AEP470 - A&EP 470 BIOPHYSICAL METHODS Manfred Lindau...

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A&EP 470 BIOPHYSICAL METHODS Manfred Lindau page 1 Biophysical Methods A&EP 470 – BIONB 470 – VETMM 470 – BME 570 (Fall Semester only ) By Manfred Lindau School of Applied and Engineering Physics Cornell University 217 Clark Hall Ithaca, NY 14850 e-mail: ml95@cornell.edu With thanks to Kassandra Kisler for the TIRF section
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A&EP 470 BIOPHYSICAL METHODS Manfred Lindau page 2 Table of Contents: Summary 4 Modern microscopy 5 Abbe’s resolution criterion 5 Point Spread Function and Contrast Transfer Function 6 Fourier optics 11 Examples of 2D Fourier transform pairs. 14 Out-of-Focus Blur 17 Deconvolution of Images 21 Confocal microscope 24 Signal-To-Noise Ratio and Background Suppression: 27 The origin of noise 30 Multi-photon microscopy 35 Total Internal Reflection Fluorescence (TIRF) Microscopy 41 Optical Trapping 50 Forces on dipoles, some electrostatics 50 How big is the force? 52 Phase contrast microscopy 69 Electron Microscopy 72 Magnetic lenses 73 Chromatic aberration 76 Spherical aberration 76 Phase contrast electron microscopy 77 Diffraction Methods 87 Diffraction is a Fourier transform 87 The convolution theorem – again 88 Wave functions 91 Waves represented by complex numbers 92 The Ewald sphere 93 Structure determination 95 Membranes and ion channels 98 Lipid bilayers and membrane capacitance 99 Membrane conductance 99 The Nernst equation 100 The nerve impulse – action potential 106 Voltage clamp 107 Voltage-dependence of activation 110 Hodgkin-Huxley analysis of voltage-clamp currents 115 Ligand-gated channels 120 Fluctuation analysis 123 Single channel recording and the patch-clamp technique 127 Current, voltage and electrodes - some advanced considerations. 130 Structure of ion channels 134 Ion channel expression in oocytes 136 Expression of Ach receptor channels 136
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A&EP 470 BIOPHYSICAL METHODS Manfred Lindau page 3 Functional studies of mutations in α− subunits: 138 Voltage-dependent channels 141 Subunits of voltage–dependent K + channel: Toxins, mutations and oocyte expression146 Foundations of Spectroscopy 150 Spectroscopic properties of rhodopsin 152 Linear Dichroism 153 Circular Dichroism 157 Fluorescence Spectra 161 The Ca 2+ indicator fura-2 161 Principle of a set-up to measure [ Ca 2+ ] in a single cell and a recording. 164 Green fluorescent protein 165 Förster (or Fluorescence) Resonance Energy Transfer (FRET) 167 Appendices Protein Crystallography by Steve Ealick A1 Protein NMR Spectroscopy by Robert E. Oswald A2 Caged Compounds and Rapid Kinetics by George P. Hess A3 Capacitance Measurements of Exocytosis and Endocytosis by M. Lindau A4
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A&EP 470 BIOPHYSICAL METHODS Manfred Lindau page 4 Summary An overview of the diversity of modern biophysical experimental techniques used in the study of biological systems at the cellular and molecular level as will be found in articles published in the Biophysical Journal. Topics covered will include methods that examine both structure and function of biological systems. Topics include light microscopy, fluorescence microscopy, Fourier
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This note was uploaded on 03/29/2009 for the course A&EP 470 taught by Professor Lindau during the Fall '08 term at Cornell University (Engineering School).

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AEP470 - A&EP 470 BIOPHYSICAL METHODS Manfred Lindau...

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