Aurox Conference on Microscopy 2020

Leigh will provide a brief welcome to the delegates and explain some housekeeping, for instance "how to ask a question" on the webinar platform.This will be followed by a brief introduction to Aurox.

Although fluorescence microscopy has been a powerful technique for imaging intracellular molecules with high specificity, the target is limited to relatively large molecules, such as proteins because of the size of the fluorescent probes. We present the Raman-tag imaging technique that utilizes a tiny alkyne for tagging small molecules. The unique Raman shift by alkyne allows us to specifically detect and image tagged molecules in live cells. In the seminar, we introduce the principle of Raman microscopy and Raman tag imaging with the applications for imaging small molecules, such as nucleic acids, lipids, and drugs. We also present a technique to improve the detection sensitivity of the alkyne tag by using surface-enhanced Raman scattering and demonstrate the detection of drug dynamics in living cells.

The State Key Lab of Applied Optics, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun, China

Hooke Instruments Ltd., Changchun, China

Raman spectroscopy is a non-invasive and label-Cells, the fundamental units of life, are the basis for understanding the processes of life, as well as the mechanism of diseases. Single cell research is an emerging field of biology which promises new insight into phenotypic heterogeneity in isogenic cell population. However, there are a great many of challenges, such as non-invasive cell analysis and in situ single cell sorting from complex samples, in front of single cell biotechnology. Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free vibrational spectroscopy technique, which can provide intrinsic chemical fingerprint of an individual cell, possesses tremendous potential applications in every fields of life sciences.

Recently we developed a new type of single cell sorting system which combines Raman spectroscopy and cell separation based on laser-matter interaction which has advantages of visualization, pinpoint accuracy and label-free. We had successfully recognized and isolated various kinds of cells from a series of complex biological samples, such as seawater, soil and gut microbiome. Furthermore, the isolated cells can be used for sequencing, culture and other follow-up studies. To combine single cell sorting with single cell sequencing technology could build the relationship between cell phenotypes and genotypes, which will help us to uncover fundamental biological mechanisms. We have also successfully achieved single cell culture of isolated cells, which provides an innovative strategy for engineering cell screening and uncultured microorganisms studies. Single cell sorter can also be coupled with mass spectrometry to shorten the detection period of pathogens by skipping culture step which often takes several days. In consideration of the wide applications of single cell Raman and sorting technique, it will surely bring significant breakthroughs in all branches of life sciences and biomedicine.

We demonstrate large scale high sensitivity optical diffraction tomography (ODT) imaging of zebrafish. We make this possible by three improvements. First, we optimize the field of view by using a high magnification over numerical aperture ratio ODT set-up with phase stepping. Second, we decrease the noise in the reconstructed images by off-axis sample placement, numerical focus tracking, and acquisition of a large number of projections. Third, we optimize the tissue clearing procedure to prevent scattering and refraction. In addition, we demonstrate combined phase and polarization contrast optical diffraction tomography imaging. The polarization sensitive ODT measurement is performed in a similar fashion with the same set-up, but with cross-polarizer acquisition of the optical field. One polarizer is placed in front of the sample, and a second at 90 degrees, relative to the first, is placed behind the sample. For the polarization contrast projections, the amplitude of the cross-polarized component is calculated and from this a scaled birefringence is calculated. We use the phase and amplitude of the digital hologram to reconstruct the refractive index and (scaled) birefringence, respectively. We developed a model to obtain the birefringence both for small and large polarization angle rotation. Birefringence contrast imaging is demonstrated on zebrafish and shows high contrast images of the muscle tissue, something that is not well visible in conventional phase-based optical diffraction tomography. A striking result is the high contrast obtained in the polarization contrast ODT projections compared to the phase projections.

Holotomography: The next frontier in live cell, label free Imaging

Holotomography (HT) enables researchers to image cell morphology in 3D without the use of labels. In addition, long term Live Cell Imaging (LCI) is possible due to the extremely low amount of light required for HT as there is no phototoxicity to the cell.

HT also facilitates precise measurement of Refractive Index (RI) at nanoscale, allowing the visualization of sub-cellular organelles and estimations of protein and lipid concentration and their changes in real time.

High speed capture of the data set allows for dynamic imaging, with 3D capture rates of 2fps and 2D imaging at over 150fps for membrane fluctuation assessments.

With the new HT2 instrument, Fast, Quantitative, Label free imaging can be combined with 3D fluorescence to allow correlative imaging with specificity. To minimize phototoxicity from fluorescence imaging, acquisition of HT and FL are decoupled allowing independent strategies.

Keywords: cryo-EM, cryo-ET, vitrified biological samples, cryo-fluorescence (cryo-FM), cryo-CLEM, cryo-TEM, Single Particle Analysis (SPA), protein molecule structure, X-ray crystallography (XRC).

Cryo-imaging of biological samples embedded in vitrified ice has unique benefits and the development and implementation of cryo-EM and SPA has led to the Nobel Prize in 2017 [1]. One benefit is that the vitrification preserves proteins, cellular assemblies and cells in a fully hydrated and near native state. Vitrified samples are compatible with the vacuum conditions required for EM and ultra-structural preservation is considered the gold standard. More and more protein structures are solved with SPA, which complements or even partly replaces the established XRC and crucially, does not require the step of crystallization.

One challenge for cryo-ET and SPA is, however, the consistent and repeatable preparation of vitrified samples and mastering cryo-specific sample handling protocols. To make such workflows for SPA, thin cells or lamella cutting in FIB-SEM more widely accessible, Linkam and LUMC have together developed an automated robotic plunge freezer, which is now in beta-testing phase. Here the paper-blotting step, a typical feature of current cryo-plunger devices, has been replaced with a programmable controlled suction method which allows real-time optically monitoring by a built-in microscope and control of the liquid film thickness prior to plunging.

Because the grid handling, glow discharge, sample application, plunging and storage are all automated in the robotic system (image below) the preparation of cryo-samples is simplified and more repeatable.

A benefit of cryo-preparation and imaging techniques in LM is the significantly reduced photo-bleaching in cryo-fluorescence: CLEM allows to map and target regions of interest without EM beam damage and by using complementing labelling techniques. Reduced bleaching and improved photon statistics aids the localization precision for super-resolution techniques.

In this talk we give an overview on the workflows and capabilities of the plunger system under development and show how the new plunger system interfaces with the cryo-fluorescence stage CMS196.

[1] www.nature.com/news/cryo-electron-microscopy-wins-chemistry-nobel-1.22738

[2] Celler et al., NATURE COMM. (2016), 7:11836 | DOI: 10.1038/ncomms11836

Cryo-correlative imaging approach for subcellular characterisation

Zubair Ahmed Nizamudeen, Alexander Kondrashov, David Scurr, Julie Watts, Andrew Hook, Christopher Parmenter, Banafshe Larijani, Morgan Alexander, Chris Denning, Kenton Arkill

Division of Cancer and Stem Cells, School of Medicine, University of Nottingham
School of Pharmacy, University of Nottingham
Department of pharmacy and pharmacology, Department of Physics, University of Bath

Conventional cryo-correlative light-electron microscopy (CLEM) approaches are limited to ultrastructural analysis of subcellular components. Here we demonstrate two advancements of the CLEM technology which can be applied to study chemical properties at subcellular scale. Using the newly developed cryo-3D HYBRID ORBI Secondary ion mass spectrometry (SIMS) platform, fluorescently labelled cryo-preserved cells were correlated using Time of flight (TOF) mode, which revealed 3D correlation of multiple lipid species. The use of ORBI mode in the system allowed improved resolution of higher mass chemical species from correlated cells in the same platform at subcellular level. Next, we correlated cryo-preserved fluorescent cells under the 3D cryo-Focused ion beam scanning electron microscopy (FIBSEM) platform to site-specifically lift subcellular regions which were then used for transcriptomic analysis. qPCR analysis for 18s TaqMan probes confirmed the presence of RNA material from lift-outs. Pilot work shows how further development of these two approaches coupled with conventional cryo-CLEM techniques can be applied in multiple fields of biology including nuclear membrane dynamics, localised transcriptomics, and extracellular vesicles.

Rapid cryo-preservation of biological specimens is the gold standard for ultra-structural analysis. However, current methods for fluorescence imaging molecules under cryogenic conditions are limited to low resolutions of about 400 nm. We have developed CryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging at 200 nm resolution using structured illumination microscopy. CryoSIM dramatically increases the usefulness of correlative imaging with cryo electron microscopy or cryo soft X-ray tomography by reducing the resolution gap. The microscope is easily reproducible, as it is assembled from commodity parts and controlled by the open source Python Microscope and Cockpit software.

One of the major obstacles for the successful implementation of super-resolution cryo-CLEM is the risk of devitrification of the sample during super-resolution data acquisition. Typically, relatively high laser intensities are required for the photo-switching of fluorescent molecules to achieve resolution improvement. Cryo-SOFI is a super-resolution cryo-FM concept based on low laser intensities, which is fully compatible with cryo-EM and does not require unusual sample preparation. Due to its simplicity, it can be implemented in any existing cryo-FM system.

The Golgi apparatus is the main sorting station of the cell and despite decades of research we are still far from fully understanding how it functions. While the machinery and molecular interactions involved in cargo sorting have been extensively investigated in vitro, there is a lack of understanding of the dynamics and nanoscale organization in living cells. To be able to image molecular processes in the intrinsically crowded perinuclear Golgi area, we have developed a labelling strategy for dual-color live-cell stimulated emission depletion (STED) super-resolution imaging. In the lab we apply gene editing techniques (CRISPR/Cas9) to generate knock-ins to avoid over-expression and multi-color live-cell nanoscopy to understand how cargo sorting is realized at the nanoscale level and in living cells.

In this talk I will focus on STED (Stimulated Emission Depletion) technology from Abberior Instruments, the leading innovator, developer and manufacturer of STED super resolution microscopes. Their instruments are designed by the inventors of the method, including Abberior’s co-founder, Nobel Laureate Prof. Stefan W. Hell. They offer STED-microscopes with unprecedented resolutions down to 20 nm. Abberior Instruments has a strong focus on custom microscopy solutions and is committed to providing extensive and long-term upgrades for its instruments. For example the STEDYCON, which is a completely new class of nanoscope, converts your conventional epifluorescence microscope into a versatile four-colour confocal (405nm, 488nm, 561nm, 640nm) and STED (775nm) system, whilst being both compact and extremely easy to use. I will also talk about the features of the newly released Facility Line, including easy3D STED and DyMIN, as well as the MINFLUX technology.

Single Molecule Localization Microscopy (SMLM) is a powerful imaging technique that combines quantitative information with the highest resolution achievable in light microscopy. Since its still recent discovery, it has been a game changer in many biological studies aiming to visualize structures at the nanoscale.

Abbelight is the result of 10 years of academic research on cutting-edge detection methods in fluorescence microscopy. Our unique offers are designed to provide the best instruments, software, and scientific expertise to speed-up the entire imaging workflow - from sample preparation, to image aquisition and analysis - within a wide range of research applications in biology, including in neuroscience, immunology, or mechanobiology...

Recently, Abbelight has implemented new cutting-edge features to its solution: ultra-fast acquisition and spectral demixing. By combining them with the 3D detection technology and the NEO software suite, this makes possible the use of simultaneous multicolor imaging at the speed of a confocal microscope with the precision of single molecule detection. SMLM then becomes an essential tool for biological research not only providing the finest imaging details but at the highest speed.

During this talk, we will present our nanoscopy solutions and focus onto those features, from the theory to practical implementations by our customers.

The spatial resolution of light microscopy is continuously being pushed with the development of new technologies. The significant milestones have been Confocal, 4Pi, Two-photon microscopy, STED, Zero-point STED, SIM and SMLM. Recently, correlative light microscopy techniques (MINFLUX, SIMFLUX, etc) have further pushed the spatial resolution down to a few nanometers.

Though the promise of resolution (~1 nm, Hell’s molecular limit) is attractive, not many biological applications need this resolution. Moreover, the test structures (microtubules, NPCs) are in the 20-150 nm range, raising concerns on the actual biologically meaningful resolution attained. In addition, the microscope designs are complex and require a significant investment, often from public funds.

The fact remains that new biology still happens in 20-200 nm range and correlative methods are needed so that the findings from one method can be independently validated by another, with an equivalent resolution and under identical imaging conditions. Here, I propose a framework to do sequential STED, SIM and SMLM on the same sample using the same fluorophore.

Are your live-cell fluorescence imaging experiments suffering from illumination overhead?

Alex Kiepas, Elena Voorand, Firas Mubaid, Peter M. Siegel and Claire M. Brown
Department of Physiology, McGill University
Goodman Cancer Research Centre, McGill University
Department of Biochemistry, McGill University
Department of Medicine, McGill University
Advanced BioImaging Facility (ABIF), McGill University

Fluorescence illumination can cause phototoxicity that negatively affects living samples. In widefield applications, many researchers are unaware of the presence and negative impact of “illumination overhead” (IO) on photobleaching and phototoxicity in live-cell experiments. IO occurs due to hardware and software delays in the microscope imaging set up. If light sources such as fast-switching LEDs can be triggered directly using transistor-transistor logic (TTL) circuits IO can be essentially eliminated. However, lamp based light sources cannot be triggered with TTL circuits and IO occurs due to hardware delays. In addition, software delays can be present due to delays in image acquisition and storage during live cell acquisition experiments. Here data is shown demonstrating the contribution of IO on widefield and spinning disk microscopes. Data is also shown demonstrating that using diffuse light delivery (DLD) or longer exposure times with lower fluorescence excitation light can be used to minimize the impact of IO without the need for hardware or software upgrades. Information on using mitochondrial morphology and cell migration as sensitive and relative fast readouts for cell health will also be presented. Finally, a workflow is presented showing how to determine the maximum exposure time that can be used.

The MACSima™ instrument is a fully automated imaging system based on fluorescence microscopy. It's MICS (multiparameter imaging cell screen) technology united with a broad spectrum of available antibodies, enables you to analyse hundreds of markers within a single sample, multiple samples at a time, while it is still convenient and easy to use.

https://www.miltenyibiotec.com/GB-en/products/macs-imaging-and-microscopy/ultra-high-content-imaging.html#gref

Department of Bioengineering, Temple University

Cancer Biology Program, Fox Chase Cancer Center

Tumor cell structures that have long been hypothesized as necessary for metastasis are invadopodia, invasive protrusions rich in structural and adhesion proteins, as well as metalloproteases. Using our unique intravital imaging approaches (Perrin et al, 2019 Cancer Rep; Bayarmagnai et al, 2018 Meth Mol Biol.), we previously demonstrated that invadopodia in vivo are necessary for intravasation and consequent lung metastasis (Gligorijevic et al, Plos Bio 2014). In primary breast carcinoma, we found that cells which assemble invadopodia migrate at slow speeds, in perivascular niches where the ECM is cross-linked. Outside of these niches, no invadopodia were observed and cells migrated at high speeds, via contact guidance along collagen fibers. The invadopodia-driven motility can be switched to contact guidance by reducing the ECM cross-linking or by knocking down Tks5, which in turn reduces intravasation and metastasis. We next deduced that invadopodia-driven motility consists of two oscillating states: i. Invadopodia state, in which a cell is relatively sessile while it assembles invadopodia and degrades ECM; ii. Locomotion state. State balance is regulated by integrin β1 activation levels (Esmaeili et al 2018 Biophys J). Importantly, the Invadopodia state only occurs in early G1, whereas the Locomotion state can be seen throughout the entire cell cycle, suggesting that the cell cycle controls invadopodia assembly. Using FUCCI markers (Esmaeili et al 2018 APL Bioeng), we next show that Invadopodia state occurs during the G1 phase of the cell cycle (Bayarmagnai et al, 2019 J Cell Sci). A close look at the regulators of G1 revealed that the cell cycle regulator p27kip1 localizes to the sites of invadopodia assembly and overexpression of p27kip1, causes faster turnover of invadopodia and increased ECM degradation. Taken together, these findings suggest that invadopodia assembly, which occurs in the perivascular niche, is necessary for lung metastasis and function is controlled by the cell cycle.

This talk will present a personal history of scanning optical (confocal) microscopy and will illustrate how developments in other technologies have made the transformation from the (sometimes difficult to use) early laboratory instruments into today’s much more versatile user friendly instruments.

Deep Learning (DL) methods are increasingly recognised as powerful analytical tools for microscopy. Their potential to outperform conventional image processing pipelines is now well established. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources, install multiple computational tools and modify code instructions to train neural networks all lead to an accessibility barrier that novice users often find difficult to cross. Here, we present ZeroCostDL4Mic, an entry-level teaching and deployment DL platform which considerably simplifies access and use of DL for microscopy. It is based on Google Colab which provides the free, cloud-based computational resources needed. ZeroCostDL4Mic allows researchers with little or no coding expertise to quickly test, train and use popular DL networks. In parallel, it guides researchers to acquire more knowledge, to experiment with optimising DL parameters and network architectures. We also highlight the limitations and requirements to use Google Colab. Altogether, ZeroCostDL4Mic accelerates the uptake of DL for new users and promotes their capacity to use increasingly complex DL networks.

https://www.biorxiv.org/content/10.1101/2020.03.20.000133v1

Modern sample preparation, molecular probes and imaging technologies enable researchers to image at high spatial and temporal resolutions. While it is relatively easy to generate large numbers of high-quality image data, it is hard to efficiently extract knowledge from them. This is due to a critical limitation in state-of-the-art image analysis tools. They require a user to have a solid understanding of image processing algorithms and master several user-facing parameters before one can efficiently use the tools. To overcome this limitation, we have designed Aivia, a full image analysis solution offering all the advantages of machine learning technology on top of standard segmentation, tracking and neuron tracing tools. This smart association will allow you to achieve quantitative studies in few steps and let you focus on more important biological and scientific questions. Furthermore, we recently released Aivia Community version, a free to use software, allowing anyone to visualize up to 5D datasets, create scientific content, e.g. snapshots, videos, and prepare annotations for machine learning image restoration and segmentation. The mission of this tool is to support the efforts of the microscopy community at no cost, so that it can foster the use of cutting-edge technologies for faster scientific discoveries.

Cell classification is an important issue in bioimaging. With the new super-resolved microscopes coupled to microfluidic devices, this classification can be done based on images rather than on the classical cytological approaches.

Fluorescent bio-markers can be detected in single cells individually (pointillist approach) or collectively as a texture depending on the quality of the microscope impulse response. The density and organization of these markers can reflect the healthy or pathological state of cells. We will present a study, based on synthetic and real microscopy images to address, with machine learning approaches, the detection of changes in spatial density or in spatial clustering depending on the microscopy image resolution (PSF).

Marine plankton includes viruses, prokaryotes and eukaryotes and many of them live in intimate symbiotic relationships. Recent oceanic biodiversity surveys have unveiled a large unknown genetic diversity in microbial eukaryotes1, which remain to be described. While microscopy can provide valuable insights into their morphology and interactions, imaging environmental samples is challenging due to their broad size, abundance, and complexity ranges and the necessity for a robust taxonomic annotation.

We present a 3D-fluorescence imaging pipeline to study the morphological complexity and symbiotic interactions of uncultivated aquatic organisms:

1. Automated multicolor confocal microscopy provides the necessary high resolution and information content of the image data.
2. Morphological features of each imaged cell are extracted, providing quantitative data about cellular ultrastructures and even interactions.
3. Based on these features, accurate automated taxonomic classification can be performed by means of a supervised machine learning classifier.
4. For further expert validation, the EcoTaxa3 web application provides an environment for collaborative curation and as well deep learning-based image classification tools4.

Key words: Adaptive Optics, Software

Optical aberrations significantly reduce the achievable image quality and resolution of microscopy systems and are intrinsic to any biological imaging process.[1] Implementing adaptive optics (AO) has been shown to significantly reduce the aberrations present thereby improving both the image resolution and the achievable imaging depth of a microscope.[2] However, implementing adaptive optics is a complex task with many stages, including calibration, wavefront sensing, aberration detection and aberration correction. Control software for doing any of these is often esoteric and not easily transferred between systems. There is a need for a robust, accurate, easy-to-use, general implementation of adaptive optics for microscopy.[3]
Here we present Microscope-AOtools, a Python addition to the Microscope hardware control software. Microscope-AOtools provides an implementation of AO techniques using deformable mirrors that is transferable between systems and easily extensible. We also demonstrate several different correction strategies on multiple different imaging systems and samples. Crucially, Microscope-AOtools can be used effectively to allow general biological scientists to perform AO correction and image quality improvement without extensive training or supervision.

[1] John M Girkin, Simon Poland, and Amanda J Wright. "Adaptive optics for deeper imaging of biological samples". In: Current opinion in biotechnology 20.1 (2009), pp. 106-110.
[2] Booth, Martin J. "Adaptive optical microscopy: the ongoing quest for a perfect image". In: Light: Science & Applications 3.4 (2014): e165.
[3] Na Ji. Adaptive optical fluorescence microscopy". In: Nature methods 14.4 (2017), p. 374.

The history and emergence of LED technology and itÍs use in fluorescence microscopy, moving onto the growing popularity and wide scale acceptance of LEDs as a convenient and reliable light source for imaging. We will cover metrics such as LED spectrum, FWHM and how intensity (irradiance) should be measured at the sample plane. Current state of the art, and a discussion on new benefits of cell viability and signal to noise in fluorescence microscopy will be presented.

3D printing a.k.a additive manufacturing offers a relatively cheap and accessible method to make one off parts like stage inserts and specimen holders to support the varied needs of customers in a core facility. Here I present how 3D printing is used in the Light Microscopy Unit at the Institute of Biotechnology in the University of Helsinki and give some tips and tricks to people who want to start a similar service.

Prior Scientific Instruments has been a stalwart of the microscope automation market for 30 years, supplying high precision stages, focusing mechanisms and robotic loading systems. Now, in 2020, we are looking ahead to support the increasingly specialised imaging equipment of the future. Building on this component-based expertise, we are now offering motorised microscope frames, which can be configured with existing or modified products, and even bespoke components designed with specific applications in mind. Having acquired Queensgate, experts in nanopositioning, in 2018, we are now able to explore customised hardware solutions with sub-nanometre resolution and offer a new range of nanopositioning focusing devices. We will also cover some of our latest products in order to illuminate how we can provide the hardware to make your customised imaging solution a reality.

During CNS development immature oligodendrocytes (OL) extend a complex network of process arbors whose terminals make numerous contacts with axonal targets. As myelination progresses a subset of these contacts are converted into myelin bearing internodes, while others are resorbed, leading to a final mature OL in which all process arbors support myelin forming terminals. A clearer understanding of these events, and the mechanisms governing them, could aid the development of myelin repair therapies. However, aside from those regions of the outer neocortex that lie within reach of in vivo 2-photon imaging, they remain largely unresolved in the intact mammalian CNS. Organotypic slice cultures containing deeper lying white matter regions provide an exciting opportunity to image these events directly and extend our knowledge of mammalian myelination. Towards this goal we have developed a protocol involving laser-free confocal imaging that is capable of observing the differentiation and myelination of immature OL in neocortical slice cultures. This talk will describe the challenges involved in achieving long-term OL imaging from slice cultures grown using the interface method, and present some of the preliminary results we have obtained by imaging myelination of subcortical white matter with this new method.

A brief summary of the impacts of the RMS over the past year and upcoming events. We will look at the how the RMS has helped over 100ꯠ school children, the medal series that celebrates the achievements of new through to established microscopists across the world, as well as the many other activities that have been run and supported by the RMS. Finally, the summary will end with actions being taken to mitigate the problems being encountered through the Covid-19 outbreak and look forward to the upcoming events in the future.

I will introduce tools the Imaris team offers to help our users and the community work from home. These include flexible licensing options, the free Imaris Viewer, learning resources online, and additional ways to get the most out of your image analysis when away from the lab.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.

Now: Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, U.K.

Understanding the architecture of bacterial communities is crucial for developing novel methods of eradication and reducing their burden on public health. However, conventional imaging techniques are limited by sacrificing either the size of the imaging volume or the spatial resolution. Here we use the Mesolens, an optical microscope with a unique combination of a low magnification (x4) and a high numerical aperture (0.47) which can image volumes exceeding 100 mm3 with lateral resolution of 700 nm and axial resolution of 7 μm. We report the formation of an intra-colony channel system which forms as an emergent property of colonial growth of Escherichia coli on a solid surface. We visualise the internal architecture of mature E. coli biofilms using widefield and confocal laser scanning mesoscopy and assess the functional role of intra-colony channels using fluorescent microspheres and nutrient sensing. Using an arabinose biosensor, we characterise the role which these structures play in nutrient uptake and delivery to the centre of mature E. coli biofilms. These findings illustrate a novel mechanism by which E. coli organise to form complex structures to promote their survival and could be exploited to develop novel methods for biofilm eradication.

Chlamydia trachomatis is a major health concern with over 200 million people with active urogenital or ocular infection each year worldwide. Chlamydia are obligate intracellular bacteria with a unique biphasic developmental cycle. Chlamydia are tightly packed in a parasitophorous vacuole, termed an inclusion, and are too small to be spatially separated with conventional light microscopy. Their small size and requirement of a host to replace has made analysis of the development gene expression on individual Chlamydia extremely difficult. We aimed to develop a method that would allow most universities to be able to perform developmental gene expression studies with nothing more than a confocal microscope and an image analysis workstation. We utilized Expansion Microscopy and pixel classification to determine gene expression, size and quantity of Chlamydia. This method allows us to compare gene expression measured over time for chlamydial inclusions vs single Chlamydia during critical developmental time points.

The ORCA-Fusion BT camera is the pinnacle of scientific CMOS performance. The specs are without compromise: ultra-low read noise, CCD-like uniformity, fast frame rates and back-thinned enabled high QE. But the beauty of the ORCA-Fusion BT is what this combination of exceptional photon detection and collection can do for you. See the dimmest whisper of signal, acquire visually stunning, high SNR images from the fewest photons, capture previously unresolved temporal events and perform computational methods with confidence. The ORCA-Fusion BT is the camera that makes the difficult experiments easy and previously impossible experiments possible.

Balles M, Zantl R, Schwarz J

Ibidi GmbH, Martinsried, Germany.

In recent years 3D organoid and spheroid based assays drastically improved our understanding of cellular behavior in vitro and in vivo. However, the nutrient supply in 3D cell cultures in combination with high quality imaging over a long period of time remains challenging, limiting their use in high throughput applications. We developed a perfusable channel system with numerous cell aggregate adhesion sites on a highly passivated coverslip. Spheroids can be directly generated by injection of cell suspension using a self-sorting process or preformed spheroids or organoids can be homogenously tethered in a large number. The quality of the imaging bottom and the local tethering of cell aggregates enable high throughput live cell microscopy combined with defined shear stress, metabolite analysis, toxicological screenings or co-culture of multiple spheroid types.