Using new methods of multiphoton microscopy to examine molecular organization in fibrillary assemblies of macromolecules

Professor Sophie Brasselet visited the Biophysics Group within the Physics Department at the University of Exeter during the academic year 2016–2017. The activities covered different topics related to bio-imaging, where Prof Brasselet contributed her knowledge in microscopy imaging and more particularly the use of light polarization. The interaction with the staff at the host institution has been extremely fruitful in both directions.

Project 1: Structural imaging of the fibrous proteins of the extracellular matrix 

Multiphoton imaging and spectroscopy is a major tool in research into the physical properties of the biopolymers which form the extracellular matrix, providing insights into changes in these properties which may underlie diseases as diverse as atherosclerosis, osteoarthritis and diabetes. Our aim, during Prof Brasselet’s visit, was to develop knowledge of the structural organisation of elastin and collagen fibres, which play an essential, complementary role in the mechanical properties of biological tissues. In particular, we wanted to understand the relationship between the molecular scale organization of these proteins and their elastic properties. To help us do this, we used a new imaging technique, initially developed at Marseille, based on the control of light polarization. Prof Brasselet’s implementation of polarisation-sensitive measurements yielded unexpectedly rich insights (Fig. 1). We are preparing this work for publication, but in addition this work is forming the basis of a younger colleague’s fellowship application, and a component of a major project in optometry at the University of Cardiff.

Fig. 1 (a) Principle : the polarization rotation induces a signal modulation from which we deduce the angular disorder. (b) Zooms depict sticks (p, mean orientation of emission dipoles for each measured pixel) which color represents the local molecular order (S2) in elastin embedded in water (left) or in ethanol (right). (c) Similar measurement on a bovine tendon under 25% strain. 

Project 2: Brillouin microscopy as a tool for probing fibre and tissue mechanics

Prof Brasselet worked with Dr Francesca Palombo, a specialist in vibrational and infra-red spectroscopy in biological tissues, to implement Dr Palombo’s plans for a novel Brillouin microscopy system. (Brillouin microscopy makes use of the interaction between light and material waves to determine the mechanical properties of the medium.) Together, Prof Brasselet and Dr Palombo built a microscope from scratch which allowed the recording of Brillouin signals in biological samples, opening new possibilities for mechanical and structural investigations. Prof Brasselet’s long experience of optical engineering was invaluable in this process and she has left Exeter’s Physics Department with a facility which is at present unique in the UK and promises to be a focus both for fundamental research on biopolymer physics and, we hope, the development of new tools for clinical investigation.

Fig. 2 Brillouin microscopy established at Exeter University. (a) Principle : the optical excitation couples with phonon modes in the matter to provoke inelastic scattering with frequency shifts proportional to its longitudinal modulus. The implemented set-up is based on epi-microscopy, which allows future imaging modalities. (b) Measured specrum in water (left), extracted from a spectrum projected in a sCMOS camera based on two virtual imaging phase array (VIPA). 

Project 3: Novel solutions for fluorescence microscopy in scattering media 

Prof Brasselet collaborated with Prof Christian Soeller, who uses super-resolution tools for biological imaging, and Dr Jacopo Bertolotti, a specialist in imaging methods in scattering media such as biological tissue (where light encounters nanoscale refractive-index inhomogeneities and so “scatters”, making it hard to obtain a clear image). Together, the three developed a new set-up to image fluorescent objects placed behind a scattering medium, using a standard epi-wide field microscope (Fig. 3). 

Figure  3. (a) Inverted microscope in wide-field mode. (b) Scheme of the excitation and fluorescence light diffusion. (c) Fluorescent object consisting of deposited micrometer beads, imaged without diffuser (d) Normalized scattered light detected below the diffuser. (e) Windowed autocorrelation of (d). (f) Reconstructed object. 

While both of the first two projects were outlined in our original application, we had not anticipated this third one on scattering media. This came about largely through casual conversations which brought together two of my colleagues and resulted in a manuscript which has significant implications for super-resolution microscopy, which is a major field of research in Exeter, and in other areas of optics.

All three projects will be of enduring benefit to Exeter’s Biophysics Research Group and collaborators throughout the University and beyond.

In addition to this hands-on research, Prof Brasselet interacted widely with our own undergraduates to whom she gave a series of introductory lectures on biomedical optics; graduate students to whom she gave a series of seminars; and with new staff appointed to the Living Systems Institute, researching and teaching at the interface between physical and biomedical science. These activities will have a formative influence on our future teaching plans. During the year, Prof Brasselet also promoted the research being undertaken at Exeter with presentations at the University of Cardiff; the Institut Curie, Paris; Université Paris Sud; and a plenary conference at the Physics French Society Meeting.

Prof Brasselet’s visit leaves the Physics Department at Exeter a legacy of novel tools for biophysical research. Together, members of the Biophysics Research Group and Prof Brasselet have discussed many new problems, which we hope will form the bases of continuing collaborations.

Publications in preparation
M. Hofer, C. Soeller, S. Brasselet, J. Bertolotti. Speckle correlations enable fluorescence imaging behind scattering media in wide-field epi microscopy.
S. Brasselet, E. Green, J. Mansfield, P. Winlove. Molecular order in elastin probed by polarized 2-photon fluorescence microscopy.
S. Brasselet, J Mansfield, P Winlove. Polarisation second harmonic microscopy as a probe of micromechanics.

Professor Sophie Brasselet
Aix Marseille Université
Visiting Professorship

Professor Peter Winlove
University of Exeter