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Localisation Microscopy

22th March 2018.  
Susan Cox, University College London      

The 7th seminar of the series on Light Microscopy entitled Localisation microscopy will be given by Susan Cox of University College London on the 22nd of March at 2.00 pm in the College lecture theatre. The poster of the series can be downloaded here.

 

 

 

Lasers and Advanced Imaging

21th March 2018.  
Gail McConnell, University of Strathclyde       

The 6th seminar of the series on Light Microscopy entitled Lasers and advanced imaging methods will be given by Gail McConnell of the University of Strathclyde on the 21st of March at 4.00 pm in the College lecture theatre. The poster of the series can be downloaded here.

 

 

 

 

Super Resolution Microscopy

20th March 2018.  
Rainer Heintzmann, University of Jena          

The 5th seminar of the series on Light Microscopy entitled Super resolution microscopy will be given by Rainer Heintzmann of the University of Jena on the 20 March, 4.00 pm in the College lecture theatre. The poster of the series can be downloaded here.

Abstract
In the past decade revolutionary advances have been made in the field of microscopy imaging, some of which have been honoured by the Nobel prize in Chemistry 2014. One high-resolution method is based on transforming conventionally unresolvable details into measurable patterns with the help of an effect most people have already personally experienced: the Moiré effect.  If two fine periodic patterns overlap, coarse patterns emerge. This is typically seen on a finely weaved curtain folding back onto itself. Another example is fast moving coarse patterns on both fences of a bridge above a motorway, when approaching it with the car. The microscopy method of structured illumination utilizes this effect by projecting a fine grating onto the sample and imaging the resulting coarser Moiré patterns containing the information about invisibly fine sample detail. With the help of computer reconstruction based on several such Moiré images, a high-resolution image of the sample can then be assembled. Another way to obtain a high-resolution map of the sample is to utilize the blinking behaviour inherent in most molecules, used to stain the sample. Recent methodological advances (Cox et al., Nature Methods 9, 195-200, 2012) enable us to create pointillist high-resolution maps of molecular locations in a living biological sample, even if in each of the required many individual images, these molecules are not individually discernible. Examples will be shown as a film of a cell at 30 millionths of millimeter resolution and 6 seconds between the individual movie frames. 

Two further recently developed modes of lightsheet imaging will also be presented. Lightwedge microscopy aims at mesoscopic imaging of fixed and optically cleared samples at 1 µm isotropic resolution without the need for sample rotation. The key-idea is to focus a lightsheet at an unusually high NA (thus the name “lightwedge”) and still obtain a large field of view due to refocusing of the lightwedge and stitching the multiple small regions of thin illumination back together. This has been simplified by electrical tunable lens technology, which has become available recently. The second mode is hyperspectral Raman imaging in a lightsheet illumination configuration [1]. To recover the spectral information a full-field Fourier-spectroscopic approach has been chosen. The difficulty here is that in a Michelson approach, it would be technically very hard to maintain the angular stability and common path approaches usually tolerate a relatively low product of étendue and maximal optical path difference. We thus developed an optically stable Mach-Zehnder like scheme based on the use of retro-reflecting corner cubes, which is inherently stable. This enabled us to obtain full spectrally-resolved Raman images consisting of over four million spectra in about 10 minutes. Advantages over the conventional Raman imaging are the reduced maximum power on the sample and out of focus heating, the lightsheet-inherent good suppression of crosstalk from the illumination side and the avoidance of glass close to the sample mounting. Light sheet illumination for Raman imaging at few specific wavelengths was previously reported [2]. With a total laser power of 2W at an illumination wavelength of 577 nm, we obtained images (2048 × 2048 pixels) of polystyrene beads, zebrafish and a root cap of a snowdrop at a spectral resolution of 4.4 cm-1 with only few minutes of exposure. The olefinic and aliphatic C-H stretching modes, as well as the fingerprint region are clearly visible along with the broad water peak of the embedding medium. Spectrally resolved spontaneous Raman microscopy therefore promises high-throughput imaging for biomedical research and on-the-fly clinical diagnostics.

[1] W. Müller, M. Kielhorn, M. Schmitt, J. Popp, R. Heintzmann, Light sheet Raman micro-spectroscopy, Optica 3, 452-457, 2016.
[2] Ishan Barman, Khay Ming Tan, Gajendra Pratap Singh, “Optical sectioning using single-plane-illumination Raman imaging”, J. Raman Spectrosc., 41, 1099–1101 (2010)

 

 

 

 

Inhibiting Protein Aggregation

25th July 2018.  
Daniel Segal, School of Molecular Cell Biology & Biotechnology, Tel-Aviv University

 On Wednesday the 25th of July 2018 Daniel Segal of the School of Molecular Cell Biology & Biotechnology at Tel-Aviv University will give a seminar entitled Inhibiting aggregation of amyloidogenic proteins as a strategy for treating neurodegeneration at 12.00 noon in the College Lecture theatre. Protein misfolding and aggregation are crucial steps in the onset of several, common neurodegenerative diseases and inhibiting the formation of toxic protein aggregates process may have considerable potential for therapy.  D Segal will report a series of landmark studies in which he and his colleagues have been able to identify small molecules that disrupt the formation of toxic protein aggregates, at least iunder laboratory conditions. Te poster of the lecture can be downloaded here.

Abstract
My laboratory is interested in intrinsically disordered proteins, in particular disease-associated amyloidogenic proteins, and in developing means for inhibiting their harmful aggregation. Insights obtained about structural determinants that facilitate self-assembly of these proteins, such as the role of aromatic residues, serve us for developing small molecules and peptidomimetics to inhibit aggregation of these amyloidogenic proteins and disassemble of pre-existing aggregates. We evaluate efficacy of the candidate inhibitors using a series of in vitro methods, cell-based assays, and studies in transgenic animal models – Drosophila and mice. I will illustrate this approach using three examples: Tryptophan-modified naphthoquinone small molecules towards amyloid-beta and tau involved in Alzheimer’s disease; β-synuclein derived peptidomimetics towards α-synuclein involved in Parkinson’s; and a naturally occurring molecule, Mannitol, against α-synuclein with unexpected lead towards clinical trial. If time permits I will describe recent results of High Throughput screening for novel inhibitors, and the use of computational tools for understanding their mechanism of action.

Biography
Daniel Segal obtained his PhD in Genetics from the Hebrew University, Jerusalem, under the supervision of Prof. Raphael Falk, studying Drosophila developmental genetics. He did his postdoctoral studies at Harvard University, with the late Prof. William Gelbart, working on molecular genetics of the decapentaplegic developmental gene complex in Drosophila. He moved back to Israel as a scientist at the Weizmann Institute working on oncogene homologs in the fruit fly and on neurogenetics. Since 1987 he is a faculty member at Tel-Aviv University, where he is the Head of the School of Molecular Cell Biology & Biotechnology. His lab focuses on intrinsically disordered proteins, in particular disease-associated amyloidogenic proteins, and on developing means for inhibiting their harmful aggregation.

Image
NMR structure of a-synuclein fibrils (pdf accession 2n0a).Tuttle MD et al Nat Struct Mol Biol 23:409, (2016).

 

 

 

Proteins at Interfaces

5th June 2018.  
Marek Cieplak, Institute of Physics of the Polish Institute of Sciences           

On Tuesday the 5th of June 2018 Marek Cieplak of the Institute of Physics of the Polish Institute of Sciences will give a seminar on Proteins at fluid-fluid interfaces at 11.00 am in the College Lecture theatre. The seminar will address theoretical and experimental advances in our understanding of how physical interfaces (water-water, water-oil, water-air) affects protein structure and function and the crucial role of these envirnoment-induced, conformational changes in cell and tissue physiology and pathology.  The poster of the lecture can be downloaded here.

Abstract
Phase transitions in protein environments have increasingly become important as they are thought to play an important role in protein aggregation and misfolding.
We study the behavior of five proteins (protein G, egg-white lysozyme, hydrophobin, tryptophan cage and LTP1) at the air–water and oil–water interfaces by all-atom molecular dynamics. The proteins are found to change orientation and get distorted when pinned to the interface. This behavior is consistent with the empirical way of introducing the interfaces in a coarse-grained model through a force that depends on the hydropathy indices of the residues. Proteins couple to the oil–water interface stronger than to the air–water one. They diffuse slower at the oil–water interface but do not depin from it, whereas depinning events are observed at the other interface. The reduction of the disulfide bonds slows the diffusion down. We use the empirical coarse-grained model to study properties of protein layers at the air-water interface. In particular, we demonstrate existence of glassy effects as evidenced by slowing down of diffusion with increasing concentration of proteins. We also show that layers of two barley proteins, LTP1 and its ligand adduct LTP1b, flatten out at the interface and can make a continuous  and dense film that should be responsible for  formation and stability of foam in beer. The degree of the flattening depends on the protein - the layers of LTP1b should be denser than those of LTP1 – as well as on the presence of glycation and the degree of reduction in the number of disulfide bonds. We also show that the interfacial forces can untie proteins with shallow knots, but they can also make knots in proteins that are natively unknotted. The physics of proteins at the air-water interface can be captured by a simple lattice model which allows for larger statistics of the pinning-depinning processes and an analysis of a Marangoni-like effect induced by a temperature gradient.

[1] M. Cieplak, D. B. Allen, R. L. Leheny, D. H. Reich, Langmuir 30:12888-96 (2014).
[2] Y. Zhao, M. Chwastyk, M. Cieplak, Sci. Rep. 7:39851 (2017).
[3] Y. Zhao, M. Cieplak, Langmuir 33:4769-4780 (2017).
[4] Y. Zhao, M. Cieplak, Phys. Chem. Chem. Phys. 19: 25197-25206 (2017

 

Image
Surface representation of the electrostatic potential of the Brichos domain of human lung surfactant protein C (pdb accession: 2yad).

 

 

 

Convergence of Circadian Clock and Cancer

11th December 2017.  
Chi Van Dang, Ludwig Institute for Cancer Research, Philadelphia              

Chi Van Dang, Director of the Ludwig Institute for Cancer Research and Head of the Ludwig Institute Unit located at the Wistar Institute in Philadelphia, will give the opening lecture of the PhD Programme of the University of Pavia on Monday the 11th of December in Aula Magna (Strada Nuova) at 10.30.  The lecture, entitled Convergence of Circadian Clock and Cancer: Time as an Inconvenient Truth, will tackle a new area in cancer research that may greatly extend our understanding of the disease.  The poster of the lecture can downloaded here and all College students of Biology, Biotechnology and Medicine are strongly encouraged to attend.  Chi Van Dang will be a guest of the College during his stay in Pavia.

Abstract
Cancer metabolism as a field of research was founded almost 100 years ago by Otto Warburg, who described the propensity for cancers to convert glucose to lactate despite the presence of oxygen, which in yeast diminishes glycolytic metabolism known as the Pasteur effect. In the past 20 years, the resurgence of interest in cancer metabolism provided significant insights into processes involved in maintenance metabolism of non-proliferating cells and proliferative metabolism, which is regulated by proto-oncogenes and tumor suppressors in normal proliferating cells. In cancer cells, depending on the driving oncogenic event, metabolism is re-wired for nutrient import, redox homeostasis, protein quality control, and biosynthesis to support cell growth and division. In general, resting cells rely on oxidative metabolism, while proliferating cells rewire metabolism toward glycolysis, which favors many biosynthetic pathways for proliferation. Oncogenes such as MYC, BRAF, KRAS, and PI3K have been documented to rewire metabolism in favor of proliferation. These cell intrinsic mechanisms, however, are insufficient to drive tumorigenesis because immune surveillance continuously seeks to destroy neo-antigenic tumor cells. In this regard, evasion of cancer cells from immunity involves checkpoints that blunt cytotoxic T cells, which are also attenuated by the metabolic tumor microenvironment, which is rich in immuno-modulating metabolites such as lactate, 2-hydroxyglutarate, kyneurenine, and the proton (low pH). As such, a full understanding of tumor metabolism requires an appreciation of the convergence of cancer cell intrinsic metabolism and that of the tumor microenvironment including stromal and immune cells.

Biography
Chi Van Dang oversees the execution of Ludwig’s scientific strategy, with a special focus on the operations and staffing of the Lausanne, Oxford and San Diego Branches of the Ludwig Institute for Cancer Research. He also manages the alignment of their efforts with those of the six independent Ludwig Centers across the US to further cultivate collaboration within Ludwig’s global research community. As a researcher, Chi Van Dang is best known for his work on the molecular signaling pathways and mechanisms that govern the unusual metabolism of cancer cells, which require vast quantities of energy and molecular building blocks to sustain proliferation. Chi Van Dang's laboratory was the first to show that a master regulator of gene expression named MYC—a gene whose mutation or aberrant expression is associated with many types of cancer—alters the utilization of a key sugar in cancer cells. This body of work bolstered the hypothesis that cancer cells can become addicted to their reengineered metabolic signaling and that disrupting these pathways could be a powerful approach to treating cancer. Chi Van Dang currently leads a Ludwig laboratory housed at The Wistar Institute in Philadelphia. Prior to joining Ludwig, he served as Director of the Abramson Cancer Center at the University of Pennsylvania Perelman School of Medicine, where he launched a series of Translational Centers of Excellence to develop novel interventions for various cancers. He began his career in medicine and research at Johns Hopkins University School of Medicine, where he was Director of the Division of Hematology and eventually became the Johns Hopkins Family Professor in Oncology Research, the Vice Dean for Research and Director of the Hopkins Institute for Cell Engineering. He has authored over 250 scientific and medical articles, book chapters and two books and am a member of the National Academy of Medicine (Institute of Medicine), American Academy of Arts & Sciences and chair the National Cancer Institute’s Board of Scientific Advisors.

New Directions in Optical Microscopy

21th March 2018.  
W Brad Amos, MRC Laboratory of Molecular Biology, Cambridge              

Abstract 
The fourth seminar of the Light microscopy series will be given by William B Amos of the MRC Laboratory of Molecular Biology in Cambridge in the College lecture theatre on Wednesday the 21st of March at 2.00 pm and will address New directions in optical microscopy. The seminar will cover: Multiphoton microscopy on the one hand and Super-resolution methods on the other.  The latter inclu Optical (structured Illumination) and Photochemical (Stimulated Emission Depletion STED, Stochastic optical reconstruction, STORM and Photoactivation Light Microscopy PALM)discuss standardised distances in compound microscopes, lens aberrations, diffraction in the light microscope, Rayleigh resolution and Fourier synthesis. The poster of the series can be downloaded here.

Biography
William Brad Amos was trained as a zoologist, researched in cell biology and is now a designer of optical instruments. With John White, Mick Fordham and Richard Durbin in Cambridge, he developed an instrument that has set the standard of modern confocal microscopes.  Derivatives of this instrument are now made by many companies and are in use throughout the world. His scientific work is now carried out done in collaboration with Gail McConnell in the University of Strathclyde. This collaboration has resulted in several novel applications of optical physics in microscopy, including what is arguably the greatest design change in microscope objectives  for 100 years. This is called the Mesolens, the name signifying that it has the wide field of a photographic macro lens and the high resolution of a microscope objective. 

 

 

 

 

Polarisation and Interference Methods

20th March 2018.  
W Brad Amos, MRC Laboratory of Molecular Biology, Cambridge              

Abstract 
The third seminar of the Light microscopy series will be given by William B Amos of the MRC Laboratory of Molecular Biology in Cambridge in the College lecture on Tuesday tthe 20th of March at 2.00 pm and will address Polarisation and Interference Methods. The seminar will discuss the nature of polarised light, the way in which polarised light interacts with crystals and biomolecules, differential interference contrast using polarisation and the use of fluorescence in microscopy including fluorescence correlation spectroscopy, fluorescent lifetime measurements and Forster resonance energy transfer.

Biography
William Brad Amos was trained as a zoologist, researched in cell biology and is now a designer of optical instruments. With John White, Mick Fordham and Richard Durbin in Cambridge, he developed an instrument that has set the standard of modern confocal microscopes.  Derivatives of this instrument are now made by many companies and are in use throughout the world. His scientific work is now carried out done in collaboration with Gail McConnell in the University of Strathclyde. This collaboration has resulted in several novel applications of optical physics in microscopy, including what is arguably the greatest design change in microscope objectives  for 100 years. This is called the Mesolens, the name signifying that it has the wide field of a photographic macro lens and the high resolution of a microscope objective. 

 

 

 

 

Ray and Wave Optics and Practical Microscopy

19th March 2018.  
W Brad Amos, MRC Laboratory of Molecular Biology, Cambridge              

Abstract 
The second seminar of the Light microscopy series will be given by William B Amos of the MRC Laboratory of Molecular Biology in Cambridge in the College lecture theatre on Monday the 19th of March at 4.00 pm and will address Ray and wave optics and practical microscopy. The seminar will discuss standardised distances in compound microscopes, lens aberrations, diffraction in the light microscope, Rayleigh resolution and Fourier synthesis. The poster of the series can be downloaded here.

Biography
William Brad Amos was trained as a zoologist, researched in cell biology and is now a designer of optical instruments. With John White, Mick Fordham and Richard Durbin in Cambridge, he developed an instrument that has set the standard of modern confocal microscopes.  Derivatives of this instrument are now made by many companies and are in use throughout the world. His scientific work is now carried out done in collaboration with Gail McConnell in the University of Strathclyde. This collaboration has resulted in several novel applications of optical physics in microscopy, including what is arguably the greatest design change in microscope objectives  for 100 years. This is called the Mesolens, the name signifying that it has the wide field of a photographic macro lens and the high resolution of a microscope objective. 

 

 

 

 

Resolution and the Nature of Optical Images

19th March 2018.  
W Brad Amos, MRC Laboratory of Molecular Biology, Cambridge              

Abstract 
The first seminar of the Light microscopy series will be given by William B Amos of the MRC Laboratory of Molecular Biology in Cambridge on Monday the 19th of March in the College lecture theatre at 2.00 pm and will address Resolution and the nature of optical images. The seminar will discuss the nature of images obtained the light microscope, Abbe's equation for resolution as well as recent developments such Interferometric Fluorescent Superresolution Microscopy (iPALM) and Coherent anti-Stokes Raman Spectroscopy (CARS). The poster of the series can be downloaded here.

Biography
William Brad Amos was trained as a zoologist, researched in cell biology and is now a designer of optical instruments. With John White, Mick Fordham and Richard Durbin in Cambridge, he developed an instrument that has set the standard of modern confocal microscopes.  Derivatives of this instrument are now made by many companies and are in use throughout the world. His scientific work is now carried out done in collaboration with Gail McConnell in the University of Strathclyde. This collaboration has resulted in several novel applications of optical physics in microscopy, including what is arguably the greatest design change in microscope objectives  for 100 years. This is called the Mesolens, the name signifying that it has the wide field of a photographic macro lens and the high resolution of a microscope objective. 

 

 

 

 

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