Southampton Bio Imaging Symposium

Friday 6th October 2017

Sponsored by ZEISS

Hosts: The event is proudly hosted by Dr Dave Johnston, Biomedical Imaging Unit (BIU), University Southampton Hospital, and Prof Sumeet Mahajan, The Institute for Life Sciences (IfLS) of the University of Southampton.

Aim of the meeting: This bio imaging symposium aims to bring together distinguished life science researchers from universities across England to share and discuss their research. The day, made up of numerous talks from well-respected speakers, will show how various imaging technologies have enabled biological breakthrough in recent years.

Agenda

10:00 - 10:20 Registration, tea/coffee 10:20 - 10:25 Welcome speech by Prof Sumeet Mahajan, Institute for Life Sciences,

University of Southampton and Dr Dave Johnston, Biomedical Imaging Unit, University of Southampton and University Hospital Southampton

Session 1 Chair: Sumeet Mahajan 10:25 - 10:55 Dr Gopi Shah, CRUK, University of Cambridge;

‘Light sheet microscopy: Prospects and challenges of high-speed volume imaging’

10:55 - 11:25 Dr Simon Lane, Institute for Life Sciences, University of Southampton; ‘The Spindle Assembly Checkpoint in mouse oocytes, a live cell imaging approach’

11:25 - 11:55 Dr Stephen D. Thorpe, Queen Mary University London; ‘Super-resolution structured illumination microscopy: a versatile tool resolving primary cilium structure and protein interactions in the nucleus’

11:55 - 12:25 Dr Verena Kriechbaumer, Oxford Brookes University; ‘The “new nature” of the plant endomembrane system with high resolution Airyscan imaging’

12:25 - 13:30 Lunch, discussion Session 2 Chair: Dave Johnston 13:30 - 14:00 Dr Claire Jackson, University of Southampton and University Hospital

Southampton PCD group; ‘Diagnosis of Primary Ciliary Dyskinesia by Microscopical Methods’

14:00 - 14:30 Prof Sue Vaughan, Oxford Brookes University; ‘Using SBF-SEM to investigate cell cycle changes in organelle distribution in Trypanosome brucei’

14:30 - 15:00 Prof Rohan Lewis, University of Southampton; ‘Multiscale three dimensional imaging of the human placenta’

15:00 - 15:30 Coffee break Session 3 Chair: Katherine Lau 15:30 - 16:00 Dr Ulrich Sauer, Carl Zeiss Microscopy;

‘Laser Microdissection – bridging the gap between Microscopy and Molecular Analysis’

16:00 - 16:30 Dr Peter Gordon, Kings College London; ‘A multi-modal, multi-scale imaging approach to visualising immune interactions in the tumour draining lymph node’

16:30 - 17:00 Prof Sumeet Mahajan, Institute for Life Sciences, University of Southampton; ‘Multimodal Non-Linear Label-Free imaging for Biomedical Applications’

17:00 - 17:05 Closing speech, Dr Katherine Lau, Carl Zeiss Ltd, Microscopy

10:25 - 11:25 Light sheet microscopy: Prospects and challenges of high-speed volume imaging

Gopi Shah, CRUK Cambridge Institute, University of Cambridge Light sheet microscopy is one of the fastest fluorescence imaging technologies available today. In the last decade, it has emerged as an ideal technique for visualizing biological processes occurring at various time and length scales: rapid three-dimensional processes such as the beating zebrafish heart can be captured at >400 frames/sec, large samples such as the developing zebrafish embryo (~0.7-1mm) can be imaged in toto at high resolution through multi-view imaging and delicate samples such as in vitro cultured 3D organoids can be monitored over days owing to its non-invasive nature. Nonetheless, the ever-increasing speed and resolution of such modern microscopes make the storage, analysis and visualisation of image data challenging. To address this, we have developed customised light sheet microscope with real-time image-processing engine that projects the 3D image volume onto a 2D map, reducing the data generated as well as providing a panoramic view of the sample. In my talk, I will discuss this and other multi-dimensional imaging approaches, which integrate whole-embryo live imaging, genetic information and analysis of an ensemble of specimen to understand how large-scale tissue movements shape various morphogenetic processes.

10:55 - 11:25 The Spindle Assembly Checkpoint in mouse oocytes, a live cell imaging approach

Simon Lane, Institute for Life Sciences, University of Southampton Oocytes, along with sperm, are the key cells that allow us to reproduce. Since the genetic material is passed on through these two cells alone it is critical that they have the correct number of chromosomes, each contributing precisely half of the full set. Strangely in the oocyte it has been found that they are poor at dividing their chromosomes, often leading to eggs with incorrect chromosome compliments. Here I show how 3D confocal timelapse imaging in oocytes has revealed the behaviour of the chromosomes and the Spindle Assembly Checkpoint (SAC) that guards against chromosome division errors. I investigate the effect of oocyte volume on the SAC and show that the SAC has the unusual property of detecting DNA damage in oocytes.

11:25 - 11:55 Super-resolution structured illumination microscopy: a versatile tool resolving primary

cilium structure and protein interactions in the nucleus

Stephen D. Thorpe, Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London, London, UK Recent technological advances in optical microscopy have facilitated the study of cellular organisation in both fixed and live cells at a resolution below the diffraction limit of light (~200 nm). The three most widespread super-resolution techniques are structured illumination microscopy (SIM), stimulated emission depletion (STED) and photoactivation localisation microscopy/stochastic optical reconstruction microscopy (PALM/STORM). Research in our lab has utilised SIM to provide insight into the organisation, structure and localisation of cellular structures and proteins. SIM involves illumination of the sample with spatially patterned light. The information contained in these interfering Moiré patterns is used to extract details in the image which are too small to be resolved by the microscope, increasing the xy resolution by a factor of 2. I will present data from our lab where SIM has facilitated observation of features that escape detection by conventional microscopy. First, in characterising the organisation of, and protein interactions within, the primary cilium—a tubulin-based structure approx. 200 nm in diameter involved in many biological signalling processes—SIM allowed us to resolve the specialised plasma membrane on each side of the cilia separately from the microtubule axoneme within. We have also used 3D-SIM to examine chromatin-modifying histone-protein interactions within the nucleus of differentiating stem cells and chondrocytes. These studies illustrate the versatility of SIM as a super resolution technique which retains the sample preparation methods, ease of use and flexibility of conventional microscopy.

11:55 - 12:25 The ‘new nature’ of the plant endomembrane system with high resolution Airyscan

imaging

Verena Kriechbaumer, Biological and Medical Sciences, Oxford Brookes University, Oxford The plant early secretory pathway comprises the endoplasmic reticulum (ER) and the cisternal stacks of the Golgi apparatus. The ER is a multifunctional organelle involved in a plethora of aspects of plant life. The polygonal network of the cortical ER consists of motile tubules that are capable of morphing into small cisternae, mainly at the three-way junctions of the ER network. Plant reticulon family proteins (RTN) for example tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. A novel family of Lunapark proteins on the other hand is involved in ER sheet formation. Transport between ER and cis-Golgi cisternae is best characterised on ER exit sites, specialized regions of the ER where secretory proteins are concentrated and leave the ER for export to the Golgi bodies. High resolution Airyscan imaging delivers new aspects and raises new question in regards to substructure of and dynamics between ER tubules and sheets, Golgi and ER exit sites.

13:30 - 14:00 Diagnosis of Primary Ciliary Dyskinesia by Microscopical Methods

Claire Jackson1,2, Janice Coles1,2, James Thompson1,2, Patricia Goggin1, 3, Jane Lucas1,2

1 Primary Ciliary Dyskinesia Centre, University Hospital Southampton NHS Foundation Trust and Clinical and Experimental Sciences Academic Unit, University of Southampton Faculty of Medicine, Southampton, UK; 2

NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK; 3 Biomedical Imaging Unit, University Hospital Southampton NHS Foundation Trust, Southampton, UK. http://www.uhs.nhs.uk/OurServices/Childhealth/Primary-ciliary-dyskinesia/pcd.aspx

Primary Ciliary Dyskinesia (PCD) is a rare inherited condition affecting motile cilia. Approximately 1:15,000 (higher in consanguineous populations) of the UK population are affected and Southampton is a national centre. Patients with impaired mucociliary lung clearance suffer recurrent and chronic upper and lower respiratory tract symptoms from birth and an early diagnosis is important for their accessing care. There is no ‘gold standard’ PCD test, and genetic testing is not yet sensitive enough. Therefore a multidisciplinary diagnosis is made on strong clinical history and combinations of specialist tests, which include several microscopical methods (Figure 1): high-speed video microscopy (HSVM), transmission EM (and tomography in difficult cases), and multichannel confocal fluorescence that we will discuss. Microscopic methods are key to PCD diagnostics, complimentary to genetic testing for disease phenotyping, and are continually evolving through research.

Figure 1: Microscopical methods of cilia imaging

A} HSVM B) TEM C) SEM

D} Multichannel confocal fluorescence

14:00 - 14:30 Using SBF-SEM to investigate cell cycle changes in organelle distribution in

Trypanosome brucei Sue Vaughan, Prof. of Cell & Molecular Biology, Trypanosome Cell Biology Group Leader, Oxford Brookes University, OX3 0BP Oxford The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle.

14:30 - 15:00 Multiscale three dimensional imaging of the human placenta

Rohan Lewis, Professor of Placental and Integrative Physiology University of Southampton, Faculty of Medicine, Southampton The three-dimensional structure of the placental villi and the complex spatial relationships of the cells they contain are central to placental function. Multiscale imaging techniques including micro-CT, wholemount confocal, light sheet and serial block-face scanning electron microscopy now allow three-dimensional imaging of whole placentas and regions of placenta down to the nm scale. This talk will illustrate these approaches and how they can inform computational and molecular studies and advance our understanding of placental function. Using these techniques, we have identified structures and visualised cell-cell relationships which could not have been possible thin tissue sections. These approaches will help us to understand fundamental aspects of placental biology as well as the changes leading to placental dysfunction.

15:30 - 16:00 Laser Microdissection - bridging the gap between Microscopy and Molecular Analysis

Ulrich Sauer, Senior Application Specialist Light Microscopy, ZEISS Microscopy Customer Center Europe, Carl Zeiss Microscopy. Oberkochen, Germany Pure sample preparation is an essential precondition for reliable results in molecular research. Amongst various options to achieve homogeneous material, only non-contact LCM (Laser Capture Microdissection) offers high-resolution control of sample composition by selecting or rejecting individual cells. Tissue preparation and extraction protocols allow the use of micro samples for qualitative and quantitative molecular and proteomic analyses like, e.g., PCR and RT-PCR amplification and microarray analysis. The PALM MicroBeam from Carl Zeiss combines Laser technology with high quality robotic tools for precise microdissection of specimens, whilst the patented method of lifting up against gravity allows for non-contact collection with no impairment to the recovery of DNA, RNA or protein. The MicroBeam offers all standard wide-field imaging methods like phase contrast PlasDIC and multichannel fluorescence. The integration of image analysis software into the microscope fully automates screening, identification and finally high-throughput sample handling. Laser Microdissection is also the method of choice to isolate single specific, e.g. transfected cells out of a living cell culture for subsequent analysis or even for re-cultivation. This could be shown for a murine stem cell line. In this field LCM opens new approaches in establishing homogenous cell populations from adherent cell and tissue cultures in order to characterize and expand defined cell types.

16:00 - 16:30 A multi-modal, multi-scale imaging approach to visualising immune interactions in the

tumour draining lymph node Peter Gordon, Fabian Flores-Borja, Tony Ng, Division of Cancer Studies, Room 2.32 New Hunt's House, King's College London, London It is becoming more evident that interactions between adaptive and innate immune cells are a key determinant of cancer progression. It is therefore important to understand which cells and signalling molecules are involved in this process. The aim therefore was to develop and combine cell isolation and multiple imaging techniques, which allow us to look at specific cellular interactions and image at the macro and micro scale to obtain a total picture of the lymph node (LN) environment. Here we use flow cytometry to separate cell sub-populations of interest, such as type 3 innate lymphoid cells (ILC3) and transitional B cells (TBC) for labelling and subsequent transfer into a recipient mouse. We then utilise a titanium suction window for 2-photon in vivo imaging of the ILC3 and TBC kinetics/interactions in the draining inguinal LN. After in vivo imaging, the LN is dissected and, to analyse changes in architecture, the tissue is labelled whole mount for B cells and follicular dendritic cells (FDC), followed by optical clearance. This cleared tissue is imaged using optical projection tomography (OPT), creating a whole organ view of B cell follicles organisation and disruption along with tumour development. With the use of fiducial markers, areas of interest in the OPT images can be located for high resolution imaging on a confocal microscope, whereby the relationship between ILC3, TBC, B cell follicles and FDC can all be assessed at the single cell level. This combination of different methods allows both micro and macro analysis of immune interactions within the tumour draining LN. This approach will allow us to define specific cell types and cell markers allowing us to dissect the exact mechanisms to which therapies can be targeted.

16:30 - 17:00 Multimodal Non-Linear Label-Free imaging for Biomedical Applications

Sumeet Mahajan, Professor of Molecular Biophotonics & Imaging, Department of Chemistry & Institute for Life Sciences, Highfield, University of Southampton, SO17 1BJ. In this talk I will present our latest work on the development and application of multimodal non-linear label-free imaging (MNLI) approaches to image live cells, tissue and organisms. MNLI approaches allow understanding of biochemical processes in a non-perturbative and non-invasive manner. They are less phototoxic, allow deeper penetration and are much more amenable to 3D imaging than conventional optical techniques. In particular we have focussed on quantitative MNLI platform approaches that consists of novel and emerging techniques of coherent Raman microscopy (CRM) combined with the more established non-linear imaging techniques of second harmonic microscopy (SHM) and Two-photon excited fluorescence (TPEF).1,2 CRM allows imaging of lipids, proteins, glycans and other biomolecules while SHM allows imaging of collagen fibrils. TPEF can be used to perform functional imaging through the autofluoroscence of metabolites such as FAD and NADH. We have applied them to understand the effect of drugs in nematodes3 and to assess the development of bioengineered cartilage on differentiation of skeletal cells4. In context of the tumour microenviroment MNLI techniques allow the generation of a comprehensive picture of the extracellular matrix and understanding matrix-cell interaction phenotypes (see Figure). We have further developed quantitative and objective classification algorithms based on spectral and textural features for analysing multidimensional imaging data provided by MNLI. The development and application of MNLI approaches and tools that enable quantitative evaluation and objective analysis can have a widespread impact across biomedical research, to aid understanding and can provide a step change in stratification, digital histopathology and diagnostics.

References: 1. JP Smus, CC Moura,...S Mahajan (2015). Tracking Adipogenic Differentiation of Skeletal Stem Cells by Label-free Chemically Selective Imaging, Chem. Sci., 2015, 6, 2282-2286 2. Alan N Hunt, Anagha Malur, Tual Monfort, Pavlos Lagoudakis, Sumeet Mahajan, Anthony D Postle, and Mary Jane Thomassen, "Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis", Am J Respir Cell Mol Biol. 2017 May 10. doi: 10.1165/rcmb.2016-0242OC. 3. Justyna P. Smus, Elizabeth Ludlow, Nicolas Dalliere, Sarah Luedtke, Tual Monfort, Catherine Lilley, Peter Urwin, Robert J. Walker, Vincent O'Connor, Lindy Holden-Dye and Sumeet Mahajan, "Coherent anti-Stokes Raman scattering (CARS) spectroscopy in Caenorhabditis elegans and Globodera pallida: Evidence for an ivermectin-activated decrease in lipid stores", Pest Management Science 2017 DOI: 10.1002/ps.4707. 4. Catarina Costa Moura, Stuart A. Lanham, Tual Monfort, Konstantinos N. Bourdakos, Rahul S. Tare, Richard O. C. Oreffo, Sumeet Mahajan, “Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging” (communicated)

Figure: Preliminary data with multimodal non-linear label-free imaging (MNLI) and analysis. A) shows a tiled scan image of a tumour area on a HNSCC tissue sample. The composite image shows distribution of different chemical components imaged (red: lipids, green: proteins, cyan: glycans and magenta: collagen. B) shows the result of our segmentation algorithm using principal component and discriminant analysis incorporating texture parameters. The tumour areas (dark red) are segmented with high precision.

Important Information: Directions to the venue The symposium will be held at the Somers Lecture Hall at the The Centre for Human Development: Stem Cells and Regeneration. Full Address: Centre for Human Development, Stem Cells and Regeneration Faculty of Medicine University of Southampton Duthie Building (MP 808) Southampton General Hospital Tremona Road Southampton SO16 6YD https://www.southampton.ac.uk/medicine/research/ids/ids-location.page Due to limited parking available on site, please use public transport where possible. Travelling By Bus: The unilink U6 and U9 buses stop at Southampton General Hospital. Timetables and route maps are available on the unilink website. Travelling By Rail: From both Southampton Central Station and Southampton Airport Parkway, you can get the unilink bus U1 to Highfield Campus Interchange, where you can then get the unilink bus U6 to Southampton General Hospital (£3.50 for an all-day pass). Travelling By Car: Postcode for satnav: SO16 6YD Visitor parking: There is a multi-storey car park off Tremona Road at the northern end of the site

and further parking off Coxford Road. Disabled parking is by the main entrance, accessed via Tremona Road.

From the M3: exit at junction 14 (Southampton A33). From the M27 West: exit at junction 3 (M271 City Centre and The Docks). Pub reservation after the Symposium If anyone would like to continue the discussion after the symposium, we would like to welcome you to join us at the Malvern Pub about 15 minutes’ walk from the Southampton General Hospital. The full address for the Malvern Pub is: 290 Winchester Road, Southampton, SO16 6TU