Research Techniques in Bioscience

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1 MSc Biophotonics The MSc Biophotonics programme is the first cross-disciplinary programme in the UK that has been designed to offer advanced training at the interface between laser optics, cell biology and medicine. Whether you are an emerging researcher thinking of undertaking a PhD or are planning a future in a biophotonics-related industry we can provide the fundamental understanding and hands-on experience necessary for work in this rapidly developing field. The programme is jointly taught by expert scientists in the School of Physics & Astronomy and the School of Biosciences. The course will use world-class research and teaching facilities to cover topics including advanced light microscopy, laser-based analysis, nanoparticles as optical bio-labels, biosensors, and the use of biophotonics in the field of medicine. 2

2 Research Techniques in Bioscience Module aim: 1. Provide an understanding of the major research disciplines in modern bioscience. 2. Teach a range of state-of-the-art biological research techniques. 3. Develop a knowledge of how these research techniques can be combined in specific biological research projects. Recommended Reading: Alberts, B. et al (2007) Molecular Biology of the Cell (5 th Edition): Reference Edition, Garland Pub. ISBN: The following may also be useful as a starting point for further reading: Primrose, SB. and Twyman, RM. (2006) Principles of Gene Manipulation (7 th Edition), Blackwell Pub. ISBN: Hammes, GG. (2005) Spectroscopy for the Biological Sciences, John Wiley & Sons ISBN: Recombinant DNA technology: including detection and analysis methods, cloning and site-directed mutagenesis. 2. DNA analysis and sequencing: including DNA sequencing methods, SNP analysis and high throughput genomic sequencing. 3. Cell manipulation and transgenic animals: including genetic manipulation of cells and whole organisms, gene ablation and RNAi technology 4. Field and population sampling: including research in biodiversity related research fields. 5. Neuroscience techniques: including electrophysiology, patch clamping and ion channel analysis. 6. Cell and tissue imaging: including light microscopy techniques, electron microscopy and MRI. 7. Protein Biology: including separation techniques, immunological detection, enzymology, Mass Spectrometry and structural analysis. 8. High throughput and array based technologies: including microarrays and proteomics. Scopes, RK. (1994) Protein Purification: Principles and Practice (3 rd Edition), Springer ISBN:

3 Mathematical Tools in Photonics and Biology Module aim: 1. Introduce and explain relevant mathematical tools in an accessible way to students coming from both physical and biological sciences. 2. Allow students to use the mathematical formalism relevant in optics and photonics. 3. Allow students to understand and execute a variety of quantitative methodologies essential to modern biological data handling and statistics. 4. Support students mathematics and data analysis skills as they move towards being independent research scientists. Riley, KF., Hobson, MP. and Bence, SJ. (2006) Mathematical Methods for Physics and Engineering (3 rd edition), Cambridge University Press ISBN: Bowker, DW. and Randerson, PF. (2006) Practical Data Analysis Workbook (2 nd edition), Pearson Education Ltd ISBN Vectors algebra and operators. Complex numbers and functions. Differentiation and integration. 2. Differential equations. Harmonic functions. The Fourier transform. 3. The Gaussian and Lorentzian function. Convolution and Correlation integrals. 4. Variability and one-way analysis of variance: understanding variability and comparing means, transformations, assumptions, comparing means after ANOVA. 5. Multi-factorial analysis of variance including two-way analysis and beyond, crossed and hierarchical factors, fixed and random factors, mixed models. 6. Bivariate regression, understanding evenness, assumptions, transformations predicting values and confidence limits. 7. Multiple regression including equation construction, links to ANOVA, finding the best prediction equation with different methods, stability of solutions. 8. Ordination: multivariate data, quantitative and presence/ absence data, principal component analysis, variable reduction and interpretation. 9. Cluster analysis: consideration of distances and similarity, clustering methods, uses and limitations, beyond clustering. 5 6

4 Optics and Light Spectroscopy including Optical Properties of Biomolecules Module aim: 1. To introduce and explain major concepts of modern optics and laser spectroscopy at a level accessible to students coming from both physical and biological sciences 2. To enable students to realize various aspects of the instrumentation involved in optical experiments, including light sources, optical elements and detectors 3. To give practical demonstrations of the working principle of important optical elements as well as to enable students to acquire practical experience with important optical elements 4. To explain the optical properties of important biomolecules Hecht, E. (2001) Optics (4 th Edition), Pearson Education Ltd ISBN: Meschede, D. (2007) Optics, Light and Lasers: The Practical Approach to Modern Aspects of Photonics and Laser Physics (2nd Edition), Wiley VCH ISBN: Properties of the light field 2. The laws of reflection and refraction 3. Foundations of geometrical optics, optical imaging and aberrations 4. Introduction to Fourier optics 5. Elements and examples of interference and diffraction phenomena 6. Optical properties of anisotropic crystals and the principles of nonlinear optics 7. Incoherent light sources and Laser light sources 8. Instrumentation for light spectroscopy analysis and detection. 9. Introduction to the quantum mechanical treatment of light-matter interaction 10. Optical transitions and transitions probabilities 11. Width and profile of spectral lines 12. Optical properties of biomolecules and organic dyes Demtroder, W. (2008) Laser Spectroscopy, Vol. 1 Basic Principles; Vol. 2 Experimental Techniques (4 th Edition), Springer ISBN: Hammes, GG. (2005) Spectroscopy for the Biological Sciences, John Wiley & Sons ISBN:

5 Modern Light Microscopy Techniques Module Aims: 1. To introduce and explain the concepts of modern optical microscopy techniques used in the biosciences. 2. To enable students to understand the properties of these techniques and the employed technical elements. 3. To give practical demonstrations and hands-on experience for selected techniques. Murphy, DB. (2001) Fundamentals of Light Microscopy and Electronic Imaging, John Wiley and Sons ISBN: Goldstein, DJ. (1999) Understanding the Light Microscope: A Computer-Aided Introduction, Academic Press ISBN: Bradbury, HSM. and Bracegirdle B. (1998) Introduction To Light Microscopy, Garland Science ISBN: Slayter, EM. and Slayter HS. (1993) Light and Electron Microscopy, Cambridge University Press ISBN: Elements of a Microscope a. Objective b. Illumination c. Imaging Detectors (Human Eye, Film, Digital cameras) 2. Special microscopy setups (Stereomicroscopy, 4-Pi microscopy) 3. Contrast due to absorption & refractive index in various illumination/collection techniques: a. Brightfield b. Darkfield c. Total internal reflection d. Oblique illumination e. Hoffman modulation contrast f. Phase contrast g. Differential interference contrast h. Polarization contrast i. Optical coherence tomography j. Stains for Biological material 4. Fluorescence a. Excitation sources b. Fluorescent Markers c. Detection d. Contrast methods (Intensity, Lifetime, Time-correlation spectroscopy, Photobleaching) 5. Scanning microscopy techniques a. Scanning optics & detectors b. Confocal c. Multiphoton d. Raman and coherent Raman e. Near-field 6. Three-dimensional imaging 9 10

6 Advanced Optical Bio-sensing Methods Medical Biophotonics Module Aims: 1. To give on overview of optical methods utilized in biosensing applications, with emphasis on state-of-the-art developments. 2. To show the biophysical working principles behind modern commercially-available label-free biosensors based on evanescent field coupling and discuss examples of their applications. 3. To introduce the state-of-the art research on label-free biosensors based on evanescent field, beyond commercially-available methods. 4. To develop a practical understanding through experimental demonstrations. Ligler, FS. and Rowe Taitt, CA. (2002) Optical Biosensors: Present and Future, Elsevier ISBN: Prasad, PN. (2003) Introduction to Biophotonics, John Wiley & Sons ISBN: Overview of optical biosensor methods 2. Working principle and applications of commercially-available labelfree biosensors based on evanescent waves: Surface plasmon resonance (SPR) and dielectric waveguide methods 3. The research advances on label-free optical biosensors based on whispering gallery modes in microresonators 4. Fluorescent labelling and the mechanism of fluorescent resonant energy transfer (FRET): applications to biosensors 5. Raman-based biosensors Module aims: 1. To introduce the student to the use of biophotonics in the field of medicine. 2. The general principles of the interaction of light with cells and tissue will be covered and specific examples of medical biophotonics applications reviewed. 3. A substantial element of practical demonstration will be included based in the research laboratories in the schools of physics and medicine. The reading list will be an up-to-date list of scientific references and will be given at the beginning of the module 1. Introduce the basic physical mechanisms of light interaction with molecules. Extend this understanding to interaction with cells and tissue highlighting the physical characteristics used in the applications to follow (e.g. absorption, fluorescence, scattering, spectral response) 2. Study the use of flow cytometry for cellular analysis and medical diagnostics. 3. Tissue engineering with light - Tissue ablation and laser surgery 4. Optical techniques in drug discovery - Fluorophore development and functionality, fluorescence microscopy of the cell cycle and optical signalling during drug interaction 5. Photo-activation of drugs - Photo-dynamic therapies, molecular caging 6. Biochip platforms Microfluidic transport systems, electro-kinetics, optics on-a-chip, semiconductor sources and micro-lenses 11 12

7 Nanostructures and Optical Manipulation Module aims: 1. To introduce students to the basic physics of quantum wells and quantum dots and methods of fabrication. 2. To describe the use of wells and dots in light sources with particular reference to use in medicine and bioscience. 3. To describe the requirements for quantum dots as labels. 4. To introduce students to the basic physics of optical trapping 5. To describe typical systems for optical trapping and manipulation with reference to bioscience applications. Silfvast, WT. (2004) Laser Fundamentals (2 nd Edition), Cambrige University Press ISBN: Prasad, PN. (2003) Introduction to Biophotonics, John Wiley & Sons ISBN: Review of the solution of Schrodinger s equation for semiconductor nanostructures: Quantum wells and dots 2. Description of methods of epitaxial growth for wells and self assembled dots, methods of formation of colloidal dots. 3. Review of optical properties. 4. Principles of light emitting diodes and lasers: 5. Typical device structures characteristics: wavelengths, output power etc. 6. Applications, eg photodynamic therapy, Optical coherence tomography, Optical Biochips 7. Colloidal dot materials and structures 8. Optical properties of colloidal dots, Metallic nanoparticles 9. Basic concept of optical trapping: origin of the forces. 10. Realisation of optical trapping Coldren, LA. and Corzine SW. (1995) Diode lasers and Photonic Integrated Circuits (Chapters 1,2, 4), John Wiley & Sons ISBN: Fox, AM. (2001) Optical Properties of Solids, Oxford University Press ISBN:

8 Examples of Biophotonics Projects Characterization of Quantum Dot Based Light Sources for Optical Coherence Tomography Optical coherence tomography (OCT) is a non-invasive, optical diagnostic technique, which enables the in vivo cross-sectional tomographic imaging of biological tissue at approximately 100 times the resolution of conventional ultrasound. This is achieved by employing a broad bandwidth light source in combination with an interferometric measurement technique. The aim of this project is to characterize alternative quantum dot (QD) based light sources. This has the potential to significantly increase OCT resolution, thereby improving the clinical feasibility of OCT in a variety of medical fields. Implanting Optoelectronics for in vivo Cytomics Tumour cells can be grown in vivo using hollow fiber 3-D cell culture systems. These hollow fibers are porous polymer tubes of ~ 1mm diameter which trap cells but allow transport of small molecules (e.g. drugs) through their outer wall. It is therefore possible to achieve contained growth of tumour cells within animal hosts without damage to the animal. The aim of this project is to image cells growing in this 3-D environment. Light from a laser source will excite fluorescent molecules within the cell to allow optical analysis via optical fibres. The fluorescent signals will then be analysed to inform on the rates of cell proliferation within the hollow fibre. Binding Affinities Measured by FRET The development of new techniques for measuring binding affinities between proteins by Fluorescence Resonance Energy Transfer (FRET) will advance our understanding of biological functions like signal transduction, where protein protein interactions are very important. FRET describes the energy transfer mechanisms occurring between two fluorophores, so called donor and acceptor, when in close proximity to each other. The aim of this project is to measure protein binding affinities via FRET using fluorophore-labelled proteins attached to a surface through tether molecules of controlled length, as opposed to volume solutions, which should greatly reduce the amount of material needed. Moreover, FRET will be measured via transient fluorophore dynamics using time-resolved detection, which will enhance the sensitivity of the technique 3-D Organisation of Microtubules The mechanical function of most connective tissues is intimately associated with the orientation of collagen within their extracellular matrix. Actin plays a role in the secretion of a number of highly oriented matrices and preliminary data shows that this also occurs in early developing bone. The aim of this project is to use fluorescent labelling techniques allied to confocal laser scanning microscopy and 3-D modelling to describe the organisation of the three major cytoskeletal elements (microtubules, actin and intermediate filaments) in bone cells and their relation to early collagen deposition. Microsphere Biosensors One of the most widely utilized optical biosensors used to detect molecular interactions is based on the phenomenon of surface plasmon resonance (SPR), where an evanescent light field travelling along a planar interface can be used to probe a target molecule on the surface. The aim of this project is to investigate the detection limits and the response dynamics of a prototype biosensor that has the potential to achieve unprecedented sensitivity for molecular detection beyond SPR. The device utilizes the frequency resonances (called whisperinggallery modes) of light travelling by total internal reflection in 30-50µm dielectric microspheres. Using this technique, protein binding to antibodies immobilized on the microsphere surface can be detected by a shift in the frequency resonances. Using Raman Spectroscopy to Achieve High Resolution Detection of Protein Phosphorylation Conventionally the enzymatic process of protein phosphorylation is monitored using 32 P-labelled phosphates. The aim of this project is to use vibrational (Raman) spectroscopy to investigate protein phosphorylation without the use of radioactive labels. Raman spectroscopy is chemically sensitive to the endogenous constituents of a reaction; Raman spectral differences can therefore be elucidated between identical synthetic peptides that differ only by the presence of an additional phosphate. The method will be developed further with the aim of monitoring the phosphorylation of a substrate by purified protein kinases and also the phosphorylation of proteins present in cell extracts