Skip to content
What's on

SIMposium: recent advancements in structured illumination microscopy

Event

Location

Zoom webinar

Overview

Theo Murphy international scientific meeting organised by Dr Kirti Prakash, Dr Benedict Diederich, Professor Lothar Schermelleh, Dr Stefanie Reichelt and Professor Rainer Heintzmann.

Credits: Lothar Schermelleh, University of Oxford.

Structured illumination microscopy (SIM) has emerged as an essential super-resolution technique for 3D and live-cell imaging. However, to date, there has not been a dedicated symposium/workshop covering different aspects of SIM, from biological applications, use of commercial instruments, to bespoke hardware and software development. The meeting aims to recap recent developments as well as outline future trends.

More information about the schedule of talks and speaker biographies will be available soon. Speaker abstracts will be available closer to the meeting date. Recordings of the presentations will be available on this page after the meeting has taken place. A two-part Philosophical Transactions A journal issue accompanies this meeting. The first volume can be read here, and the second volume will be published in early 2022. 

Attending the event

This meeting is intended for researchers in relevant fields.

  • Free to attend
  • Advance registration essential
  • Live subtitles will be available

Enquiries: contact the Scientific Programmes team.

Event organisers

Select an organiser for more information

Schedule of talks

21 February

Session 1 08:45-13:45

Elements of SIM

8 talks Show detail Hide detail

Chairs

Dr Kirti Prakash, The Institute of Cancer Research, UK.

08:45-09:00 Welcome by the Royal Society & lead organiser

09:00-09:30 Thoughts on structured illumination - past, presence and future

Professor Rainer Heintzmann, Institute of Physical Chemistry, Friedrich-Schiller-Universität Jena, Germany

Abstract

This talk presents answers to fundamental questions related to structured illumination (SIM) and more generally superresolution microscopy, based on the speaker personal views on superresolution in light microscopy. He will discuss the definition of superresolution, Abbe's resolution limit and the classification of superresolution methods into nonlinear-, prior knowledge- and near-field-based superresolution. A further focus is put on the capabilities and technical aspects of present and future SIM methods.

Show speakers

09:30-10:00 SIM: a counterfactual history

Dr James Manton, MRC Laboratory of Molecular Biology, Cambridge, UK

Show speakers

10:00-10:30 Studying chromatin organisation and RNA dynamics by 3D-SIM

Dr Lothar Schermelleh, University of Oxford, UK

Show speakers

10:30-11:00 Coffee

11:00-11:30 Structured illumination microscopy and image scanning microscopy: A comparison

Dr Colin Sheppard, University of Wollongong and Italian Institute of Technology, Australia and Italy

Abstract

Image formation in structured illumination microscopy (SIM) and image scanning microscopy (ISM) is compared. Image scanning microscopy is a confocal microscopy technique using a detector array in place of the usual confocal pinhole with a single element detector. Both techniques can result in a doubling of spatial frequency bandwidth compared with conventional fluorescence microscopy. The performance of SIM is usually presented in a way that includes digital reconstruction, which makes comparison with other techniques difficult. Here, the researchers consider the resolution of SIM based on the optical properties alone, and compare it with ISM using pixel reassignment. SIM results in a superior resolution. Of course, in both cases digital processing can result in further improvements in resolution. The main advantage of ISM over SIM is the optical sectioning property, which allows ISM to penetrate better through thick specimens. 

Show speakers

11:30-12:00 Overcoming physical resolution limits of fluorescence microscopes with sparse deconvolution

Dr Liangyi Chen, Peking University, China

Abstract

To enable live-cell long-term super-resolution (SR) imaging, Chen’s group have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimised SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging in live cells. Thereafter, they take advantage of a priori knowledge of the sparsity and continuity of fluorescently labeled biological structures, and develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

Show speakers

12:00-12:45 Panel discussion: Open challenges in SIM data acquisition and processing

Professor Rainer Heintzmann, Institute of Physical Chemistry, Friedrich-Schiller-Universität Jena, Germany
Dr James Manton, MRC Laboratory of Molecular Biology, Cambridge, UK
Dr Marcel Müller, Bielefeld University, Germany
Dr Reto Fiolka, The University of Texas Southwestern Medical Center, USA
Dr Colin Sheppard, University of Wollongong and Italian Institute of Technology, Australia and Italy

Show speakers

12:45-13:30

Lunch

Session 2 13:30-17:45

New SIM developments - hardware and software

9 talks Show detail Hide detail

Chairs

Dr Lothar Schermelleh, University of Oxford, UK

13:30-14:00 Light-sheet microscopy with multi-directional structured illumination

Dr Reto Fiolka, The University of Texas Southwestern Medical Center, USA

Abstract

Structured illumination microscopy (SIM) doubles the spatial resolution of a microscope without requiring high laser power or specialised fluorophores. In its 3D form, SIM illuminates the entire sample, which can lead to increased photo-bleaching and reconstruction artifacts due to out-of-focus blur. In contrast, light-sheet fluorescence microscopy (LSFM) mostly avoids exciting out-of-focus fluorescence and thereby drastically lowers photo-bleaching. While LSFM excels at long-term or high-speed volumetric imaging, its spatial resolution is modest. Therefore, combining LSFM with SIM appears attractive, as it promises gentle live cell imaging at doubled resolution. Unfortunately, combining the two is rather complex, as SIM requires illuminating the sample with three different pattern orientations. Applied to LSFM, this would require up to three illumination objectives to deliver differently oriented structured light-sheets. Here the researchers implement SIM in oblique plane microscopy (OPM), a light-sheet technique that requires only one objective for illumination and fluorescence detection. Rotation of the structured light-sheet is facilitated via a high-speed image rotator, which also de-rotates the fluorescence light for subsequent detection. This allows the researchers to double the resolution of OPM and image at up to 2Hz volumetric rate. They present imaging of the cytoskeleton, mitochondria dynamics and clathrin mediated endocytosis using this new microscope.

Show speakers

14:00-14:30 High-speed and cost-efficient super-resolution structured illumination microscopy enabled entirely by fiber optics

Professor Thomas Huser, University of Bielefeld, Germany

Abstract

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fastest and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high and the physical size of the instruments is still prohibitively large. Here, Professor Huser will present his and his team's latest efforts towards realising a new generation of compact, cost-efficient and high-speed SR-SIM instruments based entirely on fiber-optics. He will discuss the technical approaches which we have taken in order to realize a highly robust from of 2D- and TIRF-based SR-SIM, and show applications of cell biological data obtained with this instrument. He will also discuss the influence of the modulation transfer function of different types of sCMOS cameras on the overall system performance, in particular the spatial resolution that can be obtained with such an instrument.

Show speakers

14:30-15:00 Multi-SIM via deep learning algorithm for super-resolution live imaging

Professor Dong Li, Institute of Biophysics, Chinese Academy of Sciences, China

Abstract

Biology research is desired to characterise the intracellular dynamics at high spatiotemporal resolution with low photobleaching and phototoxicity effects, which therefore allow continuously resolve the delicate structures and behaviors of the engaged organelles over the whole biological process. However, the trade-offs between spatial and temporal resolution, and low phototoxicity/photobleaching always compromise the practical performance of current super-resolution imaging techniques. To achieve these normally opposing goals, in this talk Professor Dong Li will discuss his and his team's latest developments in multi-modality structured illumination microscopy (Multi-SIM) and lattice light sheet SIM microscopy (LLS-SIM), which enable high-speed super-resolution live-cell imaging for thousands of time-points spanning over hours of time-lapse. Recently, they further developed a deep-learning algorithm for super-resolution image reconstruction, termed deep Fourier channel attention network (DFCAN), which further extends the applicability of Multi-SIM and LLS-SIM into more challenging imaging conditions. 

Show speakers

15:00-15:30 Tea

15:30-16:00 Title to be confirmed

16:00-16:20 Digital micromirror devices for cost-effective and fast multi-color structured illumination microscopy

Dr Marcel Müller, Bielefeld University, Germany

Abstract

Modern structured illumination microscopes (SIMs) often rely on spatial light modulators (SLMs) to allow for a fast and robust implementation of the SIM technique. Of the different SLM technologies available, digital micromirror devices (DMDs) feature many advantages, such as high speed, low cost, and optical properties such as wavelength range and polarisation. However, they also pose a challenge, as their jagged surface introduces a blazed grating effect, that has to be carefully accounted for when using these devices with the coherent light sources needed for SIM. In this talk, both a simulation framework and the experimental implementations of DMDs for the use in SIM will be discussed. The simulation framework is used to find wavelength combinations suitable for multi-color DMD-based SIM, and to assess the systems alignment parameters. An experimental implementation showcases how these results can be used to create a fast, robust and very cost-effective dual- and multi-color DMD-SIM system.

Show speakers

16:20-16:40 GPU-accelerated real-time reconstruction in Python of three-dimensional datasets from structured illumination microscopy with hexagonal patterns

Dr Hai Gong, Imperial College London, UK

Abstract

The researchers present a structured illumination microscopy system that projects a hexagonal pattern by the interference among three coherent beams, suitable for implementation in a light-sheet geometry. Seven images acquired as the illumination pattern is shifted laterally can be processed to produce a super-resolved image that surpasses the diffraction-limited resolution by a factor of over 2 in an exemplar light-sheet arrangement. Three methods of processing data are discussed depending on whether the raw images are available in groups of seven, individually in a stream or as a larger batch representing a three-dimensional stack. The researchers show that imaging axially moving samples can introduce artefacts, visible as fine structures in the processed images. However, these artefacts are easily removed by a filtering operation carried out as part of the batch processing algorithm for three- dimensional stacks. The reconstruction algorithms implemented in Python include specific optimisations for calculation on a graphics processing unit and we demonstrate its operation on experimental data of static objects and on simulated data of moving objects. They show that the software can process over 239 input raw frames per second at 512×512 pixels, generating over 34 super-resolved frames per second at 1024×1024 pixels.

Show speakers

16:40-17:00 mmSIM: an open toolbox for accessible structured illumination microscopy

Dr Michael Shaw, National Physical Laboratory and Department of Computer Science, University College London, UK
Dr Craig Russell, EMBL-EBI, UK

Abstract

Over the past two decades structured illumination microscopy (SIM) has proven to be a powerful method for high-speed fluorescence imaging beyond the classical diffraction limit. This presentation will describe a simple open source approach, termed mmSIM, for controlling SIM hardware and acquiring SIM images based on the popular MicroManager software package. By complementing existing hardware designs and open source image reconstruction software, mmSIM supports the keen microscopist to develop and run their own custom-built SIM system. The configuration and performance of two mmSIM-controlled spatial light modulator-based SIM systems will discussed along with results from various bioimaging studies performed using the devices, including measurement of the kinetics of in vitro protein fibrillogenesis and visualisation of intracellular uptake.

Show speakers

17:00-17:45 Panel discussion: Biological application of SIM - from experimental design to quantitative evaluation

Dr Lothar Schermelleh, University of Oxford, UK
Professor Thomas Huser, University of Bielefeld, Germany
Dr Liangyi Chen, Peking University, China
Dr Michael Shaw, National Physical Laboratory and Department of Computer Science, University College London, UK
Professor Dong Li, Institute of Biophysics, Chinese Academy of Sciences, China
Dr Jennifer Lippincott-Schwartz, Janelia Research Campus, USA

Show speakers

22 February

Session 3 09:00-12:45

SIM and SMLM

8 talks Show detail Hide detail

Chairs

Dr Benedict Diederich, Leibniz Institute of Photonic Technology, Germany

09:00-09:30 Title to be confirmed

Dr Sandrine Lévêque-Fort, CNRS, Université Paris Saclay, France

Show speakers

09:30-10:00 Title to be confirmed

10:00-10:30 At the molecular resolution with MINFLUX?

Dr Kirti Prakash, The Institute of Cancer Research, UK.

Abstract

MINFLUX is a promising new development in single-molecule localisation microscopy, claiming a resolution of 1-3 nm in living and fixed biological specimens. While MINFLUX can achieve very high localisation precision, quantitative analysis of reported results leads us to dispute the resolution claim and question reliability for imaging sub-100-nm structural features, in its current state.

Show speakers

10:30-11:00 Coffee

11:00-11:20 Upscaling SIM reconstruction via Deep Learning

Mr Miguel Boland, Imperial College London, UK

Abstract

Structured Illumination Microscopy (SIM) is a widespread methodology to image live and fixed biological structures smaller than the diffraction limits of conventional optical microscopy. Using recent advances in image up-scaling through deep learning models, we demonstrate a method to reconstruct 3D SIM image stacks with twice the axial resolution attainable through conventional SIM reconstructions. We further demonstrate our method is robust to noise and evaluate it against two-point cases and axial gratings. Finally, we discuss potential adaptions of the method to further improve resolution.

Show speakers

11:20-11:40 Polarised Illumination Coded Structured Illumination Microscopy (picoSIM): Experimental Results

Dr Kai Wicker, ZEISS Innovation Hub Dresden, Germany

Abstract

The need for acquiring at least three images to reconstruct an optical section of a sample limits the acquisition rate in structured illumination microscopy (SIM) for optical sectioning. In polarised illumination coded structured illumination microscopy (picoSIM) the three illumination patterns are encoded in a single polarised illumination light distribution. This distribution consists of linearly polarised light, with the polarisation orientation varying with the position in the focal plane. If the sample exhibits sufficient fluorescence anisotropy, this linear polarisation will still be present to a certain degree in the emitted light. Splitting the emission light and filtering the different parts with polarisation analysers of differing orientation thus allows the acquisition of the complete SIM data in a single exposure. In this presentation Dr Wicker describes the theoretical background of picoSIM and presents an experimental set-up and first experimental results.

Show speakers

11:40-12:00 Modular Microscopy – not just a Toy

Dr Benedict Diederich, Leibniz Institute of Photonic Technology, Germany

Show speakers

12:00-12:45 UC2 - an open source system for optics (The power in your pocket)

Dr Benedict Diederich, Leibniz Institute of Photonic Technology, Germany
Mr Haoran Wang, Leibniz-Institut für Photonische Technologien, Germany

Show speakers

12:45-13:30

Lunch

Session 4 13:30-17:45

New avenues for super-resolution microscopy

10 talks Show detail Hide detail

13:30-14:00 Looking under the hood of cells: from single molecule dynamics to whole cell organelle reconstructions

Dr Jennifer Lippincott-Schwartz, Janelia Research Campus, USA

Abstract

Powerful new ways to image the internal structures and complex dynamics of cells are revolutionising cell biology and bio-medical research. In this talk, Dr Lippincott-Schwartz will focus on how emerging fluorescent technologies are increasing spatio-temporal resolution dramatically, permitting simultaneous multispectral imaging of multiple cellular components. In addition, results will be discussed from whole cell milling using Focused Ion Beam Electron Microscopy (FIB-SEM), which reconstructs the entire cell volume at 4 voxel resolution. Using these tools, it is now possible to begin constructing an 'organelle interactome', describing the interrelationships of different cellular organelles as they carry out critical functions. The same tools are also revealing new properties of organelles and their trafficking pathways, and how disruptions of their normal functions due to genetic mutations may contribute to important diseases.

Show speakers

14:00-14:20 Title to be confirmed

14:20-14:40 Extended mechanical force measurements using structured illumination microscopy

Dr Kseniya Korobchevskaya, University of Oxford, UK

Abstract

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, the researchers present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies.

Show speakers

14:40-15:00 Quality control of image sensors using Gaseous Tritium Light Sources

Abstract

Cameras and other detectors are indispensable tools for modern light microscopy. Performance may, however, vary from device to device and they can show signs of damage or ageing which can, in turn, affect the quality and reproducibility of data. Despite this, many labs do not regularly perform quantitative quality control checks on their instruments. One explanation could be a relative lack of convenient and low-cost calibration sources. Work by David McFadden, Brad Amos and Rainer Heintzmann proposes to tackle this problem using inexpensive tritium radioluminescent tubes (betalights). The mechanical design is easily reproducible and can be 3D-printed. Another major advantage of the design is that the calibration allows for a plug and play approach with automatic image analysis based on the photon transfer method. The calibration yields results for the photon conversion factor and read noise as well as the detector quantum efficiency. The intensity is suitable for calibrating detectors at very low light levels, characteristic especially of single-molecule-localisation microscopy.

15:00-15:30 Tea

15:30-15:50 Laser-free super-resolution microscopy

Dr Kirti Prakash, The Institute of Cancer Research, UK.

Abstract

A new single-molecule localisation microscopy (SMLM) configuration termed laser-free super-resolution microscopy (LFSM) is presented. LFSM enables high-density single-molecule super-resolution microscopy with a conventional epifluorescence microscope set-up and a mercury arc lamp. The setup allows single molecules to be switched on and off (a phenomenon termed as ‘blinking’), detected and localised. The use of a short burst of deep blue excitation (350–380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, stimulated emission depletion (STED) microscopy and LFSM are demonstrated on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution.

Show speakers

15:50-16:10 Enabling single-molecule localisation microscopy in turbid food emulsions

Dr Johannes Hohlbein, Wageningen University, The Netherlands

Abstract

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity. The number of accessible microscopy frameworks that are suitable for dim samples or measurements in turbid food systems, however, has remained low. The researchers therefore developed the miCube: a versatile super-resolution capable microscope, which combines high spatiotemporal resolution, good adaptability, and straight forward installation. They further enabled ultrafast data analysis using a phasor-based localisation algorithm. In the second part, Dr Hohlbein will present results on studying food-related emulsions using super-resolution microscopy. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, the researchers employed adaptive optics (AO) to compensate aberrations and to modulate the emission wavefront enabling point spread function (PSF) engineering. As a model system for a non-transparent food colloid, they designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. They targeted phosvitin with fluorescently labelled primary antibodies and obtained two- and three-dimensional images of phosvitin covered oil droplets. Their data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, their work paves the way for localising biomacromolecules at heterogeneous colloidal interfaces in food emulsions.

Show speakers

16:10-16:30 Open microscopy documentation for the real world: interactive tools for quality, reproducibility and sharing value of imaging experiments based on community specifications

Dr Caterina Strambio De Castillia, The University of Massachusetts Chan Medical School, USA

Abstract

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. In this talk Dr Strambio De Castillia will discuss highly interoperable, open-source software tools that were designed in the context of burgeoning global bioimaging community initiatives to facilitate the documentation of microscopy experiments as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, these tools visual nature of these tools make them well suited for teaching users about the intricacies of image acquisition and how it impacts the results of their experiments.

Show speakers

16:30-16:45 clesperanto: Open-source GPU-accelerated image processing across programming languages and software ecosystems

Dr Robert Haase, University of Technology TU Dresden, Germany

Abstract

The optimised computing power of graphics processing units (GPUs) is changing the way we do image analysis in the life sciences. Not just new deep learning approaches but also GPU-accelerated classical image processing techniques are becoming available to end-users with minimal coding skills. This ongoing revolution is an opportunity to synchronise image processing operations and workflows as many of them have to be rewritten for new GPU-based computing architectures. The clesperanto project is paving the path for image-analysts working with Fiji, Icy, ImageJ, Matlab, napari, Jython, Python and others to use the same language for formulating their scientific image analysis workflows in a cross-platform fashion and thus, connects the communities of multiple software ecosystems. 

Show speakers

16:45-17:45 Panel discussion - Open Microscopy: Quo Vadis?

Dr Johannes Hohlbein, Wageningen University, The Netherlands
Dr Benedict Diederich, Leibniz Institute of Photonic Technology, Germany
Dr Robert Haase, University of Technology TU Dresden, Germany
Dr Caterina Strambio De Castillia, The University of Massachusetts Chan Medical School, USA

Show speakers
SIMposium: recent advancements in structured illumination microscopy

21 – 22 February 2022

other
Was this page useful?
Thank you for your feedback
Thank you for your feedback. Please help us improve this page by taking our short survey.