Leeds integrated atomic force and confocal microscopy for life science applications

Lead Research Organisation: University of Leeds
Department Name: Physics and Astronomy

Abstract

Seeing is believing: imaging techniques have been instrumental for progress in biology since the inception of modern science. This project provides researchers at the University of Leeds with a unique instrument that integrates two types of advanced microscope for recording images and making mechanical and optical measurements at very small scales. With this capability, biological problems can be solved that have proven intractable with conventional techniques.

Optical microscopes (OMs), continuously developed since the time of Galileo, are now extremely powerful and versatile. Their ability to magnify enabled a clearer view of biological structures and this capacity remains much appreciated today to visualize live cells and the structures inside them. However, fluorescent tags that can absorb and emit light, and methods to measure the time between absorption and emission with exquisite resolution, have given OM a new dimension. With time-resolved fluorescence, the dynamics of biomolecular motion, the chemical environment of biomolecules, and the interactions and distances between biomolecules can now be studied in great detail.

Some fundamental limits remain, however. Physical properties such as mechanical forces and elasticity are also vital for the function of biological systems, from single biomolecules to cells and tissues, yet these are not accessible with optical microscopes. Since its invention three decades ago, atomic force microscopy (AFM) has emerged as a unique technique to directly measure mechanical properties at length scales down to individual molecules. Based on a tiny tip that scans across the sample, it is also able to produce height ('topographical') images with a resolution that is superior to that of optical microscopes, of less than one nanometer (1/1000 the size of a typical bacterium). AFM, as OM, can be applied in liquid environment and so biological samples can be probed alive. OM and AFM are complementary techniques, because they measure distinct physical parameters. Combining them into one device and characterizing the same sample at the same time with both techniques affords the ability to correlate data and gain new insight that cannot be obtained with either technique alone, or even with the two techniques applied separately one after the other.

On a larger scale, cells and tissues can be studied in unique ways. For example, we will characterize the structure and mechanical properties of the perineuronal net, an insulation sheath that covers the surface of neurons and modulates how neuronal connections form. Such insight may help to develop ways to delay memory loss in dementia or to repair spinal cord injury. On an intermediate scale, molecular assemblies can be probed, for example how blood clots are structured on the nanometer scale and what the mechanical properties of the individual fibres are that make up the clot. This will help to better understand thrombosis and its resolution, key to the prevention and treatment of heart attack. On yet smaller scales, we will study biological membranes, fascinating structures made from many loosely interacting molecules (lipids and proteins) forming an ultrathin film that is highly dynamic and key to cellular communication yet stable enough to compartmentalize cells and tissues. With the combined instrument, we will be able to study the structure and dynamics of biological membranes, for example to understand lipid organization (important for cell signaling), crystallization of membrane proteins (to facilitate their structural analysis, an important step for drug development), and how light-harvesting plant membranes work, for 'next-generation' bio-inspired energy generation methods.

Technical Summary

We are requesting funds for an integrated atomic force microscope (AFM) and optical microscope (OM) for life science applications at the University of Leeds. The new instrument seamlessly combines a time-resolved inverted confocal microscope with a high-end top-down AFM designed for work with biological samples. It will be unique to the UK and well-placed in our existing successful multi-user AFM facility. It will enable directly-correlated mechanical and topographical mapping of biological samples down to the level of single molecules (by AFM) with the spatial, dynamic and spectroscopic data accessible with state-of-the-art time-resolved OM. The particular AFM design offers low-noise/ high-stability scanning for high resolution measurements, fast-scanning for recording dynamic biological samples, sample environments and extended 3D scan range for imaging live cells and tissues, and 'quantitative imaging' for fast force and mechanical property mapping. The single-photon sensitive OM configuration enables fluorescence lifetime imaging for high sensitivity probing of molecular environments, Förster resonance energy transfer (FRET) analysis to detect molecular interactions and measure intermolecular distances, and fluorescence correlation spectroscopy (FCS) to determine molecular mobility, associations and related dynamics. This combination of specifications allows a step-change in our capabilities for probing challenging soft, dynamic, hydrated, biological samples. It will enhance a range of cross-disciplinary and cross-faculty research within BBSRC remit, including: Defining micromechanical and ultrastructural determinants of cell-matrix interactions on neurons, oocytes and immune cells, and of blood clots; Designing therapeutic microbubbles for targeting cancer cells; Understanding extremophile protein adaptation, bacterial protein assembly, photosynthetic membranes, membrane protein crystallization, biomembrane compartmentalization, and lipid membrane dynamics.

Planned Impact

The public demand to maintain a healthy population is becoming an increasingly complex challenge. The demographic ageing of the Western world is leading to increased rates of cancer and degenerative diseases. The change in our health needs is driving research into new ways of tackling disease states and conditions. The new instrument will give unique functionality to the researchers at Leeds to support the bioscience and biotechnological research that underpins the long-term development of new diagnostic, preventive and therapeutic methods and ultimately benefits public health.

Examples of health-related applications that will be facilitated by the proposed research are (i) new methods to modulate neuronal plasticity (relevant in ageing and dementia, and spinal cord injury), (ii) new materials for sperm selection in reproductive medicine (relevant for assisted reproduction, in humans and in agriculture), (iii) new methods for tumour cell targeting (www.microbubbles.leeds.ac.uk), (iv) new methods to control immune and inflammatory responses, and to resolve blood clotting in the vasculature, (v) new bioinspired hydrogels for biomaterial applications, and (vi) new methods to target bacterial infection. In addition, technological applications that may derive from the bioscience research are (i) bioinspired systems for 'green' energy conversion and (ii) compartmentalization methods that enable more efficient chemistries. From an economic perspective, these applications will benefit the growth of pharmaceutical, biotechnology and agriculture industries. From a societal perspective, they may impact in diverse ways on the improvement of public health and well being.

The new equipment interlinks several research groups with international reputation with their associated world-class and multi-disciplinary research facilities across the University of Leeds. Therefore, it supports an exceptional environment for training the next generation of scientists on cutting edge cross-disciplinary projects.

The University of Leeds runs regular outreach events such as science fairs for school children (Discovery Zone, www.stem.leeds.ac.uk/events/lfos) and public lectures and exhibitions of its research and technologies (Astbury Conversation, www.astburyconversation.leeds.ac.uk). The team will use these and other opportunities, to showcase the applicability of imaging techniques to challenging biological questions, and to teach and influence the public on the impact of our research on society and the related career opportunities.
 
Description This grant has provided funds for the aquisition of an instrument that combines atomic force and confocal microscopy. The device has become fully operational in 2018. It is now an important research tool at the University of Leeds. A number of research projects have started that are using the device. Among them, a few projects have already provided tangible scientific discoveries:

Bano, ..., and Richter (see publication in Biophysical Journal, 2018) have measured the unbinding forces between the extracellular matrix polysaccharide hyaluronan and its binding proteins at the single molecule level. Implications for the molecular mechanism of unbinding of hyaluronan-protein bonds under force are discussed, which underpin the mechanical properties of hyaluronan-protein complexes and hyaluoronan-rich extracellular matrices. Chen and Richter (see publication in Interface Focus, 2019) have used the device to study models of glycan-rich extracellular matrices which play an important role in many physiological (e.g. embryogenesis, innate immunity, reproduction) and pathological (e.g. inflammation, cancer) processes. In particular, the study showed that calcium ions are ten times more potent than sodium ions in affecting the properties of matrix models made from the polysaccharide hyaluronan.

Hancock et al (see publication in Nanoscale, 2019) have used the device towards the development of bio-hybrid light-harvesting nanomaterials.
Exploitation Route The findings may help to develop new methods to modulate physiological or pathological processes such as neuronal plasticity or new materials for sperm selection in reproductive medicine.
Sectors Energy,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Understanding hyaluronan crosslinking mechanisms in ovulation and inflammation: CryoEM structural and interaction analysis of HC-HA/PTX3 complexes
Amount £326,437 (GBP)
Funding ID BB/T001631/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2019 
End 10/2022
 
Description University of Leeds AFM Facility
Amount £136,770 (GBP)
Funding ID EP/R043337/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 10/2020
 
Title FLIM/AFM combined analysis method of model light harvesting membranes 
Description preliminary investigation has shown that Fluorescence Lifetime Imaging Microscopy (FLIM) and Atomic Force Microscopy (AFM) allows the quantification of energy transfers between photosynthetic proteins and synthetic dye molecules and the mapping of membrane topography. This allow structural information (nanoscale topographic mapping) to be related to functional information (quenching of fluorescence). This appears to allow the direct spatial correlation of nanoscale membrane arrangement with photo-protective energy dissipation in LHCII. This is expected to lead to future publication(s). 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact This tool can be used to directly correlate the organization and function of photosynthetic proteins or other molecular FRET pairs, i.e. their nanoscale membrane arrangement and their energy transfer. 
 
Title
Description  
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Description Industry collaboration - PepsiCo 
Organisation PepsiCo
Country United States 
Sector Private 
PI Contribution Dr. Simon Connel, Co-I on this grant, along with Dr. Josephine Orfila and Dr. Johann Mattson (both University of Leeds) have engaged with PepsiCo for a joint PhD project (funded by EPSRC and PepsiCo) that aims at developing new approaches to understand the role of the plant cell wall on potato processing and quality. The combined AFM/FLIM technology in essential tool in this ongoing project.
Collaborator Contribution Project supervision, provision of experimental samples, access to experimental plant processing equipment.
Impact This collaboration is multidisciplinary involving food science, plant biology and physics.
Start Year 2018
 
Description Industry collaboration - Tissue Tech 
Organisation Tissue Tech Inc.
Country United States 
Sector Private 
PI Contribution Dr. Ralf Richter, PI on this grant, has engaged in a collaboration with Tissue Tech for a research project which aims at developing new methods to study complexes of the extracellular matrix polysaccharide hyaluronan with proteins. The combined AFM/OM technology in essential tool in this ongoing project.
Collaborator Contribution TissueTech funded a postdoctoral research associate (6 months) and provided samples for this study.
Impact This collaboration is multidisciplinary involving extracellular matrix biology, biophysics and regenerative medicine.
Start Year 2018
 
Description Royce@Leeds PhD Winter School 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Half a day of AFM lecture, lab tour and demonstration with around 20 attendees from different universities in the UK.
Year(s) Of Engagement Activity 2020