cost-efficient open source laser engine for microscopy
Schroeder D, Deschamps J, Dasgupta A, Matti U, Ries J.
Biomedical Optics Express doi: 10.1364/BOE.380815
Previously on bioRxiv doi: 10.1101/796482
Corresponding GitHub repository


Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates
Elfarrash S, Møller Jensen N, Ferreira N, Betzer C, Thevathasan JV, Diekmann R, Adel M, Mansour Omar N, Boraie MZ, Gad S, Ries J, Kirik D, Nabavi S, Jensen H.
Acta Neuropathologica Communications doi: 10.1186/s40478-019-0865-5
Previously onbioRxiv doi: 10.1101/681064
Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy
Rieger B, Stallinga S, Heydarian H, Schueder F, Jungmann R, Ries J, Przybylski A, Bates M, Keller-Findeisen J, van Werkhoven B.
bioRxiv doi: 10.1101/837575
Topological data analysis quantifies biological nano-structure from single molecule localization microscopy
Pike JA, Khan AO, Pallini C, Thomas SG, Mund M, Ries J, Poulter NS, Styles IB.
Bioinformatics doi: 10.1093/bioinformatics/btz788
Previously on bioRxiv doi: 10.1101/400275
Photoactivation of silicon rhodamines via a light-induced protonation
Frei MS, Hoess P, Lampe M, Nijmeijer B, Kueblbeck M, Ellenberg J, Wadepohl H, Ries J, Pitsch S, Reymond L, Johnsson K.
Nature Communications doi: 10.1038/s41467-019-12480-3
Previously on bioRxiv doi: 10.1101/626853
Nuclear pores as versatile reference standards for quantitative superresolution microscopy
Thevathasan JV, Kahnwald M, Cieśliński K, Hoess P, Peneti SK, Reitberger M, Heid D, Kasuba KC, Hoerner SJ, Li Y, Wu Y, Mund M, Matti U, Pereira PM, Henriques R, Nijmeijer B, Kueblbeck M, Sabinina VJ, Ellenberg J, Ries J.
Nature Methods doi: 10.1038/s41592-019-0574-9
Previously on bioRxiv doi: 10.1101/582668
Type-I myosins promote actin polymerization to drive membrane bending in endocytosis
Manenschijn HE, Picco A, Mund M, Rivier-Cordey AS, Ries J, Kaksonen M.
eLife doi: eLife.44215
Previously on bioRxiv doi: 10.1101/490011
Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically‐Encoded Probes for DNA‐PAINT
Schlichthaerle T, Strauss MT, Schueder F, Auer A, Nijmeijer B, Kueblbeck M, Sabinina VJ, Thevathasan JV, Ries J, Ellenberg J, Jungmann R.
Angewandte Chemie International Edition doi: 10.1002/anie.201905685
Previously on bioRxiv doi: 10.1101/579961
A tessellation-based colocalization analysis approach for single-molecule localization microscopy
Levet F, Julien G, Galland R, Butler C, Beghin A, Chazeau A, Hoess P, Ries J, Giannone G, Sibarita JB.
Nature Communications doi: 10.1038/s41467-019-10007-4
Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization
Li Y, Wu Y, Hoess P, Mund M, Ries J.
Biomedical Optics Express doi: 10.1364/BOE.10.002708
Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software
Sage D, Pham T, Babcock H, Lukes T, Pengo T, Chao J, Velmurugan R, Herbert A, Agrawal A, Colabrese S, Wheeler A, Archetti A, Rieger B, Ober R, Hagen GM, Sibarita J, Ries J, Henriques R, Unser M, Holden S.
Nature Methods doi: 10.1038/s41592-019-0364-4
Previously on bioRxiv doi: 10.1101/362517
















Ries Lab

EMBL Heidelberg
Meyerhofstr. 1
69117 Heidelberg

Room 402
Phone number: +49 6221 3878199
E-Mail: ries(at)embl.de

Ries Lab website at www.embl.de
European Molecular Biology Laboratory