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Biophotonics & Biomedical Microscopy

Wednesday 10th October - Theatre 4 - Jaguar Suite

The conference programme comprises presentations by invited speakers from academia, selected updates from industry and there will be a ‘poster session’ of contributed papers from early career scientists who are researching novel and exciting new techniques and unique applications - MORE>


09:00 Registration opens in the atrium
10:15 Welcome and opening remarks
Professor Sumeet Mahajan

  Chair: Professor Sumeet Mahajan
10:20 Quantitative 3D imaging of unconstrained model organisms using light field microscopy
Dr Mike Shaw, National Physical Laboratory, UK

Many advances in our understanding of biological systems and processes, from the function of specific genes and proteins to neural activation and signalling, have come from the study of model organisms. However, our ability to measure the properties and responses of these models is limited by conventional microscopy techniques which are unable to capture suitable 3D image information at sufficient speed.

In this presentation I will describe how light field microscopy, in which a microlens array enables simultaneous recording of both transverse spatial and angular information about image forming rays, allows fast volumetric imaging of the microscopic nematode C. elegans. By analysing light field images using computational depth estimation techniques originally developed for photography we are able to reconstruct and parameterise the 3D posture and motion of the organism.

We show how this approach can be used to investigate the behaviour of unconstrained C. elegans in 3D environments and quantify differences between mutant strains.

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10:40 Use of gentle 3D fluorescence imaging techniques – Lightsheet and Airyscan – to avoid photodamage and photobleaching in living biological samples
Dr Katherine Lau, Zeiss Microscopy, UK

The ability to image living biological samples presents significant advantages over end-point experiments. Biological processes can be observed over time using the same samples, rather than extrapolating answers based on snap shots of different samples at discreet time points. Apart from keeping the environment stable and resembling physiological conditions, the challenges of avoiding photobleaching and photodamage in living samples are well known. The key to success is the use of low light dosage together with a high sensitivity detection method. 

ZEISS Airyscan imaging is a patented superresolution technology, which simultaneously provides 4-8x increase in sensitivity and 1.7 times increase in (x,y,z) spatial resolution compared with confocal imaging. The high sensitivity enables the significant reduction in light dose for excitation, while the 120 nm lateral and 350 nm axial resolution help resolve intricate details in biological samples.

Lightsheet Z.1 is ideal for providing gentle and oblique illumination at sub-micron resolution. Only the sample plane being observed is illuminated avoiding unnecessary light exposure. The highly sensitive sCMOS cameras enable Z-stacks to be acquired at high speed. The sample can freely rotate to enable the optimal angle of imaging, or sampling from multiple angles, further improving the resolution in x,y and z.

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11:00 Detecting structural changes in the lipid bilayers of cells by light scattering
Dr Andrew Hudson, University of Leicester, UK

The talk will describe a measurement technique combining optical tweezing and light scattering that reports on rapid changes in the bending modulus and fluidity of vesicle lipid bilayers. As a consequence of these changes, we can make deductions concerning the formation, or disappearance, of heterogeneities in lipid-packing order.

Individual unilamellar vesicles are first isolated by optical tweezing and then, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of the lipid bilayer are revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers.

Observing the heterogeneities in lipid ordering has enabled us to provide information on how lipid-lipid and lipid-protein interactions change during the self assembly of protein oligomers in bilayers.

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11:20 In room break
  Chair: Dr Amanda Wright
11.30 Optical manipulation and sensing using machined chips & fibres
Dr Lynn Paterson, Heriot-Watt University, UK

There is a requirement from the biomedical and life sciences community to rapidly manipulate and detect single cells for the purposes of cell sorting and cell identification. We address this need using light.  Complex, buried, 3D microfluidic channels and integrated waveguide structures have been written in fused silica to generate a monolithic device capable of controlled particle manipulation. The device was fabricated using ultrafast laser inscription followed by selective chemical etching. Particles flowing through the device, such as microspheres and cells, are controllably manipulated first by hydrodynamic flow focusing then by the optical scattering force from the laser light emitted by the embedded waveguide. The device is capable of both passive and active separation of particle species, and the routing of particles to required outlets demonstrates potential for cell sorting.

SERS substrates have been laser-written within the channels and initial results show promise for particle identification. Multicore optical fibre has been machined using focused ion beam etching to create an optical tweezer on the end of the fibre enabling manipulation of single cells under any analytical microscope. Combining these approaches, we hope to create miniaturized cell sorters and sensors that will find many applications in the life sciences and as point of care devices.

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11:50 Spectral standing wave microscopy-contour mapping the cell
Mr Ross Scrimgeour, University of Strathclyde, UK

Standing Wave microscopy (SWM) is an axial super-resolution technique in which two counter-propagating waves interfere to produce planes of constructive (anti-nodes) and destructive (nodes) interference. Only fluorophores on a specimen whose axial location coincide with the anti-nodal planes undergo excitation and are spaced at regular intervals of λ/2n and provide an axial resolution of λ/4n [1], [2]. Previously, we have applied widefield SWM to generate a contour map of red blood cell membrane dynamics in real time with an axial resolution on the order of 90 nm [3]. More recently, we have extended the SWM technique through the application of multi-wavelength modalities to fluorescently-labeled eukaryotic cells adhered to a mirrored surface. As a result, depending on the wavelengths selected, we observe colour-coding which contains additional three-dimensional geometric information about the cellular structure encoded within the two-dimensional images. SWM can be easily implemented in a standard widefield epi-fluorescence and confocal microscopes with only the simple addition of a mirror to provide axial super-resolution.

References -
[1] B. Bailey et al., 1993 Nature366, p644 - 48
[2] Amor et al., 2014. Sci. Rep., 4, p7359
[3] Tinning et al., 2018 Biomed Opt Express., 9, p1745-1761

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12:10 Three minute presentations by presenters of posters.
12:30 Break
13:30 POSTER SESSION in exhibition hall (see below)
  Chair: Dr Brian Patton
14:00 Live cell imaging using a super resolution quantum diamond microscope
Professor Melissa Mather, University of Nottingham, UK

Speaker Biography
Melissa is a professor of Biomedical Imaging in the Optics and Photonics Research group, Faculty of Engineering, University of Nottingham. She carried out her postgraduate studies in the Centre for Medical and Health Physics, QUT. Following completion of her PhD she moved to the UK to take up a research position at the University of Nottingham. During this time, she worked on the development of sensing in monitoring techniques for use in Regenerative Medicine, and concurrently held a Strategic Research Fellowship in the Biomaterials group at the National Physical Laboratory.

In 2011 she was awarded an EPSRC Career Acceleration Fellowship and in 2013 was appointed as the Deputy Director of the Institute of Physics, Imaging and Optical Sciences, University of Nottingham. I

In August 2015, she moved to Keele University where she held the position of Professor of Biomedical Imaging in the Institute of Science and Technology for Medicine.

Melissa was awarded a European Research Council Consolidator Research Grant which commenced in September 2016. This fellowship provides five years of funding to develop a new technique to study transmembrane proteins in their natural state using optical detection of magnetic resonance. In August 2018 she returned to the University of Nottingham where she is currently working.

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14:30 Adaptation of Expansion Microscopy and DNA-PAINT to study the 3D molecular arrangements in healthy and failing hearts
Dr Izzy Jayasinghe, University of Leeds, UK
Signalling ‘nanodomains’ are small (50-300 nm) sub-cellular structures which specialise in evoking or relaying fast intracellular signals. These signals form some of the basic underpinnings of the human physiology (e.g. the heartbeat, muscle contraction, nerve communication and secretion of insulin in the pancreas). Nanodomains often comprise of intimately arranged ion channels embedded within the membranes of a compartment, regulatory proteins and various chemical groups which act as ‘molecular switches’. Often the intimate nature of the co-clustering of these components makes it impossible to understand the intra-nanodomain communication of these components, even with conventional super-resolution methods such as SIM, dSTORM or PALM. In this talk, I will outline how the adaptation of DNA-PAINT and Expansion Microscopy (ExM) have afforded us superior spatial resolution for examining nanodomains of the heart. With this improved vision, we have mapped the three-dimensionally complex topologies of these protein arrays and even the phosphorylation state of the principal calcium release channels within them – the Ryanodine receptors. Applying this method to examining healthy and failing hearts has revealed nanoscale spatial and chemical remodelling of the nanodomains which can explain the deficit in the intracellular calcium signalling that is observed in such pathologies. 

Sheard, T., White, E., Hurley, M., Hou, Y., Narayanasamy, K., Pervolaraki, E., Kirton, H.M., Norman, R., Yang, Z., Hunter, L., Shim, J., Steele, D., Colyer, J., Colman, M., Jayasinghe, I.
14:40 Microscopic evaluation of light-driven unidirectional molecular machines as targeted photodynamic therapy agents
Dr Robert Pal, University of Durham, UK

An important need for personalised therapeutics is the effective targeted in vivo destruction of selected cells and cell types, that are currently being highlighted by emergence of powerful optogenetic strategies. Using a new generation of light activated uni-directional molecular nanomachines (MNM) we have demonstrated their application to expedite cell death using single photon excitation in the UV domain (Nature, 548, 567, 2017).

We embarked to promote our “proof of principle” technology into in vivo biomedical applications using two photon (2PE) activation, as UV light activation in vivo has significant limitations associated with shallow tissue penetration and non-uniform excitation/activation, limiting the 3D precision required for therapeutics. This direction in activation not only allow deeper tissue penetration to realise in vivo photodynamic therapy (PDT) development, but also remove UV radiation as a confounder of biomedical efficacy. Since with 2PE MNM activation will only occur in a small truly diffraction limited 3D voxel it allows the next phase of targeted photodynamic therapy protocols and methods to be designed, as with careful chemical engineering of the MNMs cell type and target receptor specific binding can be facilitated with experimental therapeutic precision as small as a single cell. We demonstrated that by scanning a safe dose of NIR light in a 3D raster pattern for a predetermined period of time and repetition, only the surface bound MNM bearing cancerous cells are lysed, whereas all ‘healthy’ neighbouring cells remain intact and unaffected.

Once fully developed and validated 2PE activation of cell type and condition specific MNMs could be potentially adopted as a new form of extremely high optical precision, facile and non-invasive Type IV PDT for cancer treatment.

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15:10 Battling Big Data with correlative microscopy in bioimaging
Dr Martin Jones, Francis Crick Institute, UK

Several different microscopy modalities are widely used in biology, including fluorescence, electron and x-ray microscopy. Each of these gives different types of functional and structural information about a sample. New correlative workflows allow us to combine their outputs in ways that can provide new insights that are not accessible to the individual methods alone. By utilising the strengths of each method, we are able to perform more targeted imaging to reduce acquisition time and data footprint, as well as allowing us to more efficiently extract information from datasets that can reach into the terabyte regime.

Our facility has built several custom hardware systems to allow us to implement different correlative workflows to optimise our experiments for a range of scientific questions.

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15:30 Concluding comments and poster awards
15:40 Close of meeting
  The exhibition remains open until 17:00
1. Hyperspectral imaging investigation of lipid metabolism in C. elegans mutations via spectral interferometric polarized CARS
Priyank Shah, King's College London
2. Optimising superoscillatory spots for far-field super-resolution imaging
Konstantinos Bourdakos, University of Southampton
3. Label free super-resolution imaging with second harmonic generation microscopy
Peter Johnson, University of Southampton
4. Characterization of Hepatic and Renal Vasculature Using Optoacoustic Spectroscopy
Bill Whelan, University of Prince Edward Island
5. Lasers in microscopy: trends and challenges
Syed Asad Hussain, University of Oxford
6. Coherent Raman Imaging Approaches for Monitoring Intracellular Drug Actions
Jack Taylor, University of Southampton
7. 3D Histology & Deep Learning for Investigating Tumour Microvasculature
Natalie Holroyd, University College London

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Prof Sumeet Mahajan
Professor of Molecular Biophotonics & Imaging, University of Southampton

Dr Brian Patton
Royal Society University Research Fellow, Department of Physics, University of Strathclyde

Dr Amanda Wright
Associate Professor, Optics & Photonics Group, University of Nottingham