Aurox On-line Conference on Microscopy 2021

The talk shall review aspects of the design, construction and applications of a confocal microscope equipped with both Raman and Brillouin spectrometer. Whilst the Raman detection allows identification of chemical bonds in samples and in turn the organic compounds making up the biomatter of samples, Brillouin spectrometric detection allows measurement of the velocity or speed of acoustic phonons which in certain circumstances permits calculation of elastic properties of a sample. We discuss operation principles, aspects of optical and optomechnical design, and the application of this microscope to a variety of biomedical and microbiological applications.

Catarina Costa Moura¹, Ana I. Gomez-Varela², Frederico Tremoço^1ƻ, Adelaide Miranda¹, Ana G. Silva³ and Pieter A. A. De Beule<sup>1

1</sup> International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga s/n, Braga, Portugal.

² Department of Applied Physics, University of Santiago de Compostela, E-15782, Santiago de Compostela, Spain

³ Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, Caparica, Portugal

The invention of the Atomic Force Microscope (AFM) in 1986 and subsequent developments in liquid imaging of soft samples, e.g., molecules, proteins, cells, tissues, and viruses enabled the study of biological samples with nanometric resolution under almost physiological conditions. The AFM is a unique microscope for imaging biological samples, as specimens require minimum or no preparation, and AFM can be performed with temperature and CO2 control. Furthermore, AFM can quantitatively characterise nanomechanical properties such as elasticity, viscosity and adhesion. The main drawback of AFM is that imaging is limited to the sample surface. Hence, the advantages of combining microscopy approaches were soon realised. The possibility of retrieving physical, chemical and biological information in complex systems using correlative microscopy methods set the stage for the emergence of hybrid systems. Although many biologically oriented AFM systems, the so-called Bio-AFMs, include an optical microscope coupled to the AFM, the correlation of these techniques is complicated.

We will briefly present some of the capacities of present liquid mode AFM technology and discuss several of the challenges encountered in hybrid AFM – fluorescence microscopy development, with a special focus on the realization of simultaneous data acquisition. We will demonstrate through analytical and numerical modelling that AFM cantilever heating induced by fluorescence excitation light can be a real problem in case one aims to study e.g. membrane proteins at physiological conditions. Furthermore, we will discuss how fluorescence excitation light is known to complicate and sometimes entirely spoil AFM operation. In this light, AUROX technology exhibits a key advantage in enabling simultaneous colocalized operation over standard confocal microscopy. As an application, we demonstrate how correlative AFM helps to characterise different cell types in complex samples.

The RMS has been as busy as ever over the past year and had to adapt and design new ways of working to help the community. This has ranged from the open forums to support the opening and safe practices in microscopy labs, adapting many of our conferences for the virtual world as well as designing courses to enable some online teaching. There is still much to come in the coming year to look forward to, such as mmc2021 and elmi with interactive workshops, all of which will be discussed in the short overview.

Holotomography (HT) is a technology that allows researchers to image the cell morphology in 3D using no labels. Due to the extremely low amount of light needed to acquire HT images, long term Live Cell Imaging is possible as there is no phototoxicity to the cell.

The technology itself aids a precise measurement of Refractive Index (RI) at a nanoscale, granting the visualization of sub-cellular organelles. Also, RI quantification permits a precise estimation of protein and lipid concentration in the cells and their changes over time.

The cell membrane fluctuation can be assessed as the capture speed of the data sets is very fast; 3D images can be captured at 2FPS while 2D images can be acquired at over 150FPS.

The new Tomocube HT-2 microscope is a Fast, Quantitative, and Label Free instrument with the capability to acquire images that combines 3D HT and 3D Fluorescence (FL) to allow correlative imaging with specificity. The acquisition of HT and FL are decoupled to allow independent strategies, which minimize the phototoxicity from FL imaging.

Authors: Omer Wagner¹, Alexander K Winkel¹, Eva Kreysing¹ and Kristian Franze<sup>1

1</sup>Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, United Kingdom

Many biological processes are regulated by tissue mechanics, including cell migration, neuronal growth and stem cell differentiation. The increasing research on the importance of mechanics on the dynamic cellular processes demand methods that can sense and fuse data on both varying tissue stiffness and imaging of the accompanying cellular changes. Here we present a platform that can perform live imaging of samples using Fourier Ptychographic Microscopy (FPM) co-localised with Atomic Force Microscopy (AFM) measurements. We demonstrate how in that case, FPM and AFM allows enhanced imaging of biological samples.

Stephanie Rey, Nilofar Faruqui, Alex Hoose, Camilla Dondi and Maxim G Ryadnov

National Physical Laboratory, Teddington, Middlesex, TW11 0LW

The emergence of multidrug-resistant bacteria stimulates the search for antimicrobial materials capable of addressing challenges conventional antibiotics fail to address. The ability to target intracellular bacteria remains one of the most fundamental tasks for contemporary antimicrobial treatments. Here we highlight our recent progress in demonstrating this ability for engineered protein virus-like particles targeting bacteria, which are internalised in macrophages. Using single-cell electron microscopy analysis we show that these materials effectively disrupt the bacteria without affecting the host cells.

With better antibiotics inevitably leading to fitter intracellular pathogens, there is a pressing need for antimicrobial materials that may support mechanisms which are different from those of antibiotics. This study entails a promising discovery strategy by probing a principally more challenging strategy for bacteria to overcome – antibacterial virus-like forms that destroy bacteria on contact.

Endosomal trafficking in single cells is built of generation of membrane vesicles, their motor protein mediated transport, morphological alterations such as tubulation, fusion and fission, and dynamic maintenance of various identities, defined by the localization of specific proteins on them. Endosomal maturation is a major process in endosomal trafficking in which endosomes shed one specific protein and acquire another, resulting in an identity change. Individual steps listed above, that build up endosomal trafficking, including conversions are interlinked and are stochastic. Therefore, when and where events will occur within a cell cannot be precisely predicted. Here, capitalising on the rapid volumetric imaging capability of Lattice-light sheet, we capture whole-cell volumes, enabling post-acquisition analysis of all conversion events as well as other dynamic characteristics. Based on these experiments, we describe a mechanism based on EEA1 (Early Endosomal Antigen 1) that enables extracting order in ensemble endosomal conversions out of the chaotic intracellular environment. Further, using Förster Resonance Energy Transfer, we demonstrate that EEA1 undergoes conformational changes through the endosomal cycle, opposite binding ends modulate specific activity, endosomal collisions trigger conversions and clustering of EEA1 is essential. Using simulations, we show that the activity of EEA1, thus functions as a timekeeper of the endosomal maturation process.

Carcinogenesis has been described as a process by which homeostatic organ architectures and the multicellular patterns specifying tissues are lost. Using a combination of imaging, 3D cell biology and multiscale modeling, I will show that this is only part of the picture. Housed within their native extracellular matrix (ECM) environments, cancer cells re-organize themselves and remodel their microenvironments in unique ways. I will use two examples, one from breast tumor heterogeneity and the other from ovarian cancer spheroidogenesis to show the deployment of pattern formation within invasive tumor populations. The consequences of such organization on cancer invasion will also be discussed.

Python-microscope and microscope-cockpit, colloquially referred to as just Cockpit, are two recently released Python packages which allow users to control imaging systems of arbitrary scale and complexity. The Cockpit packages leverage many years of experience building bespoke optical systems and doing biological experiments, in order to bring to its users an intuitive and optimal workflow, which could be further tailored for specific applications.

To illustrate the ability of Cockpit, it is used to control a system built around the Aurox Clarity. It consists entirely of commercially available parts and it demonstrates a method of turning almost any standard widefield microscope into an instrument capable of acquiring sectioned data similar to that captured with a confocal microscope.

Within microscopy, the light source will have a significant impact on the quality of one's image. Appreciating this dependence will allow microscope users to identify appropriate light sources for their application and use them to improve the quality of their image.

This presentation will discuss the attributes most impacted by the light source, such as: Irradiance (i.e. intensity) at the specimen plane and how one can measure it, Signal-to-Noise (SNR), homogeneity, as well as various optical aberrations and artefacts.

Daniela Malide¹, Nuo Sun² and Toren Finkel<sup>2

1</sup> Light Microscopy Core, ² Center for Molecular Medicine,

National Heart, Lung, Blood Institute, NIH, Bethesda, MD, USA

KEY WORDS: in vivo mitophagy, FLIM microscopy, keima, pH indicator

Mitophagy is a cellular process that selectively removes damaged, old or dysfunctional mitochondria. Defective mitophagy is thought to contribute to normal aging and to various neurodegenerative and cardiovascular diseases. Previous methods used to detect mitophagy in vivo were cumbersome, insensitive and difficult to quantify. We created a transgenic mouse model that expresses the pH-dependent fluorescent protein mt-Keima in order to more readily assess mitophagy. Keima is a pH-sensitive, dual-excitation ratiometric fluorescent protein that also exhibits resistance to lysosomal proteases. At the physiological pH of the mitochondria (pH 8.0), the shorter-wavelength excitation predominates. Within the acidic lysosome (pH 4.5) after mitophagy, mt-Keima undergoes a gradual shift to longer-wavelength excitation [1, 2]. In addition to intensity imaging we describe here how to apply mt-Keima fluorescence lifetime microscopy (FLIM) to visualize mitophagy in live cells as well as various tissues including skeletal muscle, heart, liver, and kidney, obtained from mt-Keima transgenic mice. We observed that in control live cells mt-Keima fluorescence exhibits two components a short (0.4ns) lifetime corresponding to the mitophagic compartment and a longer (2.6ns) lifetime corresponding to normal mitochondria, in good correspondence to the intensity images. Interestingly, in the tissues the lifetime measurements reveal a heterogeneous mitophagic compartment containing in addition to the short (0.5ns) lifetime mt-Keima species an intermediary (1.2ns) longer lifetime component. Whether these 2 components correspond to different folding states, digestion products of the mt-Keima in the acidic environment remains to be elucidated. In conclusion FLIM provide a complementary approach to asses mitophagy in normal cells and tissues as well as in disease situations, or altered under environmental, genetic perturbations, or in aging.

[1]. N. Sun; J. Yun; J. Liu, D. Malide; C. Liu; II. Rovira; KM. Holmström; MM. Fergusson; YH. Yoo; CA. Combs and T. Finkel Measuring in vivo mitophagy Mol Cell. 60(4):685-696 (2015)

[2]. N. Sun; D. Malide; J. Liu; II. Rovira; CA. Combs and T Finkel. A mouse model for measurement of mitophagy. Nature Protocols 12, 1576–1587 (2017)

Optical sectioning is the basis of a range of microscopy techniques that enable three-dimensional imaging. This talk will look at a range of physical and computational methods that can reject out-of-focus light to produce a sectioned image. The mechanisms by which they work and what they can and cannot achieve are discussed.”

The quality of image visualization and analysis very much relies on the quality of microscopy data, which always suffers from noise and blur, and frequently also from chromatic and spherical aberration, crosstalk, and sample drift. All these imaging artifacts can easily be restored with the Huygens Software. Huygens is considered the gold standard for deconvolution and restoration of microscopy data from widefield, confocal, spinning disk, multiphoton, STED, and Array detector and Light Sheet systems.

In this session, you’ll see how easy Huygens can be used at any time and place, how experimental PSFs can be measured, and how different tasks can be combined for batch processing using the new Huygens Workflow Processor.

Company description:
Our Huygens Software is developed with the firm belief that reliable image processing is key in understanding the true nature of microscopic objects. For more than 25 years, we collaborate with expert microscopists around the globe to promote best imaging practices, and to further improve the user-friendliness and quality of our light microscopy software. Huygens offers with its true image deconvolution, restoration tools - for issue like crosstalk, bleaching and drift correction, and 2D-5D visualization and analysis a complete image processing workflow from microscope to high quality figures and reliable results.

PSFcheck slides are laser-written fluorescent slides that can be used as calibration standards to directly measure the point spread function (PSF) of an optical microscope. In addition they can be used to measure chromatic shift, distortion, magnification and field uniformity. The slides are compatible with widefield, confocal, SIM, multiphoton and single molecule imaging microscopies. PyCalibrate is a freely available web app that takes the image stacks of PSFcheck features (or sub-resolution beads) and automatically processes them to deliver a readout of microscope imaging performance. PyCalibrate can accept all BioFormats compatible data types and is capable of processing multiple points within the field of view. As PyCalibrate is cloud-based, it avoids all problems of software maintenance and system compatibility. For more details see: https://www.psfcheck.com/

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localisation microscopy (SMLM) techniques and 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, we demonstrate that STED and LFSM can be performed 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.

Reference:

Prakash, Kirti. "Laser-free super-resolution microscopy." Philosophical Transactions of the Royal Society A 379.2199 (2021): 20200144.

Stimulated emission depletion (STED) microscopy has proven to be an imaging technique, which provides its’ users with the possibility of generating high-quality images with a resolution down to 20 nm.

Abberior Instruments as the leading innovator, developer and manufacturer of STED super resolution microscopes has a strong focus on offering bespoke imaging solutions for customers. Their instruments are designed by the inventors of the method including Abberior’s co-founder, Nobel Laureate Prof. Stefan W. Hell. Therefore, the systems are always state-of-the-art due to a history of constantly improving technology.

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 Facility Line, combing cutting-edge microscopy with unprecedented ease-of-use including technologies like adaptive optics and the novel MATRIX detector.

Adaptive optics (AO) is used to correct aberrations when focussing deep into specimens and is particularly important in super-resolution nanoscopy methods, whose performance is particularly sensitive to aberration effects. Novel AO methods have been developed to deal with the particular challenges posed by super-resolution microscopes. We show how various three-dimensional nanoscopy applications can be facilitated using new approaches to AO. Specifically, this includes whole cell and tissue imaging using a 4Pi single molecule localisation microscope. This uses an optical system with dual opposing objective lenses and two deformable mirrors, which require new approaches to AO control to facilitate correction of specimen aberrations in both paths. We show further results from structured illumination microscopy, with adaptive illumination and aberration correction permit sub-resolution imaging in multiple fluorescence channels. We have also developed methods to combine AO stimulated emission depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS) to provide higher precision measurements inside living cells.

Advances in imaging, in particular semiconductor technology, are directly linked to new scientific achievements, especially in the life sciences. The progress of imaging technology over the last few years has accelerated rapidly. Demanding, low-light experiments that require extremely high levels of sensitivity would not be possible without these enhanced image sensors.

In this session, we will guide you through the advancements of Hamamatsu CMOS technology, including a crash course on our flagship sCMOS camera, the ORCA-FusionBT, and how this ultra-low noise, 95% QE back thinned camera can improve your images and data quality. We’ll also be dropping a hint about the next big thing to come from Hamamatsu, and how you can find out more about what our latest technology can offer.

About Hamamatsu Photonics

Hamamatsu Photonics is a world-leading manufacturer of optoelectronic components and systems. The Company’s corporate philosophy stresses the advancement of photonics through extensive research and yields products that are regarded as state-of-the-art. All products are designed to cover the entire optical spectrum and provide solutions for a wide variety of applications including analytical, consumer, industrial and medical instrumentation.

A.A. Pushkina, G. Maltese, J.I. Costa-Filho, P. Patel, A.I. Lvovsky

The resolution of optical imaging devices is ultimately limited by the diffraction of light. To circumvent this limit, modern super-resolution microscopy techniques employ active interaction with the object by exploiting its optical nonlinearities, nonclassical properties of the illumination beam, or near-field probing. Thus, they are not applicable whenever such interaction is not possible, for example, in astronomy or non-invasive biological imaging. Far-field, linear-optical super-resolution techniques based on passive analysis of light coming from the object would cover these gaps. In this paper, we present the first proof-of-principle demonstration of such a technique. It works by accessing information about spatial correlations of the image optical field and, hence, about the object itself via measuring projections onto Hermite-Gaussian transverse spatial modes. With a basis of 21 spatial modes in both transverse dimensions, we perform two-dimensional imaging with twofold resolution enhancement beyond the diffraction limit.

Reference:

https://arxiv.org/abs/2105.01743

In vivo imaging in mouse neocortex has revealed the dynamic behaviour of glial cells. Microglial processes constantly survey the local environment and respond to local injury and disease processes, while oligodendrocyte (OL) differentiate and myelinate axons in response to sensory stimuli. However, these in vivo observations are limited to outer neocortical regions falling within the reach of 2-photon imaging, and information on glial behaviours in deep lying brain regions remain unknown. In this talk I will describe how my group uses organotypic brain slice cultures to image OL behaviours in deeper lying regions including cerebellar white matter and deep neocortical grey and white matter. I will present our recent findings on activity-dependent myelination, and describe a novel method we are using to live-image myelination in subcortical white matter.

Developmental apoptosis is a physiological process that eliminates about a third of immature post mitotic neurons. In rodents, this wave of cell death occurs during the first two postnatal weeks, a period at which neurons display typical spontaneous, synchronous activity patterns. Silencing immature neuronal networks promotes cell death, indicating that these typical activity patterns are crucial for neuronal survival. Using pharmacological treatments, imaging and electrophysiology, I could show that electrical activity differentially affects the survival fate of early-born cortical neurons versus the remaining cortical neurons. I further demonstrate that the spatiotemporal pattern of apoptosis is causally related to the spatiotemporal profile of activity in the developing cortex. Altogether, these data demonstrate that electrical activity plays a crucial role in the establishment of functional neuronal networks.

Over the past decades, customization of scientific instruments has come a long way. Rather than having to choose between the two extremes “turn-key one size fits all” vs. “building from basic components”, to image. Day researchers are able to configure a turn-key system that ideally fits their specific experimental needs. The Scientifica HyperScope multiphoton microscopy platform is a perfect example of such a “customized off the shelf system”: Consisting of a range of modules that are configured to create a complete system, it is able to grow with experimental needs and funding over the years. In addition, the HyperScope platform includes interfaces to industry standard optomechanical systems to allow it to be used as a proven foundation for implementing novel imaging techniques. These factors, along with the seamless integration with Scientifica’s electrophysiology components, ensure neuroscientists can build the rig they need.

Cell culture assays with two-dimensional monolayers have been a well-established and convenient tool for investigating cell behavior and function in many areas of cell biology, such as cancer biology and stem cell research. However, these 2D assays come with certain limitations, since they do not accurately reflect the in vivo cell-to-cell interactions. Because of these limitations, 3D cell culture models have gained more and more relevance, as they have been proven to yield more accurate insights and data about cell function, structure, and morphology.

We present an introduction to straightforward three-dimensional cell culture. We will discuss surfaces and which labware is suitable for cell-based assays in 3D combined with live cell imaging. You will see experimental examples of 3D cell culture applications. Furthermore, we will give you insights into the latest ibidi innovations, which facilitate the generation and long-term cultivation of spheroids and organoids in combination with high-resolution imaging.