Biophysical Interactions and Sensing Lab
Isabel Llorente Garcia (Lecturer)
Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, UK.
Summary of research activities
We carry out research on magnetic, optical and electrical methods to manipulate microparticles in solution with the main aim of investigating interactions in biological systems.
Main research topics:
– Manipulation of graphitic microparticles in solution using electric and magnetic fields, in collaboration with Prof. Sonia Contera (Dept. Physics, Univ. of Oxford); 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 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);
– New microscopy development: combined light-sheet fluorescence microscopy and 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).
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 just published a novel 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 in 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.
Our new method enables quantification of the response of the micro-particles to orienting fields in different experimental configurations for a broad range of applications. These include energy storage devices, opto-electronic devices, synthesis of artificial materials, biological and chemical sensing applications, fundamental studies in biological and medical physics, etc. 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.
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.
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.
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.
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.
An example of 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 preliminary measurements taken on our microscopy platform when pulling on CD4 receptor proteins on the surface of mouse fibroblasts.
Left: Thee-dimensional position 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. Right: Measured preliminary 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 the range 5-20pN.
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.
Investigation of the role of physical forces in antigen extraction by B cells.
The aim of this work is 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. In this sabbatical at the Crick, I will collaborate with Dr. Pavel Tolar to investigate the role of physical forces in controlling these recognition mechanisms in B lymphocytes (a type of white blood cell in our immune system). We will carry out magnetic-tweezer experiments in live cells which will ultimately inform the development of new methods for generating protective antibodies by vaccination.
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. Its new building next to the British Library by St Pancras station in London will open in late 2016.