LIGHT 4 LIFE workshop - INTERNATIONAL DAY OF LIGHT 2024
1/1-1 - Aula "A. Rostagni"
Dipartimento di Fisica e Astronomia - Edificio Marzolo
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08:30
Registration 1/1-1 - Aula "A. Rostagni"
1/1-1 - Aula "A. Rostagni"
Dipartimento di Fisica e Astronomia - Edificio Marzolo
Entrance from via Paolotti200 -
09:00
Welcome to L4L - IDL 2024
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1
Opening talk: Photon-Resolved Microscopy: a New Microscopy Paradigm for Life-Science Research
Fluorescence optical microscopy has long been a valuable and minimally invasive tool for visualizing biological structures and functions at the cellular level and beyond.
However, understanding many fundamental biological processes crucial to human health and disease remains beyond the capabilities of conventional optical microscopy.Super-resolved microscopy, which shifts our perspective from considering fluorophores as mere passive markers to active participants in image formation, has significantly expanded the capabilities of optical microscopy, marking a new era for life sciences. Inspired by the transformative potential of shifting perspectives, we introduce the innovative concept of photon-resolved microscopy. By examining fluorescent light in terms of its most elemental components—the photons—we unveil even greater potential for fluorescence microscopy. Our exploration begins by illustrating how this fresh approach can reinvigorate one of the most widely used and traditional microscopy architectures: the confocal laser-scanning microscope. Subsequently, we reveal the synergies between photon-resolved microscopy and more advanced techniques, including super-resolved microscopy.
This change in perspective holds the promise of not only enhancing the capabilities of fluorescence microscopy but also unlocking new horizons in studying intricate biological processes.
Speaker: Dr Giuseppe Vicidomini (IIT) -
Session 1. Advanced light control
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2
Light wavefront engineering to modulate neuronal activity at cell resolution
Light wavefront engineering represents a valuable tool to control the electric field intensity distribution at the sample volume by modulating the phase and/or the amplitude of the light wavefront in a conjugated space. For imaging purposes, this approach has been traditionally adopted on one side to compensate for optical aberrations due to the sample or the medium so as to improve either the resolution or the signal-to-noise ratio; on the other side, to achieve particular illumination spatial profiles so as to increase the background rejection. Along with novel applications for imaging, in the last ten years wavefront engineering has become a fundamental tool in neuroscience research when light is used not to record but also to control the neuronal circuit activity, taking advantage from the development of light sensitive ion channels rendering light sensitive the activity of the cells in the brain. Following a brief background, I will present the methods and the application for applying these methods in neuroscience research.
Speaker: Prof. Marco Dal Maschio (Department of Biomedical Sciences) -
3
Dual-functional metalenses for the polarization-controlled generation of structured beams
The ability to generate different structured beams in a compact optical path by controlling the input polarization has been a challenge of the last few years in the optics and photonics field. In this regard, we propose designing, fabricating, and characterising new dielectric dual-functional metaoptics that generate 3D orbital angular momentum beams or vector beams along custom-define trajectories with on-demand different behaviours acting on the input light’s polarization. Our meta-optics are designed as an array of periodic subwavelength metastructures (the so-called meta-atoms) composed of silicon nanofins on a silicon substrate. Each nanorod acts like a half-wave plate that exploits both the geometrical and dynamical phases in a different way depending on its position on the entire optic. The optical elements have been fabricated in the form of phase-only metasurfaces (meta-atoms) with high-resolution electron-beam lithography and characterized with a custom-made optical bench. The main result of this work is the design of tiny high-resolution optics generating longitudinally-variant vector beams and spheres of light that are able to impart new peculiarities to the light. In particular, the proposed metaoptics could open new applications of structured light for holography, super-resolution imaging, optical trapping and particle tweezing.
Speaker: Dr Andrea Vogliardi (Department of Physics and Astronomy) -
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Adaptive optics in biological imaging: enhancing resolution and fidelity
The use of adaptive optics (AO) in biological imaging has emerged as an important technique for overcoming the inherent optical aberrations present in biological specimens. We will provide an overview of the principles, applications, and recent advancements in AO technology within the realm of biological imaging. We will discuss the implementation of AO in various imaging modalities, including confocal microscopy, two-photon microscopy, and optical coherence tomography, highlighting its capability to enhance image quality and enable high-resolution imaging. Furthermore, we explore the integration of AO with adaptive lenses in imaging techniques such as light-sheet microscopy and super-resolution microscopy.
Speaker: Dr Stefano Bonora (CNR - Institute of Photonics and Nanotechnology)
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2
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11:00
Coffee break
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Session 2. Advanced platforms and optical tools
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5
Going beyond fluorescence: bringing label-free optical spectroscopy in deep brain regions using a single thin optical fiber
Optical approaches for in vivo neural monitoring using genetically-encoded fluorescent
molecular reporters offer a precious window on brain functions, and on the mechanisms of development, ageing or disease progression. Nonetheless, the existing methods are still shortsighted with respect to the complex biomolecular alterations that accompany these physiological and pathological dynamics. As a result, our grasp of the multifaceted components of brain activity is still partial. To surpass these limitations, this talk will discuss the opportunities offered by the broad physical phenomenologies underlying light-brain interactions to capture a more comprehensive picture of neural mechanisms using label-free optical spectroscopy in deep brain regions. In particular, I will present a vibrational fiber photometry method, based on spontaneous Raman scattering, that allows monitoring the bio-molecular content of arbitrarily deep brain volumes of the mouse brain – in vivo – to gather information on molecular alterations caused by traumatic brain injury and to detect diagnostic markers of brain cancer using a single thin optical fiber. This approach, which can be employed alongside conventional photometry techniques, has the potential to empower emerging research on brain-immune and brain-cancer bidirectional dynamics.Speaker: Dr Filippo Pisano (Department of Physics and Astronomy) -
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Single-molecule force spectroscopy with optical tweezers
Optical Tweezers exploit light to manipulate objects at the micro- and nanoscale, demonstrating to be a powerful tool for investigating the biological world. Force spectroscopy measurements with optical tweezers allow the application of controlled mechanical stimuli and displacements on individual molecules of DNA, RNA and proteins, while monitoring the time evolution of the system as it undergoes biochemical reactions. In this way, it is possible to derive information on the elastic and kinetic properties of the molecule, characterizing its molecular pathways and its free energy landscape. In this talk, the working principles of optical trapping and its application to single-molecule experiments will be presented; the study we are carrying out with optical tweezers on the Thymidylate Synthase consensus RNA will be also discussed.
Speaker: Dr Annamaria Zaltron (Department of Physics and Astronomy) -
7
Droplet microfluidic platform for extracellular vesicle isolation and handling
Extracellular vesicles (EVs) are double-layered phospholipid vesicles having nanometric size that are rapidly gaining in popularity as biomarkers of various diseases, acting as cargoes of valuable information from the cell of origin [1]. Despite their value, their current use in clinical practice is still limited. Among the limiting factors, one of the most critical is their isolation. In fact, conventional approaches are characterized by low purity and throughput, or poor reproducibility [2]. Here, we propose a droplet microfluidic platform developed for EV isolation by affinity capture with magnetic beads [3]. This platform is capable of processing large sample volumes in a relatively short time. Systematic comparison with commercial methods proves that the platform leads to an improved EV capture efficiency of 2.5-fold. This is due to the fact that EVs and magnetic beads are co-encapsulated within the same droplet, which acts promoting their mixing [4]. The beads are extracted within the microfluidic system and collected for EV analysis. At first, the platform has been validated from the microfluidics point of view: throughput, automation and magnetic beads handling have been investigated. Then, the EV isolation capability has been performed by the most used techniques: confocal microscopy and flow-cytometry prove the presence of EVs captured on the beads, while scattering techniques and protein assays allow defining a capture efficiency. Finally, the miRNAs cargo has been quantified to verify the EV integrity. The remarkable improvements compare with monophasic microfluidic indicate how droplet microfluidics represent a suitable technology for EV isolation especially in case of clinical applications [5], where a few mL of starting sample is considered. To achieve this aim, preliminary validation using human plasma samples will be presented.
[1] G. Raposo, et al., Nat.Rev.Mol.Cell Biol. 20,509 (2019).
[2] F. Momen-Heravi, et al., Biol.Chem. 394,1253 (2013).
[3] A. Meggiolaro, et al. Sens. Actuators B: Chem. 409, 135583 (2024).
[4] C.N. Baroud, et al., Lab-Chip. 10,2023 (2010).
[5] A. Meggiolaro, et al. Biosensors. 13,1 (2023).Speaker: Dr Davide Ferraro (Department of Physics and Astronomy) -
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The use of light to guide neuronal connection in vitro and in vivo
The brain is the most complex and delicate organ in our body. Brain damage typically results in devastating outcomes and consequences not only for the patient's health but also for their quality of life. These effects are caused by the irreversible loss of neurons, building blocks of the brain responsible of signal transmission. Neuronal loss therefore leads to an alteration in communication between the different areas of the brain, resulting in the inability to perform specific functions. One of the most complex challenges of regenerative medicine is to find a strategy to recreate functional connections within the damaged brain, with the aim of restoring the patient's physiological activities. Currently, there is no therapy capable of regenerating lost tissue and connections between neurons. There are no drugs capable of regenerating dead cells, and cellular therapy, which exploits the use of stem cells, is unable to effectively replace lost tissue and recreate functional connections.
In this project, we aim to develop an integrated approach between bioengineering and regenerative medicine capable of controlling and guiding the formation of brain connections, in order to promote the restoration of lost brain functions following damage.
Specifically, to replace damaged tissue, we will exploit the use of brain organoids, small
hree-dimensional structures that resemble the brain in cellular composition and structural organization. To guide the formation of neuronal connections, photosensitive gels will be used, which can be manipulated with infrared light directly inside the brain of the living animal. Specifically, using multiphoton light, it is possible to create three-dimensional structures or empty channels within the biomaterial itself that guide the growth of neurons. Thanks to the ability to manufacture these structures directly within the brain in a defined anatomical site, we can then connect the neurons of the organoid implanted at the site of the lesion with the remaining neurons in the tissue of the host animal, thus reconnecting the damaged tissue. This will enable the design of a new neuronal network capable of recovering a specific function lost due to the lesion.This project therefore has the potential to create a new therapeutic strategy to promote the restoration of lost brain functions following damage (e.g., stroke, tumor resection, trauma), made possible only through the fusion of expertise from very different scientific fields: neuroscience, bioengineering, and regenerative medicine.
Speaker: Prof. Cecilia Laterza (Department of Biomedical Sciences) -
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Detecting membrane contacts and associated Ca2+ signals by reversible chemogenetic reporters
Membrane contact sites (MCSs) enable different intracellular organelles to coordinate their activities, yet the small size and the dynamic nature of these regions hinder their study by current imaging techniques. By designing a series of reversible chemogenetic reporters based on improved, low-affinity variants of splitFAST, we analysed the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrated that these versatile reporters suit different experimental setups well and identified a hitherto unknown pathway, in which calcium (Ca2+) signalling regulates the juxtaposition between endoplasmic reticulum and mitochondria. Finally, the integration of Ca2+-sensing domains into the splitFAST technology allowed us to introduce PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), an unprecedented class of reporters to simultaneously visualize MCSs and the associated Ca2+ dynamics by a single biosensor.
Speaker: Dr Riccardo Filadi (CNR - Institute of Neuroscience)
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5
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13:10
Lunch
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Session 3. Advanced techniques for microscopy
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10
Towards personalized medicine: investigating Parkinson's disease by patient-derived midbrain organoids
One of the most exciting advancements in stem cell research of the last few years has been the development of human brain organoids. This in vitro system consists of multiple cell types that can self-organize in three-dimensions representing a brain region able to recapitulate physiological and pathological relevant aspects. Compared to animal models, patient-derived organoids provide emerging prospects for testing new drugs and developing precision medicine. Human midbrain organoids (hMBOs) can mimic the sunstantia nigra, the brain region that degenerates in Parkinson’s disease (PD), including the complex interaction of dopaminergic neurons with other types of neurons and glial cells. In this work, we characterized hMBOs derived from healthy subjects and PD patients carrying monogenic mutations identified to cause PD in a highly penetrant manner. In order to address the complexity of hMBOs, we combined patch-clamp, multielectrode arrays (MEA) and two-photon microscopy, testing potential therapeutic compounds.
Speaker: Prof. Mario Bortolozzi (Department of Physics and Astronomy) -
11
Astrocyte-mediated phagocytosis: identification of novel players using a genetic screening
Astrocytes participate in the clearance of obsolete or unwanted neuronal synapses. However, the molecular machinery recruited for the recognition of synapses is not fully clarified, especially in pathological conditions. Here, we investigated the phagocytic process through individual gene silencing using a druggable gene library. Our study demonstrates that astrocyte-mediated synapse engulfment is regulated by the Atypical chemokine receptor 3 (Ackr3). Mechanistically, we have shown that Ackr3 recognizes phosphatidylethanolamine (PE)-bound Cxcl12 at synaptic terminals both in vitro and in cells, thus serving as a novel marker of synaptic dysfunction. The removal of synapses by astrocytes, dependent on Ackr3, occurs prominently in brains affected by Alzheimer's disease (AD). Notably, both the receptor and its ligand are overexpressed in post mortem AD human brains, and downregulation of the receptor in AD mouse models (5xFAD) significantly diminishes astrocyte-mediated synaptic elimination. Overall, this work unveils a novel, possibly targetable mechanism of astrocyte-mediated synaptic engulfment implicated in the most common neurodegenerative disease.
Speaker: Prof. Laura Civiero (Department of Biology) -
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Highlighting mitochondria-ER contact sites
In recent years it has become clear that intracellular organelles are not isolated entities, but rather they interact to coordinate their function. Organelles crosstalk occurs at points of proximity between their surfaces, which are kept together by proteinaceous tethers. These closely juxtaposed membrane subdomains are known as membrane contact sites (MCS). The most studied MCS are those between the endoplasmic reticulum and the mitochondria (MERCs). MERCs play an important role in many physiological and pathological subcellular processes, including lipid and Ca 2+ homeostasis, mitochondrial dynamics and response to stress stimuli: altered MERCs structure and function contribute to severe pathological conditions including Alzheimer’s and Parkinson’s disease. Hence, understanding which conditions or treatments modulate the structure of MERCs could be of relevance for both basic and translational research. To this end, we developed a FRET-based mitochondria-ER proximity probe (FEMP) for the study of MERCs dynamics in living and fixed cells, and we miniaturized it to make it suitable for high-throughput screenings.
Speaker: Prof. Marta Giacomello (Department of Biology) -
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A powerful model swims in the light blue: using the zebrafish to study neurodegeneration
The zebrafish (Danio rerio) emerged as a powerful tool for scientific research starting from the beginning of the 1980s. Its remarkable characteristics such as the high fecundity, the external fertilization and development, the elevated similarity with the human genome and the possibility to easily manipulate it, made the zebrafish an optimal animal model for basic research and translational applications. The optical clarity of the embryo during its whole development constitutes another fundamental feature that promoted the generation of multiple transgenic lines aimed at deciphering the mechanisms underlying cellular processes, tissues morphogenesis and organs functionality. The targeted expression of fluorescent proteins, indeed, allows direct visualization and scoring of manifold processes to shed light on different aspects of cellular and developmental biology and physiopathology of diseases. In our research, that we propose as an example of zebrafish use in the study of neurodegenerative processes, we exploited the optical accessibility of larvae and fluorescent transgenic zebrafish lines to unravel the impact of increased levels of glucosyl-sterols on the development and function of motor neurons and neuromuscular junctions. Unbalance in the amount of these molecules in the organism, indeed, has been associated with the occurrence of neurodegenerative outcomes similar to Parkinson’s disease and Amyotrophic Lateral Sclerosis, that involve neuronal loss and impaired functionality.
Speaker: Dr Francesca Terrin (Department of Biology)
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10
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FLASH PRESENTATIONS
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16:00
Coffee break
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Roundtable: where are we going next?
Prof. F. Mammano, Prof. F. Romanato, Prof. M. Dal Maschio, Dr. G. Vicidomini
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Closing remarks
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08:30
