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About

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Current collaborations

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In 2020, we started a spin-out company Infinite, Biomedical Technologies, devoted to animal-free safety testing of nanomaterials and chemicals. We are excited to pursue this joint journey to bridge all TRL levels from basic science to the final product/service.

Past collaborations

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LBF has a long tradition in development of technologies and infrastructure in the fields of tissue engineering, antimicrobial surfaces, biomedical and pharmaceutical applications, optimization of industrial materials and processes, and reasonable use of energy and building biophysics.

news

We are hiring!

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We are searching for talented and motivated people to join our team! Currently we are seeking staff with a physics-related backround for development of microscope-on-chip technologies.

Ana defends her MSc!

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Our MSc student Ana Jandrlić defended her thesis today at the Faculty of Pharmacy, University of Ljubljana - congratulations, and welcome to the team of our spin-out company Infinite!

publications

SDSL-ESR-based protein structure characterization

Published in European Biophysics Journal, 2009

Abstract

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

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Conformational analysis of the partially disordered measles virus NTAIL-XD complex by SDSL EPR spectroscopy

Published in Biophysical journal, 2010

In brief

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein and provided a mechanistic explanation for the high affinity of the binding reaction between the measles virus and the viral phosphoprotein.

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Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells

Published in Cell Biology International, 2010

In brief

We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping.

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Nitroxide–fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence

Published in Organic & Biomolecular Chemistry, 2011

In brief

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide–fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy.

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Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells

Published in Biomedical optics express, 2011

In brief

Here we present a development of fluorescence microspectroscopy (spectral imaging) based on a white light spinning disk confocal microscope with emission wavelength selection by a liquid crystal tunable filter. Spectral contrasting of images was used to localize polymer nanoparticles and cell membranes labeled with fluorophores that have substantially overlapping spectra. In addition, fluorescence microspectroscopy enabled spatially-resolved detection of small but significant effects of local molecular environment on the properties of environment-sensitive fluorescent probe.

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Coexistence of Probe Conformations in Lipid Phases—A Polarized Fluorescence Microspectroscopy Study

Published in Biophysical journal, 2013

In brief

In this study, bleaching-corrected polarized fluorescence microspectroscopy with nanometer spectral peak position resolution was applied to characterize conformations of two alkyl chain-labeled 7-nitro-2-1,3-benzoxadiazol-4-yl phospholipids in three model membranes, representing the three main lipid phases. The combination of polarized and spectral detection revealed two main probe conformations with their preferential fluorophore dipole orientations roughly parallel and perpendicular to membrane normal. Their peak positions were separated by 2–6 nm because of different local polarities and depended on lipid environment.

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Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution

Published in Optics express, 2013

In brief

Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores with small spectral shifts, which are commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy.

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Recovering position-dependent diffusion from biased molecular dynamics simulations

Published in J. Chem. Phys., 2014

Abstract

All atom molecular dynamics (MD) models provide valuable insight into the dynamics of biophysical systems, but are limited in size or length by the high computational demands. The latter can be reduced by simulating long term diffusive dynamics (also known as Langevin dynamics or Brownian motion) of the most interesting and important user-defined parts of the studied system, termed collective variables (colvars). A few hundred nanosecond-long biased MD trajectory can therefore be extended to millisecond lengths in the colvars subspace at a very small additional computational cost. In this work, we develop a method for determining multidimensional anisotropic position- and timescale-dependent diffusion coefficients (D) by analysing the changes of colvars in an existing MD trajectory. As a test case, we obtained D for dihedral angles of the alanine dipeptide. An open source Mathematica® package, capable of determining and visualizing D in one or two dimensions, is available at https://github.com/lbf-ijs/DiffusiveDynamics. Given known free energy and D, the package can also generate diffusive trajectories.

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Molecular Mobility of Scaffolds’ Biopolymers Influences Cell Growth

Published in ACS Appl. Mater. Interfaces, 2014

In brief

The scope of our study was thus the investigation of the correlation of fibroblast cell growth on different gelatin scaffolds with their morphological, mechanical as well as surface molecular properties. In this study, anisotropy of the rotational motion of the gelatin side chain mobility was identified as the most correlated quantity with cell growth in the first days after adhesion, while weaker correlations were found with scaffold viscoelasticity and no correlations with scaffold morphology.

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Ca2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

Published in Biochemical Journal, 2014

In brief

In the present study, we show that overall fXa activity on PS-containing membranes is sharply regulated by a ‘Ca2+ switch’ centred at 1.16 mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both Ca2+ and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor [fVa (Factor Va)] to achieve significant activity.

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Resolving Internal Motional Correlations to Complete the Conformational Entropy Meter

Published in J. Phys. Chem. Lett., 2014

Abstract

Conformational entropy (SΩ) has long been used to theoretically characterize the dynamics of proteins, DNA, and other polymers. Though recent advances enabled its calculation also from simulations and nuclear magnetic resonance (NMR) relaxation experiments, correlated molecular motion has hitherto greatly hindered both numerical and experimental determination, requiring demanding empirical and computational calibrations. Herein, we show that these motional correlations can be estimated directly from the temperature-dependent SΩ series that reveal effective persistence lengths of the polymers, which we demonstrate by measuring SΩ of amphiphilic molecules in model lipid systems by spin-labeling electron paramagnetic resonance (EPR) spectroscopy. We validate our correlation-corrected SΩ meter against the basic biophysical interactions underlying biomembrane formation and stability, against the changes in enthalpy and diffusion coefficients upon phase transitions, and against the energetics of fatty acid dissociation. As the method can be directly applied to conformational analysis of proteins and other polymers, as well as adapted to NMR or polarized fluorescence techniques, we believe that the approach can greatly enrich the scope of experimentally available statistical thermodynamics, offering new physical insights into the behavior of biomolecules.

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Cell-Scaffold Adhesion Dynamics Measured in First Seconds Predicts Cell Growth on Days Scale – Optical Tweezers Study

Published in ACS Appl. Mater. Interfaces, 2015

In brief

In our study, optical tweezers combined with confocal fluorescence microscopy were optimized to investigate the initial cell–scaffold contact and to investigate its correlation with the material-dependent cell growth. We have shown that dynamics of cell adhesion on scaffold surfaces correlates with cell growth on the days scale, which indicates that the first seconds of the contact could markedly direct further cell response and predict scaffold biocompatibility.

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Protein Corona Prevents TiO2 Phototoxicity

Published in PLoS ONE, 2015

In brief

We used fluorescence and spin trapping EPR spectroscopy to reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity of TiO2 thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface.

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Internalization and Accumulation in Dendritic Cells of a Small pH-Activatable Glycomimetic Fluorescent Probe as Revealed by Spectral Detection

Published in ChemBioChem, 2015

Abstract

DC-SIGN, an antigen-uptake receptor in dendritic cells (DCs), has a clear role in the immune response but, conversely, can also facilitate infection by providing entry of pathogens into DCs. The key action in both processes is internalization into acidic endosomes and lysosomes. Molecular probes that bind to DC-SIGN could thus provide a useful tool to study internalization and constitute potential antagonists against pathogens. So far, only large molecules have been used to directly observe DC-SIGN-mediated internalization into DCs by fluorescence visualization. We designed and synthesized an appropriate small glycomimetic probe. Two particular properties of the probe were exploited: activation in a low-pH environment and an aggregation-induced spectral shift. Our results indicate that small glycomimetic molecules could compete with antigen/pathogen for binding not only outside but also inside the DC, thus preventing the harmful action of pathogens that are able to intrude into DCs, for example, HIV-1.

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Aggregation-induced emission spectral shift as a measure of local concentration of a pH-activatable rhodamine-based smart probe

Published in Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2018

In brief

Generating activatable probes that report about molecular vicinity through contact-based mechanisms such as aggregation can be very convenient. Specifically, such probes change a particular spectral property only at the intended biologically relevant target. In this work, we show that the size of the aggregation-induced emission spectral shift at specific position on the sample can be measured by fluorescence microspectroscopy, which at particular pH allows estimation of the local concentration of the observed probe at microscopic level.

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Surface deposited one-dimensional copper-doped TiO2 nanomaterials for prevention of health care acquired infections

Published in PLoS ONE, 2018

In brief

Here we show that copper doped TiO2 nanotubes produce about five times more ·OH radicals as compared to undoped TiO2 nanotubes and that effective surface disinfection, determined by a modified ISO 22196:2011 test, can be achieved even at low intensity UVA light of 30 μW/cm2. The nanotubes can be deposited on a preformed surface at room temperature, resulting in a stable deposition resistant to multiple washings. Up to 103 microorganisms per cm2 can be inactivated in 24 hours, including resistant strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-spectrum beta-lactamase Escherichia coli (E. coli ESBL).

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Lipid Composition but Not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles

Published in Viruses, 2018

In brief

To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1–1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1–1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles’ surface.

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New coumarin- and phenoxazine-based fluorescent probes for live-cell STED nanoscopy

Published in European Biophysics Journal, 2019

In brief

We designed and tested several new orange and red fluorescent probes for live-cell STED nanoscopy. Three of the newly-synthesized probes exhibited improved resolution in STED nanoscopy with a 775 nm STED laser and two of these were also remarkably photostable, thus being perfectly suited for STED nanoscopy. The best overall results were assigned to the probe MePyr500, which was also not cytotoxic, making it an excellent candidate for live-cell STED nanoscopy.

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Two-photon retinal theranostics by adaptive compact laser source

Published in Applied Physics A, 2020

In brief

In our work, compact fiber-laser based on high-speed gain-switched laser diode has been shown to achieve adaptable/independently tunable repetition rate and energy per pulse allowing coupled fluorescence lifetime ophthalmic diagnostics via two-photon excitation and phototherapy via laser-induced photodisruption on a local molecular environment in a complex ex vivo retinal tissue.

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Background Reduction in STED-FCS Using a Bivortex Phase Mask

Published in ACS Photonics, 2020

In brief

In this paper, we investigate how different STED confinement modes reduce the spurious background contributions from undepleted areas of the excitation in 3D FCS, thus increasing the signal quality and ultimately the resolution. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume.

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Prediction of Chronic Inflammation for Inhaled Particles: the Impact of Material Cycling and Quarantining in the Lung Epithelium

Published in Advanced Materials, 2020

In brief

The prediction of diseases associated with nanomaterials is currently hampered by an incomplete understanding of the underlying mechanisms. Together with international collaborators, we discovered nanomaterial quarantining and counteracting nanomaterial cycling, which allowed us to incorporate these main modes of cellular response into a mechanistic model and predict in vivo inflammation solely on animal-free in vitro tests. The paper was published in the prestigious journal Advanced Materials and selected as the frontispiece of the journal issue.

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Revealing Inflammatory Indications Induced by Titanium Alloy Wear Debris in Periprosthetic Tissue by Label-Free Correlative High-Resolution Ion, Electron and Optical Microspectroscopy

Published in Materials, 2021

In brief

In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches: hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination.

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Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells

Published in FEBS Letters, 2021

Abstract

To disentangle the elusive lipid–protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

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How to Control Fluorescent Labeling of Metal Oxide Nanoparticles for Artefact-free Live Cell Microscopy

Published in Nanotoxicology, 2021

In brief

The assessment of a nanoparticles’ mechanism of toxicity often depends on tracking the nanoparticles in cells or tissues using non-invasive advanced optical methods, such as live-cell fluorescence microscopy. This requires stable labeling of nanoparticles with fluorescent dyes - a process which, if not carefully controlled, can result in experimental artefacts that stem from the presence of unbound fluorophores and inadvertently altered chemical and physical properties of the nanomaterial.

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research

Blood coagulation

Published:

In brief

We investigate basic molecular mechanisms of the blood coagulation cascade. In collaboration with Barry R. Lentz’s laboratory from University of North Carolina at Chapel Hill and with prof Rinku Majumders’ laboratory from the Louisiana State University School of Medicine in New Orleans, both recognized experts in the field of blood coagulation, Tilen Koklič showed heretofore yet unknown molecular mechanisms regarding regulation of factor Xa and of the prothrombinase (fXa-fVa) complex (Biochemical Journal, IF 4.40, Biophysical Journal, IF 4,32; Biochemistry, IF 3.56).

Probes

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Probes

Artificial intelligence in microscopy

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In brief

We are introducing computer vision algorithms to improve the acquisition efficiency of advanced optical microscopies. Within a national research project ARRS J7-2596, we collaborate with

resources

Clean chemical and cell laboratory

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Standardly equiped cell laboratory is placed in a clean room (ISO4/5) to ensure the most controllable environment to seed and grow cell lines. A clean chemical laboratory (ISO5) nearby ensures the most efficient hadnling during preparation and basic characterization of the samples. Finally, at the end of the air flow of the clean rooms, there is nano laboratory (ISO 6) for handling of nanomaterials and cleaning.

Clean optical laboratory

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Optical setups are placed in the clean optical laboratory (ISO 6) together with modular platform for development of medical diagnostic technologies to ensure controllable and adaptable measurement environment. A small preparation lab enables final modification of the samples before optical measurements.

Fluorescence microspectroscopy with optical tweezers

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The home-made modular spinning-disk-based confocal fluroescence microscope with the ability of spectral detection for fast spectrally resolved imaging of environmentally sensitive probes, structures and interactions on the scales around 200 nm with spectral resolution of 1 nm. NIR optical tweezers enable contactless micro-manipulation.

teaching

Molekularna biofizika

Univerza v Ljubljani, Fakulteta za farmacijo, Laboratorijska biomedicina, MSc.

Predmet se izvaja v zimskem semestru 1. letnika študijskega programa Laboratorijska biomedicina (II. bolonjska stopnja) na Fakulteti za farmacijo Univerze v Ljubljani.

team