Eli Slenders

Eli Slenders

Marie Skłodowska-Curie Actions research fellow at the Italian Institute of Technology

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Blog Post number 4

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Better ways to give medicines

Cancer is one of the major causes of death in our society. The anti-cancer drugs currently used in chemotherapy produce several side effects and, although fatal for almost all cells in a tumor, a small percentage of cells appear to resist the treatment. It is therefore urgent to design new therapies that specifically target these cells while causing no harm to healthy tissue. The resistance to multiple drugs is linked to the presence of specific molecules at the cellular plasma membrane, that actively pump out chemical drugs (efflux pumps). Additionally, some cancer cells have the ability to self-renew, thereby initiating secondary tumor growth. Multi-drug resistant cells and cancer stem cells are suggested to be the main cause of treatment failure and relapse.

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Structure of Biomimetic Materials

A large number of natural and synthetic hydrogels are currently used for tissue engineering and regenerative medicine. Over the last decade, there has been an increasing awareness of the role of material properties of the substrates in guiding cellular behaviour. This has inspired chemists to create a new generation of materials with mechanical properties closed to that of natural occurring biopolymer networks. Recently, the groups of Prof. Alan Rowan (Queens University, Australia) and Prof. Paul Kouwer (Radboud University of Nijmegen, The Netherlands) were able to develop a fully synthetic material that mimics in all aspects the gels prepared from cellular filaments. These synthetics gels are prepared from polyisocyanopeptides (PICs) grafted with oligo(ethylene glycol) chains and share structural features of biopolymers: their helical structure renders the polymer molecules relatively stiff while the interaction between the side chains enable the formation of bundles or fibrils of defined dimensions. The triethylene glycol side chains attached to the polymer backbone render the material thermo-responsive (it will gel upon heating beyond 20 °C and become liquid again upon cooling). Despite being characterized extensively in bulk, the fundamental dynamics and the relation between the macroscopic properties and the microscopic structure at cellular length scales of PIC-based hydrogels remains obscure.

Structure of the PIC polymer/monomer unit and cartoon of the polymer structure showing the helical structure.

Classically, structural characterization of materials is performed with electron microscopy or scanning probe microscopy. Despite the high spatial resolution achievable with these techniques, they are unable to measure dynamics ‘in situ’ and sample preparation can be a laborious process. In contrast, optical microscopy has the potential to unravel the dynamics in complex heterogeneous systems but has been limited to a spatial resolution of ca. 200 nm. In the past 10 years fluorescence imaging has been revolutionized by the successful development of sub-diffraction (super-resolution) microscopy modalities which can achieve resolutions down to tens of nanometers (see Molecular Organization at the Nanoscale).The various possibilities of fluorescence microscopy to probe dynamics and heterogeneities, with molecular resolution, for a wide range of time scales makes it an ideal tool to address many topics of polymer science. In this project we are using STED to image the polymer network at the nanometer scale.

STED image of PIC network.


For more information on PIC-based hydrogels:

  • Kouwer P.H.J., et al. (2013) Responsive biomimetic networks from polyisocyanopeptide hydrogels, Nature, 493, pages 651–655 (article can be found here)
  • Jasper M., et al. (2014) Ultra-responsive soft matter from strain-stiffening hydrogels, Nature Communications, 5, 5808 (article can be found here)
  • Jasper M., et al. (2016) Bundle Formation in Biomimetic Hydrogels, Macromolecules, 17(8), pages 2642–2649 (article can be found here)

Polymer reptation in 3D

Our current theoretical understanding of entangled polymer chain dynamics is based on the reptation model. First proposed by Doi and Edwards, and further expanded by de Gennes, the reptation model assumes that a polymer chain is confined by the surrounding matrix and is therefore forced to move inside an imaginary tube defined by the transient network of entangled neighboring chains. Intuitively this motion resembles that of a snake or worm. The reptation model predicts five dynamical regimes for segment diffusion, summarized in the figure below. These regimes are as follows: (0) sub-segmental processes (“glassy dynamics”) at very short times (microseconds), (I) small motion subject only to chain connectivity, (II) “local reptation”: short-distance motion within the constraints imposed by the surrounding chains (“tube”), (III) “reptation”: diffusive motion along the curvilinear tube over distances larger than the polymer size, and (IV) free diffusion.

Rheology at the micrometer scale

Due to the crucial role of physical cues in regulating cell behaviour, the mechanical properties of hydrogels are a key design parameter in tissue engineering applications. The shear elastic properties of viscoelastic materials are commonly measured by mechanical rheometers. Storage and loss moduli of a material can be measured by application of strain while measuring stress or vice versa. In contrast, recently developed optical micro-rheology techniques use nanometer- or micrometer-sized particles embedded in the material to obtain the viscoelastic response parameters. Thermal or passive micro-rheology for viscoelastic materials is based on an extension of the concepts of Brownian motion of particles in simple liquids. The movement of the embedded particles can be monitored using particle tracking. Initially developed to investigate the rheological properties of uniform complex fluids, particle tracking micro-rheology (PTM) is becoming a popular technique to analyze polymer blends and gels, as well as the deformability and elasticity within cells. However, if the beads locally modify the structure of the gel or are contained in a pore in an inhomogeneous matrix, the bulk rheological properties will not be retrieved. A solution is to use the cross-correlated thermal fluctuation of pairs of tracer particles, ‘two-point micro-rheology’. This method provides a better agreement between micro and macro-rheology, even in complex micro-structured fluids. However, technical constrains limit the wide application of this technique. One of the major limitations of two-point micro-rheology is the reduced number of trajectories that can be used for analysis. During particle tracking micro-rheology, the length of the calculated trajectories is limited by the time spent by the tracers in the field of view (x,y) and depth of focus (z). Consequently, mechanical characterization of complex polymer matrixes at the micrometer scale would benefit greatly of a new method for (fast) tracking in 3D. We are developing a new method for fast tracking of (fluorescent) beads in 3D using a multi-plane wide field microscope. This will allow a better mechanical characterization of soft materials, at the microscale.

Cellular adhesion in 3D matrices

Cells sense physical forces and the mechanical properties of the microenvironment via several distinct mechanisms and cellular components. The first step of cellular adhesion to the ECM occurs via transmembrane heterodimers of the integrin family. Once integrin molecules adhere to the ECM, they are activated and form clusters. As the number of bound molecules increases, some of the focal complexes evolve from small (0.5-1µm in diameter) transient ‘dot-like’ contacts to elongated structures (3-10µm) which couple with actin and associated proteins. The mechanical coupling between the ECM and the cell cytoskeleton is controlled by the dynamics of the focal adhesion complexes (assembly, disassembly and turnover).

Protein-Protein Interactions

Protein-protein interactions (PPIs) are intrinsic to all cellular processes, driving both metabolic and regulatory pathways. Despite the numerous techniques available, detection of transient short-lived PPIs remains challenging4. The main fluorescence microscopic techniques developed for visualizing PPIs in a cellular context are based on Föster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC)5. Both techniques detect the interaction between a pair of labeled molecules. Although highly informative, they require fine positioning of the labels and in the majority of the applications the spatial resolution achieved is limited by the diffraction of light to about 200 nm. More information concerning the use of FRET to detect PPIs can be found at Cellular Signalling).

We have use a single molecule localization based super-resolution technique to detect and map PPIs at the cell membrane. This new variant of PAINT that enables mapping of short-lived transient interactions between cytosolic and membrane-bound proteins inside living mammalian cells, at the nanometer scale. In this method the protein of interest is labeled with a light-controllable fluorescent protein and imaged under TIRF illumination, which leads to the selective activation and subsequent detection of molecules in close proximity with the plasma membrane. Interacting molecules are discriminated using a stringent fitting of the fluorescence signal recorded for every single molecule.

Organoids: models for cell communication

Nowadays, human organoids are becoming a highly promising tool to model organ development, function and especially human diseases in vitro. In general, organoids are miniature, simplified organs that can easily propagate in vitro originating from one or a few cells, typically stem cells.

Single cell manipulation by endoscopy

Nanowire-based endoscopy has attracted interest due to its ability to manipulate cells at the single-cell level with minimal cellular perturbation. High-density, vertically aligned nanowire arrays have been used as an efficient gene delivery system. Despite the high transfection rates, culturing the cells on nanowire arrays might have other influences on the cellular behaviour. For example, stem cells cultured on silicon nanowires show significantly different adhesion, proliferation and differentiation, compared with flat silicon or other control substrates. Furthermore, such arrays are not location-specific and require optimization of the nanowire density and dimension for the different the cell types. In collaboration with the group of Prof. Hiroshi Uji-i we are developing a method to delivery genetic material using a single nanowire. In contrast to the existing methods, this approach can be applied to any cell type and is extremely specific: it can target a single cell and it can deliver the genetic material exactly at the desired position, such as inside of the nucleus, with no damage to the cell. Since gene editing is a stochastic event occurring in only a fraction of the cells, the transfer of genetic material (or proteins) is of crucial importance in genome editing methods, where the nucleases must be efficiently delivered. The duration and magnitude of the nuclease expression are critical parameters for the level of both on-target and off-target nuclease activity. Additionally, the dose of donor template DNA is important to ensure efficient homologous recombination. The proposed method offers the possibility to deliver different molecules at different times, in synchronization with the cell cycle. The lab of Prof. Uji-i is one of the first (and few) groups worldwide to have developed and optimized a novel nanoscopic technique using 1D nanowires, with a diameter of less than 100 nm, for SERS endoscopic studies. It has been already proven by us that the thin diameter and 1D structure of the NW greatly reduces the damage induced to a live cell during probe insertion. Although designed for a different purpose, this nanoprobe is ideal as a starting point to develop a new NW-based gene delivery system.

Principle of nanowire-based gene delivery system.

New Drug delivery systems

In this decade, the pharmacology field has been intensively exploring different approaches to deliver multiple drugs with a single drug nano-carrier, such as liposomes, polymer nanoparticles, and inorganic nanoparticles. The advantage of nanoparticle based drug delivery is the ability to unify pharmacokinetics by simultaneous delivery of multiple drugs to specific target cells.

Ever since first reported in 2001, mesoporous silica nanoparticles (MSNPs) have manifested themselves as highly potential candidates for targeted drug delivery. They owe their popularity to their high drug load capacity, chemical stability, biocompatibility and easy functionalization. Since the diameter of the nanoparticles (100 to 200 nm) is tunable, one can obtain a size suitable for passive targeting through the hyperpermeable tumor vasculature, thereby promoting accumulation of the nanoparticles in tumor tissue due to the enhanced permeability and retention effect (EPR). Additionally, functionalization of the nanoparticles with ligands which have a high affinity for tumor cell specific surface receptors promotes more specific internalization in cancer cells. For example, hyaluronic acid (HA) has been extensively used as a targeting ligand due to its affinity for CD44, a transmembrane glycoprotein receptor that plays a critical role in malignant cell activities and, most importantly, it is overexpressed in many solid tumor cells, in metastasis and cancer stem cells.

Correlative AFM and Fluorescence Microscopy

Biological processes are often carried out in the context of macromolecular assemblies. In addition, arrangements of these complexes can be dynamic, resulting in a heterogeneous ensemble. Single molecule techniques can resolve distinct populations in heterogeneous systems, in contrast to bulk experiments where heterogeneity is averaged out. In turn, mechanistic details of bio-macromolecular interactions can be uncovered. Atomic force microscopy (AFM) is a technique that can generate 3D reconstructions of individual biomolecules and complexes thereof in a label-free fashion, and with ~ nm resolution. To this end a very sharp tip, mounted on a flexible cantilever, scans a sample surface in a raster pattern using a piezo-scanner, while keeping the interaction force between sample and tip constant. In every pixel (x,y) of the scanned area, the z-position is recorded. Consequently, a 3D representation of the surface topography can be reconstructed. An alternative way to study single molecules is by fluorescence microscopy. The molecule of interest is labeled with a fluorescent tag providing high contrast. Emission of the tag after excitation, is detected through an optical system. Due to the wave character of light, the emitted light is spread out on the detector described by the point spread function (PSF) of the optical system. This effect limits the resolution achieved with optical microscopy, referred to as the diffraction limit. However, when the signal of a single molecule is detected, the position of this molecule can be determined by fitting of the recorded fluorescence signal with a mathematical approximation of the PSF such as a two-dimensional Gaussian function. This principle underlies single molecule localization microscopy (SMLM). AFM and SMLM are highly complementary technologies: AFM can provide insight in topographic features at a nanometer resolution while SMLM is sensitive towards specifically labelled molecules in complex samples. Integrated setups combining both technologies can therefore provide orthogonal information at the single-molecule level.

Cell signalling: probes and methods

Cell signaling involves the sensing of an extracellular signal by a cell surface receptor, which then transduces this signal to an intracellular response. Despite the numerous studies performed on signaling pathways and mechanisms, little is known about the initial steps occurring at the plasma membrane: receptor pre-assembly at the molecular level and potential reorganization after ligand activation. Traditionally crystallography is used to investigate receptor multimerization. However, the crystallized state might not represent the biochemically active form due to the harsh preparation conditions and the absence of the cellular environment. Other approaches include macroscopic biochemical or biophysical methods, such as chemical cross-linking, ion-channel gating, immunoprecipitation or binding assays. Nowadays, established fluorescence imaging and spectroscopic techniques offer a versatile toolbox to study membrane receptor organization in (living) cells.

In the lab we are using fluorescence fluctuation spectroscopy to quantify physicochemical processes (mobility, binding affinity, stoichiometry, absolute concentration) occurring on a micro-to-millisecond time scale. Fluorescence experiments down to picoseconds are also commonly possible with methods such as time-correlated single photon counting (TCSPC), that allow, e.g., measuring fluorescence lifetimes and molecular tumbling. Additionally, spatially resolved microscopy with high temporal resolution also has clear benefits. For example, combined with confocal laser scanning microscopy (LSM), TCSPC allows protein-protein interactions (PPIs) to be imaged via Förster resonance energy transfer (FRET) based fluorescence lifetime imaging microscopy (FLIM). Imaging based FCS methods such as raster (RICS), number and brightness analysis (N&B) or (spatio-) temporal image correlation spectroscopy [(S)TICS] combine the quantitative analytical power of fluctuation methods with spatial information to map, among many other things, mobility and stoichiometry inside living systems. Simultaneous dual-color fluorescence imaging is possible when fast alternating excitation (alias pulsed interleaved excitation, PIE) is employed. PIE renders analysis of dual-color point FCS experiments considerably more straightforward. The combination of PIE with fluctuation imaging (PIE-FI) allows extracting the maximum amount of molecular information (mobility, stoichiometry, interactions…) from each species present in dual-color LSM images.

PIE (a), PIE-FI (b) and subsequent analyses, based on spatial/temporal auto-/cross-correlation or fluorescence lifetimes, which allow to extract the maximum amount of information of the molecules present in the imaged structure.

For more information on these methods:

  • Hendrix J., Lamb D.C. (2014) Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS. In: Engelborghs Y., Visser A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology (Methods and Protocols), vol 1076. Humana Press, Totowa, NJ (chapter can be found here)
  • Hendrix J., Schrimpf W., Höller M., Lamb D.C. (2013) Pulsed Interleaved Excitation Fluctuation Imaging, Biophysical Journal, 105(4), 848-861 (article can be found here)

Imaging single HIV virions

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. In collaboration with Prof. Zeger Debeyser (KU Leuven) and Prof Hendrix (UHasselt) we have applied different single-molecule sensitive fluorescence methods to investigate viruses, one-by-one. While this collaboration is still ongoing, there is already several publications that show-case the potential of imaging single virions.

publications

Investigation of the melting behavior of the reference materials biphenyl and phenyl salicylate by a new type adiabatic scanning calorimeter

Abstract

Simultaneously measured high-resolution enthalpy and heat capacity data are obtained by means of a novel type Peltier-element-based adiabatic scanning calorimeter that can also operate as a classical adiabatic heat-step calorimeter. Specific enthalpy and specific heat capacity results with 2% uncertainty and sub-mK temperature resolution are presented for the melting transition of the calorimetric reference materials biphenyl and phenyl salicylate. The simultaneously obtained enthalpy and heat capacity data allow for a simplified and reliable purity analysis.

Published in Thermochimica Acta, 2014

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Phase transitions of binary lipid mixtures: a combined study by adiabatic scanning calorimetry and quartz crystal microbalance with dissipation monitoring

Abstract

The phase transitions of binary lipid mixtures are studied by a combination of Peltier-element-based adiabatic scanning calorimetry (pASC) and quartz crystal microbalance with dissipation monitoring (QCM-D). pASC, a novel type of calorimeter, provides valuable and unambiguous information on the heat capacity and the enthalpy, whereas QCM-D is proposed as a genuine way of determining phase diagrams by analysing the temperature dependence of the viscosity. Two binary mixtures of phospholipids with the same polar head and differing in the alkyl chain length, DMPC + DPPC and DMPC + DSPC, are discussed. Both techniques give consistent phase diagrams, which compare well with literature results, showing their capability to map the phase behaviour of pure lipids as well as lipid mixtures. This work can be considered as a departure point for further investigations on more complex lipid mixtures displaying relevant phases such as the liquid-ordered phase and solid-lipid interfaces with biologically functional importance.

Published in Advances in Condensed Matter Physics, 2015

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Coherent intensity fluctuation model for autocorrelation imaging spectroscopy with higher harmonic generating point scatterers—a comprehensive theoretical study

Abstract

We present a general analytical model for the intensity fluctuation autocorrelation function for second and third harmonic generating point scatterers. Expressions are derived for a stationary laser beam and for scanning beam configurations for specific correlation methodologies. We discuss free translational diffusion in both three and two dimensions. At low particle concentrations, the expressions for fluorescence are retrieved, while at high particle concentrations a rescaling of the function parameters is required for a stationary illumination beam, provided that the phase shift per unit length of the beam equals zero.

Published in Physical Chemistry and Chemical Physics, 2015

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Study of Thermophysical Properties of Silver Nanofluids by ISS-HD, Hot Ball and IPPE Techniques

Abstract

In this work, the impulsive stimulated scattering technique, in a heterodyne diffraction detection configuration (ISS-HD), was used to study the dependence of the speed of sound and the thermal diffusivity on the concentration of silver nanoparticles in water, to which also d-glucose and carboxymethyl cellulose were added, in order to reduce sedimentation. The ISS-HD results, which show a slight increase of thermal diffusivity with increasing concentration, were cross-validated with results obtained by the inverse photopyroelectric method and the hot ball technique.

Published in International Journal of Thermophysics, 2015

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Biocompatible label-free detection of carbon black particles by femtosecond pulsed laser microscopy

Abstract

Although adverse health effects of carbon black (CB) exposure are generally accepted, a direct, label-free approach for detecting CB particles in fluids and at the cellular level is still lacking. Here, we report nonincandescence related white-light (WL) generation by dry and suspended carbon black particles under illumination with femtosecond (fs) pulsed near-infrared light as a powerful tool for the detection of these carbonaceous materials. This observation is done for four different CB species with diameters ranging from 13 to 500 nm, suggesting this WL emission under fs near-infrared illumination is a general property of CB particles. As the emitted radiation spreads over the whole visible spectrum, detection is straightforward and flexible. The unique property of the described WL emission allows optical detection and unequivocal localization of CB particles in fluids and in cellular environments while simultaneously...

Published in Nano Letters, 2016

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Label-free imaging of umbilical cord tissue morphology and explant-derived cells

Abstract

In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

Published in Stem Cells International, 2016

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The SlZRT1 gene encodes a plasma membrane-located ZIP (Zrt-, Irt-Like Protein) transporter in the ectomycorrhizal fungus Suillus luteus

Abstract

Zn is an essential micronutrient but may become toxic when present in excess. In Zn-contaminated environments, trees can be protected from Zn toxicity by their root-associated micro-organisms, in particular ectomycorrhizal fungi. The mechanisms of cellular Zn homeostasis in ectomycorrhizal fungi and their contribution to the host tree’s Zn status are however not yet fully understood. The aim of this study was to identify and characterize transporters involved in Zn uptake in the ectomycorrhizal fungus S. luteus, a cosmopolitan pine mycobiont. Zn uptake in fungi is known to be predominantly governed by members of the ZIP (Zrt/IrtT-like protein) family of Zn transporters. Four ZIP transporter encoding genes were identified in the S. luteus genome. By in silico and phylogenetic analysis, one of these proteins, SlZRT1, was predicted to be a plasma membrane located Zn importer. Heterologous expression in yeast confirmed the predicted function and localization of the protein. A gene expression analysis via RT-qPCR was performed in S. luteus to establish whether SlZRT1 expression is affected by external Zn concentrations. SlZRT1 transcripts accumulated almost immediately, though transiently upon growth in the absence of Zn. Exposure to elevated concentrations of Zn resulted in a significant reduction of SlZRT1 transcripts within the first hour after initiation of the exposure. Altogether, the data support a role as cellular Zn importer for SlZRT1 and indicate a key role in cellular Zn uptake of S. luteus. Further research is needed to understand the eventual contribution of SlZRT1 to the Zn status of the host plant.

Published in Frontiers in microbiology, 2017

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Dynamics of the phospholipid shell of microbubbles: a fluorescence photoselection and spectral phasor approach

Abstract

The lipid organization of microbubbles is important in many applications. By monitoring the photoselection and emission spectrum of the fluorescent probe Laurdan in perfluorobutane gas-filled DPPC microbubbles with a two-photon laser scanning microscope, we observed a transition to a more rigid lipid organization in 30 minutes to several hours.

Published in Chemical Communications, 2018

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Enantiomorphing Chiral Plasmonic Nanostructures: A Counterintuitive Sign Reversal of the Nonlinear Circular Dichroism

Abstract

Plasmonic nanostructures have demonstrated a remarkable ability to control light in ways never observed in nature, as the optical response is closely linked to their flexible geometric design. Due to lack of mirror symmetry, chiral nanostructures allow twisted electric field “hotspots” to form at the material surface. These hotspots depend strongly on the optical wavelength and nanostructure geometry. Understanding the properties of these chiral hotspots is crucial for their applications; for instance, in enhancing the optical interactions with chiral molecules. Here, the results of an elegant experiment are presented: by designing 35 intermediate geometries, the structure is “enantiomorphed” from one handedness to the other, passing through an achiral geometry. Nonlinear multiphoton microscopy is used to demonstrate a new kind of double‐bisignate circular dichroism due to enantiomorphing, rather than wavelength change. From group theory, a fundamental origin of this plasmonic chiroptical response is proposed. The analysis allows the optimization of plasmonic chiroptical materials.

Published in Advanced Optical Materials, 2018

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Image correlation spectroscopy with second harmonic generating nanoparticles in suspension and in cells

Abstract

The absence of photobleaching, blinking, and saturation combined with a high contrast provides unique advantages of higher-harmonic generating nanoparticles over fluorescent probes, allowing for prolonged correlation spectroscopy studies. We apply the coherent intensity fluctuation model to study the mobility of second harmonic generating nanoparticles. A concise protocol is presented for quantifying the diffusion coefficient from a single spectroscopy measurement without the need for separate point-spread-function calibrations. The technique’s applicability is illustrated on nominally 56 nm LiNbO3 nanoparticles. We perform label-free raster image correlation spectroscopy imaging in aqueous suspension and spatiotemporal image correlation spectroscopy in A549 human lung carcinoma cells. In good agreement with the expected theoretical result, the measured diffusion coefficient in water at room...

Published in The journal of physical chemistry letters, 2018

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Resolution in coherent and incoherent optical imaging with two–photon excitation microscopy

Abstract

Driven by both curiosity and applications in healthcare, biomedical sci-entists seek to understand the complex machinery that is the human body. As a biophysicist, I contribute to this quest by developing new lab techniques. Tissues, cells, cell organelles,... the small sizes of many biological structures explain the need for increasingly better–performing imaging systems. Because diffraction limits the resolution in conventional optical microscopes to several hundred nanometers, other methods are needed to study biological samples at the desired resolution. In this work, several possible ways to challenge the diffraction limit are presented. The thesis starts with two general chapters, the first being an introduction in microscopy, the second providing the necessary mathematics describing the image formation process. In the other chapters, of which three are based on original publications, several methods to circumvent the diffraction limit are described. Finally, a brief summary and an outlook are presented in the conclusion.

Published in University of Hasselt, 2018

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Combustion-derived particles inhibit in vitro human lung fibroblast-mediated matrix remodeling

Abstract

The continuously growing human exposure to combustion-derived particles (CDPs) drives in depth investigation of the involved complex toxicological mechanisms of those particles. The current study evaluated the hypothesis that CDPs could affect cell-induced remodeling of the extracellular matrix due to their underlying toxicological mechanisms. The effects of two ultrafine and one fine form of CDPs on human lung fibroblasts (MRC-5 cell line) were investigated, both in 2D cell culture and in 3D collagen type I hydrogels. A multi-parametric analysis was employed. In vitro dynamic 3D analysis of collagen matrices showed that matrix displacement fields induced by human lung fibroblasts are disturbed when exposed to carbonaceous particles, resulting in inhibition of matrix remodeling. In depth analysis using general toxicological...

Published in Journal of nanobiotechnology, 2018

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Ambient black carbon particles reach the fetal side of human placenta

Abstract

Particle transfer across the placenta has been suggested but to date, no direct evidence in real-life, human context exists. Here we report the presence of black carbon (BC) particles as part of combustion-derived particulate matter in human placentae using white-light generation under femtosecond pulsed illumination. BC is identified in all screened placentae, with an average (SD) particle count of 0.95 x 10^4 (0.66 x 10^4) and 2.09 x 10^4 (0.9 x 10^4) particles per mm^3 for low and high exposed mothers, respectively. Furthermore, the placental BC load is positively associated with mothers' residential BC exposure during pregnancy (0.63–2.42 ug per m^3). Our finding that BC particles accumulate on the fetal side of the placenta suggests that ambient particulates could be transported towards the fetus and represents a potential mechanism explaining the detrimental health effects of pollution from early life onwards.

Published in Nature Communications, 2019

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Two-photon image-scanning microscopy with SPAD array and blind image reconstruction

Abstract

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure ~1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of sqrt(2) – and automatic background correction is shown to boost the image...

Published in Biomedical Optics Express, 2020

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Image scanning microscopy with multiphoton excitation or Bessel beam illumination

Abstract

In image scanning microscopy, the pinhole of a confocal microscope is replaced by a detector array. The point spread function for each detector element can be interpreted as the probability density function of the signal, the peak giving the most likely origin. This thus allows a form of maximum likelihood restoration, and compensation for aberrations, with similarities to adaptive optics. As an example of an aberration, we investigate theoretically and experimentally illumination with a vortex doughnut beam. After reassignment and summation over the detector array, the point spread function is compact, and the resolution and signal level higher than in a conventional microscope.

Published in Optical Society of America A, 2020

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Monitoring indoor exposure to combustion-derived particles using plants

Abstract

Indoor plants can be used to monitor atmospheric particulates. Here, we report the label-free detection of combustion-derived particles (CDPs) on plants as a monitoring tool for indoor pollution. First, we measured the indoor CDP deposition on Atlantic ivy leaves (Hedera hibernica) using two-photon femtosecond microscopy. Subsequently, to prove its effectiveness for using it as a monitoring tool, ivy plants were placed near five different indoor sources. CDP particle area and number were used as output metrics. CDP values ranged between a median particle area of 0.45 x 10^2 to 1.35 x 10^4 μm2, and a median particle number of 0.10 x 10^2 to 1.42 x 10^3 particles for the indoor sources: control (greenhouse) < milling machine < indoor smokers < wood stove < gas stove < laser printer. Our findings demonstrate that Atlantic ivy, combined with label-free detection, can be effectively used in indoor atmospheric monitoring studies.

Published in Environmental Pollution, 2020

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Confocal-based Fluorescence Fluctuation Spectroscopy with a SPAD Array Detector

Abstract

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images–of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

Published in Light, science & applications, 2021

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Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration

Abstract

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images–of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

Published in Optical Society of America A, 2021

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Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

Abstract

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy. However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark noise, photon-detection efficiency, after-pulsing probability, and optical cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present, to our knowledge, a novel SPAD array detector with an active cooling system that substantially reduces the dark noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to −15°C significantly improves the signal/noise ratio due to a 10-fold decrease in the dark count rate compared with room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing green-fluorescent-protein-tagged proteins. For fluorescence fluctuation spectroscopy, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements for which dark noise is substantially higher than the median value. Overall, this detector represents a further step toward the integration of SPAD array detectors in any FLSM system.

Published in Biophysical Reports, 2021

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Signal strength and integrated intensity in confocal and image scanning microscopy

Abstract

The properties of signal strength and integrated intensity in a scanned imaging system are reviewed. These properties are especially applied to confocal imaging systems, including image scanning microscopy. The integrated intensity, equal to the image of a uniform planar (sheet) object, rather than the peak of the point spread function, is a measure of the flux in an image. Analytic expressions are presented for the intensity in the detector plane for a uniform volume object, and for the resulting background. The variation in the integrated intensity with defocus for an offset point detector is presented. This axial fingerprint is independent of any pixel reassignment. The intensity in the detector plane is shown to contain the defocus information, and simple processing of the recorded data can improve optical sectioning and background rejection.

Published in Optical Society of America A, 2022

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The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy

Abstract

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.

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Published in Nature Communications, 2022

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ISM-FLUX: MINFLUX with an array detector

Abstract

Single-molecule localization based on the concept of MINFLUX allows for molecular resolution imaging and tracking with a laser-scanning microscopy architecture. In MINFLUX, a doughnut-shaped structured illumination beam sequentially excites the sample, thereby minimizing the localization uncertainty for a given number of photons. However, MINFLUX has a limited field of view (FOV) and therefore requires a precise prelocalization step. We mitigate this problem by introducing the single-molecule image scanning microscopy (smISM) concept. In smISM, the point detector of the laser-scanning microscope is replaced with a small single-photon detector array, thereby leveraging the benefits of both structured illumination and structured detection. We show via simulations that by combining smISM and MINFLUX (i.e., ISM-FLUX), we obtained a localization uncertainty between 1 and 15 nm over a FOV of more than 800 nm with 100 photons. In addition, smISM allows the localization of multiple molecules simultaneously. We calculate the effect of different parameters, such as the relative position of the doughnut beams, the number of detector pixels, the number of photons, and the signal-to-background ratio, on the localization uncertainty. We predict that the combination of a good localization precision and the enhanced robustness of ISM-FLUX will help the wide adoption of MINFLUX.

Published in Physical Review Research, 2023

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Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy

Abstract

We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.

Published in Photonics, 2023

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Open-source tools enable accessible and advanced image scanning microscopy data analysis

Abstract

Thanks to the advent of high-performance detector arrays, image scanning microscopy (ISM) has transformed the field of fluorescence laser-scanning microscopy1. In particular, the development of fast, sensitive single-photon avalanche diode (SPAD) arrays2 has paved the way for robust ISM imaging with exceptional information content3. In ISM, a focused excitation beam scans the sample. For each scan point, the SPAD array collects a small image — called a micro-image — of the diffraction-limited fluorescent region. In effect, each SPAD of the detector acts as an independent and displaced pinhole, generating a confocal-like image. Thus, the ISM dataset can be seen either as a large set of small wide-field images or as a small set of large confocal images, both point-of-views opening unique ways to process the data. Tailored algorithms enable the transformation of the raw ISM dataset into a single image with enhanced spatial resolution, signal-to-noise ratio, and optical sectioning. With the recent introduction into the market of commercial microscopes equipped with a detector array — namely, AiryScan from Zeiss, PRISM from Genoa Instruments, MATRIX from Abberior, and NSPARC from Nikon — we expect ISM to become increasingly widespread in use. With this in mind, we have designed an open-source tool called BrightEyes-ISM to provide the scientific community with a computational toolbox for processing the ISM datasets with cutting-edge algorithms for image reconstruction, image quality evaluation, and simulation. BrightEyes-ISM is an open-source Python package with three different modules, each containing functions specific to data loading, data analysis, or optical simulations. The simulation module contains a point spread function (PSF) simulator (Fig. 1a–c) and a tubulin simulator. The PSF simulator is currently built upon PyFocus4, a Python package that enables the vectorial calculation of focused optical fields. The simulator generates a set of PSFs, one for each element of the detector array, with all of the relevant physical parameters that can be fine-tuned. We also built a phase calculator that enables the introduction of any aberration or a user-defined phase mask. Optionally, the simulator can introduce a third field for stimulated emission depletion (STED), enabling the simulation of STED-ISM PSFs. The dataIO module enables the user to easily read and store data and metadata in a hierarchical data format (HDF), which is particularly convenient for ISM datasets that are inherently large and multi-dimensional (Fig. 1d). The analysis module contains libraries for state-of-the-art analysis of ISM data processing. The APR library contains the functions needed to perform adaptive pixel reassignment (APR), a fast and robust method to reconstruct a super-resolution image from an ISM dataset5 (Fig. 1e). The deconvolution library contains many functions to perform image deconvolution, the most important one being the multi-image deconvolution6 that enables an excellent alternative to APR for image reconstruction (Fig. 1f). The Focus-ISM library contains all of the functions needed to perform Focus-ISM, namely, an innovative algorithm that greatly enhances the optical sectioning of ISM, effectively removing the out-of-focus light from the ISM datasets7 (Fig. 1g). The FRC library provides the tools needed to perform Fourier ring correlation (FRC) analysis on the reconstruction, enabling a quantitative method to evaluate the resolution of the images8 (Fig. 1h). Notably, we also use FRC as a tool to estimate the PSFs from experimental data and perform blind multi-image deconvolution9. BrightEyes-ISM can be found on GitHub (https://github.com/VicidominiLab/BrightEyes-ISM), together with the complete documentation. Importantly, we intended our package to be useful both for developers and microscopy users. We therefore integrated the majority of the functionalities of BrightEyes-ISM into a Napari plugin, named Napari-ISM (https://github.com/VicidominiLab/napari-ISM). Napari is a multi-dimensional image viewer that provides a quick and easy way to explore and analyse the ISM data.

Published in Nature Photonics, 2023

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teaching

Teaching experience 1

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Undergraduate course, University 1, Department, 2014

Teaching experience 2

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Workshop, University 1, Department, 2015

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Eli Slenders

ShortBio

Eli Slenders has a bachelor’s and master’s degree in physics from Hasselt University (Hasselt, Belgium) and KU Leuven (Leuven, Belgium), respectively. He graduated in 2013. From 2014 to 2018, he worked as a PhD student at Hasselt University in the Biophysics group of prof. M. Ameloot. His PhD thesis was entitled “Resolution in coherent and incoherent optical imaging with two-photon excitation microscopy”. In 2019, Eli joined the Molecular Microscopy and Spectroscopy research line at the Italian Institute of Technology (IIT, Genoa, Italy) under the supervision of Giuseppe Vicidomini, first as a postdoc, since 2021 as a Marie Skłodowska-Curie Actions research fellow. His research interests include the theoretical design and experimental validation of optical microscopy tools such as multiphoton image scanning microscopy, fluorescence correlation spectroscopy, and super-resolution microscopy.

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