Research

Biophysical Interactions and Sensing Lab

Group Leader and Principal Investigator: Isabel Llorente Garcia (Lecturer).

Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, UK.

Biological Physics Group (BioP)
Atomic, Molecular, Optical and Positron Physics Group (AMOPP)

Summary of research activities

Our group is interested in measuring molecular interactions in biological systems using of single-molecule fluorescence microscopy and force spectroscopy and through the development of new techniques based on the manipulation of microparticles in solution via magnetic, optical and electrical methods. Additionally, we work on the development of biosensors for the detection of very small concentrations of biomolecules in solution.

Main research topics:

– Manipulation of graphitic microparticles in solution using electric and magnetic fields. The aim is to develop new tools to: i) measure force and torque at the single molecule level in biological systems and ii) achieve controlled positioning and transport of micro-particles in solution for lab-on-a-chip applications.

– Investigation of molecular interactions relevant to virus entry into living cells via single molecule force spectroscopy and fluorescence microscopy, in collaboration with Prof. Mark Marsh (UCL MRC Laboratory for Molecular Cell Biology);

– Development of novel microscopy techniques: combined light-sheet fluorescence microscopy and dual-beam optical tweezers for force-sensing;

– Investigation of the role of physical forces in antigen extraction by B cells, in collaboration with Dr. Pavel Tolar (Francis Crick Institute).

– Development  of optical whispering-gallery-mode biosensors for early diagnosis of disease, in collaboration with Prof. Peter Barker and Dr. Lia Li (UCL Physics and Astronomy).

– Development of custom suites of image processing algorithms for single-particle tracking experiments, including tracking of single molecule fluorophores in living cells, rotational tracking of magnetic particles, etc.

Research topics

Manipulation of graphitic microparticles in solution using magnetic and electric fields

We aim at controlling the position and orientation of these particles in solution, developing rotational and translational traps for torque sensing and force sensing, respectively, and devising transport mechanisms for the microparticles.

For this work, we collaborate with Prof. Sonia Contera (Oxford Physics) on the fabrication of lipid-coated graphitic microflakes and on the analysis of their properties via atomic force microscopy (AFM).

Magneto-electrical orientation and rotational trapping

We have devised a magneto-electrical method for rotationally trapping and controlling the orientation of lipid-coated graphitic microparticles in solution:

Magneto-electrical orientation of lipid-coated graphitic micro-particles in solution, RSC Advances (April 2016), by J. Nguyen, S. Contera, and I. Llorente García.  DOI: 10.1039/C6RA07657B. http://pubs.rsc.org/en/content/articlelanding/2016/ra/c6ra07657b#!divAbstract

This paper presents a novel method for manipulating anisotropic, lipid coated- biocompatible graphitic micro-particles in aqueous solution. The method is based on the application of two perpendicular fields: a vertical static magnetic field and a horizontal time-varying electric field oscillating at fast frequencies (MHz). We generate rotational traps to confine the orientation of the micro-particles parallel to the plane containing both fields and use measurements of random orientational fluctuations of the particles due to collisions with water molecules to calibrate the strength of the traps and measure the torque exerted on them, as well as to measure their dependence on the frequency of the applied electric field.

In general, the controlled orientation and manipulation of carbon-based micro- and nano-particles such as graphite/graphene platelets and carbon nanotubes submerged in aqueous solution is of relevance to a number of scientic and technological challenges. We are particularly interested in the manipulation of individual graphitic micro-akes for the development of new single molecule probes for sensing biologically relevant torque. Also of interest are biological and chemical sensing and controlled uid mixing in microuidic devices for lab-on-a-chip applications, as well as applications for opto-electronic devices.

Our scheme can be applied to other carbon-based micro/nano-particles, such as graphene platelets or carbon-nanotubes, has great potential for being scaled down via micro-fabrication, and can open up new ways for trapping, transporting and separating these particles with the use of time-varying fields enabling frequency-control of the magneto-electrical manipulations.

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Schematic of magneto-electrical orientation of HOPG micro-flakes represented as oblate ellipsoids. (a) HOPG particle rotation upon application of a vertical magnetic field (B0). (b) HOPG particle rotation upon additional application of a horizontal electric field (E0).

alignedHOPGflakes
Microscope brightfield (transmission) images of lipid-coated HOPG micro-flakes: (a) with no applied fields; (b) vertically aligned in the presence of an applied vertical magnetic field; (c) sequence of rotation around a vertical Z axis upon turning on a horizontal electric field oscillating at 30 MHz.

       
Left: Tracked orientation angle of graphitic microflakes at different AC electric field frequencies as the particles rotate to align with the field. Right: orientational Brownian fluctuations of graphitic microflakes in magneto-electrical rotational traps. Stronger rotational trapping (lower spread of fluctuations) is observed for higher electric field frequencies.

       
Angular mean squared displacement (MSD) (a) and autocorrelation (b) for the orientational fluctuations of graphitic microflakes in magneto-electrical rotational traps.

Calibrated measured rotational trap stiffness (the maximum torque shown equals 1/2 of the rotational trap stiffness) in magneto-electrical rotational traps for 10 different graphitic microflakes in solution.

See videos of particle rotation in the Supplementary Info for the paper here.

Additionally, we have developed the theoretical framework for the AC electro-orientation of lipid-coated graphitic microflakes:

Orienting lipid-coated graphitic micro-particles in solution using AC electric fields: A new theoretical dual-ellipsoid Laplace model for electro-orientation, J. Nguyen, Jonathan G. Underwood and I. Llorente García. Colloids and Surfaces A: Physicochemical and Engineering Aspects 549, 237-251 (2018). https://doi.org/10.1016/j.colsurfa.2018.02.032

Electro-orientation of layered ellipsoid (lipid-coated graphitic microflake) in a horizontal, linearly polarised AC electric field in solution.

    

Left: Calculated real parts of the complex effective polarization factor (K) along the in-plane (x) and out-of-plane (z) directions of the graphitic particle for the dual-ellipsoid model developed in our paper and in comparison to calculations for a simple one-ellipsoid model. Right: Calculated maximum torque versus electric field frequency for both models.

Example of fit of measured data to theoretical model for the maximum electro-orienting torque on a graphitic microflake in solution.

 

Investigating molecular interactions relevant to virus entry into living cells

This project is in collaboration with Prof. Mark Marsh [UCL MRC Laboratory for Molecular Cell Biology (LMCB)].

Viral infections remain a serious threat to human health. For instance, the Human Immunodeficiency Virus (HIV) infects approximately two million people a year and kills a similar number of people, despite the development of effective anti-retroviral drugs.

The cell membrane is the main barrier that viruses need to overcome to penetrate living cells and cause disease. As part of their entry strategy, viruses interact with specific receptor proteins at the cell surface in ways which are not fully understood. Cell-surface receptors are nanometre-sized macromolecular protein complexes typically embedded in the plasma membrane of the cell. These proteins can enact communication between the cell interior and the cell’s surrounding environment and they can move randomly (via Brownian diffusion) in the membrane plane. Physical properties of cell-surface receptors such as their mobility and interaction with the cellular cytoskeleton (a mesh of filaments beneath the cell membrane) may importantly influence virus entry events. However, our knowledge of these receptor properties and their role in virus entry is currently very limited.

HIVentry3

HIV-1 entry into an immune cell via the interaction with receptors CD4 and CCR5/CXCR4.

We focus on the Human Immunodeficiency Virus (HIV) as a model system, given that the specific cell-surface receptors for HIV entry have been identified and are the increasingly well understood cell-surface receptors CD4 and CCR5/CXCR4. HIV particles first attach specifically to CD4 and CCR5/CXCR4 molecules on the surface of cells of the immune system (see Figure above). These receptors then redistribute and accumulate at the sites of virus attachment on the cell surface. Eventually, the virus penetrates the cell membrane and releases its genome into the cellular cytoplasm.

Our research work investigates the molecular interactions necessary for receptor-mediated virus entry into living cells by means of precision force-sensing experiments (force spectroscopy with optical tweezers) and fluorescence microscopy at the single molecule level. Our results will likely be applicable to other virus-receptor systems that exhibit similar entry mechanisms to HIV.

 

New Microscopy development

Light-sheet fluorescence microscopy and optical tweezers for force sensing

We have built an advanced microscopy platform that combines optical tweezers with two-colour, light-sheet fluorescence imaging. This platform enables force sensing at the femto-picoNewton level together with quantitative single-molecule fluorescence microscopy, featuring nanometre spatial resolution and millisecond temporal resolution.

IMG_20160602_180515247_HDR      IMG_20160602_180316848

 

Microscopy platform photos: details of optics in the setup (first 6 photos), general view of setup, sample area with objective and nanopositioning stage inside temperature-controlled box for experiments with living cells, colour splitter optics to separate fluorescence emission wavelengths on EMCCD camera sensor.

 

Light-sheet fluorescence microscopy

Molecular complexes that perform vital functions in living cells can be labelled with fluorescent tags that emit light when illuminated by the appropriate excitation laser light. Using fluorescence microscopy to image the cells then provides valuable information about the location, number and distribution of these complexes in the cell. When carried out in living cells, this technique allows dynamic monitoring maintaining the native biological context and functionality in the cell.

Example of fluorescence microscopy image: NIH-3T3 M22 mouse fibroblast cells with CD4 receptors labelled with red fluorescent Alexa568 and Lck labelled with green fluorescent Alexa 488. Nuclear DNA is labelled in blue (Hoechst 33342).

Fluorescence microscopy of tagged complexes in live cells in our lab can be performed with a spatial resolution (diffraction-limited) of about 300 nanometres (for visible light) and down to millisecond time scales, resulting in acquired videos that will help us elucidate the mechanisms and functions of the biological complexes of interest. Image processing of these videos and single-particle tracking can then lead to a localisation precision at the nanometre level. Fluorescence excitation laser light is delivered from the side to the sample in the form of a thin light-sheet that illuminates the top membrane of the cell. The fluorescence emission from the labelled molecular complexes is collected by either the top of bottom objective and reaches an EMCCD camera to form a two-colour fluorescence image.

Left: Schematic diagram of cell illuminated by laser light-sheet with cylindrical lens on the sample side. The top/bottom objectives collect the fluorescence emission. Right: Measured light-sheet intensity as aid camera is scanned along light-sheet axis.

Custom-made sample holder design for light-sheet fluorescence microscope.

 

Optical tweezers for single molecule force spectroscopy

Optical tweezers allow controlled trapping of a micro-bead in solution in a near-harmonic, three-dimensional optical trap. Once calibrated, the trap essentially behaves like a spring and obeys Hooke’s law (F=-k Δx), so that the measured/applied optical force (F) is proportional to bead displacement (Δx) away from the trap centre via the trap stiffness constant (k) obtained in the calibration. In this way, measurements of bead displacement translate into force measurements. Our optical tweezer system features nanometre-precise detection of bead and sample position (via back-focal-plane interferometry), force resolution at the fempto-picoNewton level and millisecond time resolution for fast force sensing.

Three-dimensional positions of a 1µm-diameter bead confined in the optical trap (~120mW, 1064nm) measured with nanometre precision via back-focal-plane interferometry using a quadrant photo-diode.

Interrogating receptor-ligand bonds with force in living cells

A single-molecule force spectroscopy experiment near the surface of a living cell is described as follows: a polystyrene bead (~1 micrometre in diameter) is first functionalised for specific attachment to a biological complex of interest (e.g. a cell-surface receptor protein) in a living cell; the bead is then be trapped with optical tweezers above the cell surface and brought close to the cell controllably until contact with the cell membrane is made and attachment to individual receptor molecules is achieved; the optical trap is then be moved relative to the cell sample to pull the receptor molecule while the applied forces are measured; experiments can be performed at different pulling speeds with the idea of detecting the small forces required to break molecular bonds, with the aim of uncovering molecular interactions relevant to the function of receptors such as, for instance, attachments to nearby molecules in the cell. The images below show schematics of the experiments and example measurements taken on our microscopy platform when pulling on CD4 receptor proteins on the surface of mouse fibroblasts.

 

Left: Polystyrene microsphere coated with a low density of anti-CD4 antibodies for attachment to CD4 receptors in optical tweezer force pulling experiments. Right: Schematic of sequence of events and measured force versus time when measuring receptor-ligand bond ruptures in living cell membranes.

 Examples of measured pico-Newton-level force traces when vertically pulling anti-CD4 antibody coated beads attached to CD4 cell-surface receptors on the surface of mouse fibroblast cells for virus entry investigations. Marked jumps correspond to unbinding events requiring vertical forces in up to ~10pN.

Measuring local cell-membrane mechanical stiffness (pN/µm)

The local mechanical stiffness of the cell membrane can also be measured using optical tweezers. These measurements can help elucidate the role of the physical and mechanical properties of the membrane locally influence biological function. For instance, the expression of certain proteins in cellular membranes can affect the stiffness of the membrane and influence events such as virus entry or cargo uptake.

Top: Schematic of optical tweezers experiments to measure local cell-membrane stiffness by indenting the cell membrane with an optically trapped bead. Bottom left: Measured motion of the sample stage (substrate), bead position relative to the trap centre, cell-membrane deformation and force on the cell membrane. Bottom right: Analysis of the indentation (push) region and fit of force on the membrane versus membrane deformation to obtain local membrane stiffness.

Left: Examples of various measured indentation traces with optical tweezers on cellular membranes. Right: Example histogram of measured cell-membrane stiffness values over many repetitions in fibroblast cells.

The above mentioned techniques are broadly applicable to various biomedical and biophysical problems that involve cell-surface receptors and are important to human health such as, for instance, receptor-mediated virus entry, cell growth in cancer, immune response to infections and neuronal activity. They are also applicable to measuring ligand binding strength and bond lifetime for drug discovery applications.

Investigation of the role of physical forces in antigen extraction by B cells

This project is part of Dr. Llorente-Garcia’s sabbatical attachment at the Francis Crick Institute in 2016-2017, in the group of Dr. Pavel Tolar. The collaboration with the Tolar group is still active and we share the supervision of a PhD student.

The aim of this work was to investigate the role of physical forces in antigen extraction and internalisation by B cells.

Physical forces, mechanical properties and binding affinities play a crucial role in the processes involved in the immune response of white blood cells to infection by pathogens. Crucial to this, is the specific recognition of pathogen-derived molecules by specialised molecular complexes on the surface of immune cells. We are investigating the role of physical forces in controlling these recognition mechanisms in B lymphocytes (a type of white blood cell in our immune system). To do this, the aim is to carry out magnetic-tweezer experiments in living cells to measure receptor-ligand binding.  Our results will ultimately inform the development of new methods for generating protective antibodies by vaccination.

antigenExtractionByBcell_endocytosisForce2.png

The Francis Crick Institute is a unique partnership between the Medical Research Council, Cancer Research UK, the Wellcome Trust and 3 Universities (UCL, Imperial College and King’s College). The Crick aims to play a key role in understanding the causes of disease and to find new ways to prevent or treat serious illnesses.

https://www.ucl.ac.uk/slms/newsletter/internal/13.10.15#

 

Development  of optical whispering-gallery-mode biosensors for early diagnosis of disease

Our aim is to develop sensitive and reliable diagnostic tools for the diagnosis of disease. In collaboration with Prof. Peter Barker and Dr. Lia Li (UCL Physics and Astronomy), we are developing innovative fluid biosensors based on ultrasensitive whispering-gallery-mode (WGM) optical detection of specific macromolecules in solution to go beyond the precision and sensitivity of current standard analysis techniques. The development of minimally-invasive, routine diagnostic tests for blood, urine, saliva and/or tear body fluids is of key importance to disease diagnosis. Our initial focus is on the diagnosis of neurodegenerative conditions such as Alzheimer’s disease.

 

Left: Microsphere-cantilevers fabricated in Peter Barker’s lab. Right: Optical fibre close to the microsphere to couple light onto a WGM resonance. A dip in light transmission through the fibre is seen when tuned to the WGM resonance. The resonance wavelength shifts when molecules bind to the microsphere surface, enabling protein detection.

 

Development of single-particle-tracking image processing algorithms

We develop custom-made algorithms for single particle tracking for translational and rotational motion and for single molecule fluorescence experiments.

Example of results from rotational tracking algorithms: quantitative extraction of particle rotation angle and frequency via image processing. Top: Sequence of image frames acquired for a polymeric magnetic micro-ellipsoid rotating in a rotating magnetic field (counter-clockwise, see yellow arrow). Yellow lines indicate particle orientation and green crosses indicate centre-of-mass. Bottom: Corresponding orientation angle versus time and fit of post-processed angle to obtain the particle rotation frequency.

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