Prof. Francesco Pasqualini

Synthetic Physiology Lab, University of Pavia, Italy

Background

Prof. Francesco Pasqualini is a Harvard-trained bioengineer leading the synthetic physiology laboratory at the University of Pavia, currently researching cardiac development using engineered cell culture platforms. By optimizing this platform with cell lines and then moving to human induced pluripotent stem cells, Prof. Pasqualini can get a unique perspective of the developing heart.

Prof. Pasqualini told us more, “We are studying the mechanobiology of the heart as well as investigating the use of various extracellular matrix components to control tissue and organ-level behavior. We are particularly interested in recapitulating the early phases of human heart development using defined extracellular matrix and cardiac cell types. All using live-cell microscopy, of course.”

This study uses a Nikon Ti2 microscope and a modular spinning-disk confocal module, the X Light V3 from CrestOptics.

Dr. Alessandra Scarpellini, Dr. Maria Giubettini

CrestOptics S.p.A., Via di Torre Rossa, Rome, Italy

Background

CrestOptics is a leading company in the development and manufacture of advanced systems for fluorescence microscopy, featuring products such as the X-Light series of spinning-disk confocal modules. CrestOptics has also recently launched DeepSIM, a super-resolution module for 3D samples. We discussed DeepSIM with the Head of Sales and Marketing Dr. Scarpellini, and Application Specialist Dr. Giubettini.

Dr. Scarpellini told us more, “We noticed an increase in demand for higher resolution to see more of biological samples, but many options for super-resolution imaging were not accessible due to high cost, specialized sample preparation, or incompatibility with live sample imaging. This is why we developed DeepSIM, which makes super-resolution accessible, similar to how we made spinning-disk accessible. You can work with the same sample you’d typically use, but in a variety of configurations, such as combining DeepSIM with a spinning disk confocal or as a standalone, on upright or inverted microscopes, and able to work with a full range of objectives.”

Dr. Giubettini expanded on the possible imaging configurations, “It’s super easy to change between three modalities: widefield, spinning disk, and DeepSIM, you can switch to SIM to go into more detail on a sample, all while working with the same sample preparation.”

Dr. Christopher Toepfer

Radcliffe Department of Medicine, University of Oxford, UK

Background

Dr. Chris Toepfer is a principal investigator interested in understanding how cardiac physiology changes in inherited cardiovascular conditions. The lab uses fluorescence microscopy and calcium imaging to observe how cellular contractility is affected across different heart conditions, such as those seen in professional sports.

Using human stem-cell-derived cardiomyocytes with a known genome, they then use CRISPR Cas9 to insert patient-specific mutations and GFP tags into this cell model and observe the effects; such as whether the cells beat harder or faster than normal. This is done with calcium imaging to monitor calcium flux of a cell, and imaging of the GFP-labelled sarcomeres themselves, enabling Dr. Toepfer and team to measure contractility on the fundamental unit of muscle contraction in real-time.

Dr. Yu Ting Chow, Dr. Amir Tahmasebipour

Mekonos Inc., San Francisco, California, US

Background

Mekonos Inc. is a start-up company developing a biomedical microelectromechanical system (bio-MEMS) platform based on semiconductor and microfluidic technologies, to enable single-cell transfection with high viability and a scalable workflow.

We spoke with Dr. Amir Tahmasebipour, a Senior Scientist at Mekonos Inc., to learn more, “We do a lot of experiments: prototyping, development, designing, iterating and troubleshooting devices, and as this is used for single-cell transfection we work on very small scales. Our microfluidic platform is high-throughput and uses high velocities, so we really rely on good imaging devices to be able to characterize our system, for both microfluidic and MEMS key technologies.”

“Our devices allow us to flow substances over attached cells, with the aim to manipulate and transfect single cells on several different timescales. We use imaging systems to measure the quality and quantity of transfection, as well as track where particles and cells are going.

Moritz Engelhardt, Dr. Kristin Grussmayer

Department of Bionanoscience, TU Delft, Netherlands

Background

The lab of Dr. Kristin Grussmayer develops light microscopy and analysis tools for life sciences and biophysics research, in particular addressing questions in molecular and cell biology. PhD student Moritz Engelhardt works within this group, with a project focused on studying protein aggregation in Huntington’s disease using cellular models, transfecting cells with plasmids containing the mutant Huntingtin protein and observing the effects of aggregation on cell health.

Moritz further explained his research, “We use a reconstruction algorithm that requires multiplane imaging to construct phase from brightfield images [1]. A prism splits light into different axial planes in the sample, and we can simultaneously look at different planes in the sample and use this to reconstruct the phase.”

“We also want to do subcellular segmentation in our phase images, we want to use a deep learning-based segmentation algorithm, to create the training data we use correlative fluorescence imaging. We take a phase image, then a fluorescence image to get the ground truth data of what part is the aggregate or organelle of interest.”

Prof. Daniel Cohen, Dr. Julienne LaChance

Department of Chemical and Biological Engineering, Princeton University, USA

LaChance J, Cohen DJ (2020) Practical fluorescence reconstruction microscopy for large samples and low-magnification imaging. PLoS Comput Biol 16(12): e1008443. https://doi.org/ 10.1371/journal.pcbi.1008443

Background

Fluorescence reconstruction microscopy (FRM) takes in transmitted light from a biological sample and outputs a series of reconstructed fluorescence images that predict what the sample would look like were it labeled with a certain set of fluorophores. This is done using ‘deep learning’ with a convolutional neural network (e.g. U-Net) that can be trained for FRM.

The FRM approach enables many benefits including reduced phototoxicity, freeing up of fluorescence channels, simplified sample preparation, and the ability to re-process legacy data for new insights. With the increase of computational processing power year on year, FRM may become a standard tool to augment quantitative biological imaging.

However, FRM is currently complex to implement, and FRM benchmarks are abstractions that are difficult to relate to how valuable or trustworthy a reconstruction is. In this paper, Cohen et al. relate the conventional benchmarks and demonstrations to practical and familiar cell biology analyses to demonstrate how FRM should be judged in context. This research demonstrates that FRM performs well even with lower-magnification microscopy data, often collected in screening and high-content imaging. This research specifically presents results for nuclei, cell-cell junctions, and fine feature reconstruction; provides data-driven experimental design guidelines; and provides researcher-friendly code, complete sample data, and a researcher manual to enable more widespread adoption of FRM.

Prof. Bernd Rieger

Department of Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, The Netherlands

Background

Prof. Bernd Rieger works with applied physics at TU Delft, and tells us about his research, “We are an applied physics and engineering group researching computational microscopy and building microscope systems, improving spatial resolution and temporal resolution of imaging systems. We start with empty optical tables and then build our systems.”

“Over the last two years, we have been developing new ideas, including novel structured illumination microscopy (SIM) methods such as SIMflux and SIM using single-mould fibres. Typically, in SIM you shift and rotate a pattern mechanically, which limits the speed. We have used fibres to generate these patterns and can change the pattern in the kilohertz range.”

Prof. Rieger’s work with SIM pushes the boundaries of high-speed super-resolution imaging.

Prof. Sophie Martin

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

Vještica, A., Bérard, M., Liu, G., Merlini, L., Nkosi, P. J., & Martin, S. G. (2021) Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance. PLoS biology, 19(1), e3001067

Introduction

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers a signaling cascade, which represses mating and initiates meiosis.

In this research, Prof. Sophie Martin and the team from the University of Lausanne establish a system to demonstrate that mating blocks not only safeguard zygote ploidy but also prevent cell death caused by aberrant fusion attempts. This was done using long-term imaging and flow cytometry, and we identified previously unrecognized and independent roles for Mei3 and Mei2 in yeast zygotes.

Dr. Rory Power

Advanced Light Microscopy, EMBL Heidelberg, Germany

Background

Dr. Rory Power is a staff scientist and engineer at the advanced imaging center of the EMBL headquarters in Heidelberg. Involved in a variety of projects, Dr. Power also oversees Ph.D. students involved in building custom light sheet imaging systems.

Dr. Power described their light sheet imaging system, “It’s an oblique plane microscope (OPM) that uses a single objective, which allows us to do light sheet microscopy in a more traditional inverted, epifluorescence setup with less restrictions on sample geometry and using a water dipping objective.”

“This system is for imaging of a little tentacle monster Nematostella, which are interesting from a morphological and behavioral point of view, how their muscle hydraulics and neural dynamics influence their development and motion. We are using our light sheet system to image contractile waves of motion within these animals.”

Prof. Dirk Geyer, Martin Richter (M.Sc.), Adrian Breicher (M.Eng.)

Laboratory for Optical Diagnostics and Renewable Energy (ODEE), Department of Mechanical and Plastics Engineering, Darmstadt University of Applied Sciences, Darmstadt, Germany

Background

The group of Prof. Dirk Geyer, including Ph.D. students Martin Richter and Adrian Breicher, work towards the decarbonization of energy conversion. They told us about their research, “Our main research field is the combustion of promising new fuels for the future such as hydrogen and ammonia, which, unlike methane, contain no carbon in their molecular structure and therefore produce no CO2 emissions in the combustion process.”

“In our recent work, we are looking at the co-firing of methane (CH4), the main component of natural gas, and hydrogen (H2). We have many combustion systems operating with natural gas, and we could substitute some of the CH4 for H2, but this has effects on combustion. To understand the fundamentals of these effects, we investigated laminar flames with reduced complexity, such as Bunsen flames: if we burn pure CH4 we observe a smooth flame cone, if we add certain amounts of H2 cellular structures start to appear, so we are looking into the structure of these with planar laser-induced fluorescence (PLIF).”

“PLIF can analyze molecules/species that occur during the combustion process, such as the OH radical, which tell us where reactions are happening, and we can then map other measurements onto this.”