Overview of Physics 173 BGGN 266 Prof David Kleinfeld Modern Physics Biophysics Laboratory This laboratory course consists of table top experiments in biological and solid state physics with an emphasis on spectroscopy Students choose from a variety of experiments which include 1 Fluorescent Microscopy 2 Genetic Transcription 3 Holography 4 Laser Light Scattering 5 Mossbauer Spectroscopy 6 Oocyte Electrophysiology 7 Optical Trapping 8 Chaos in a Model Electrical Circuit 9 Spin Echo NMR 10 Swept Field NMR 11 Visual Transduction in Flies 12 Zeeman Effect 13 Belousov Zhabotinskii Reaction A brief description of some off the more biologically based aspects of the course along with ideas for future extensions is given below pages 1 to 5 Summary reports and support material for all projects are found on the web site http wwwphysics ucsd edu neurophysics physics 173 273 html The general philosophy of the course is given in page 5 Electrophysiology and genetic expression The electrical dynamics of nerve cells is one of the most basic and rewarding topics in for students to learn in an instructional laboratory Past approaches to electrophysiology instruction have involved the use of invertebrates with extensive efforts expended in the preparation of desheathed ganglia We propose an complementary approach where students use oocytes as the test bed for basic electrophysiology and for the induced expression of individual channels Oocytes are readily available commercial equipment exists that allows oocytes to be current or voltage clamped with relative ease and commercial kits exists that allows genetic material to be readily synthesized In their native form frog oocytes can exhibit inositol 1 4 5 trisphosphate IP3 receptor mediated oscillations in their Ca2 currents that can be measured via a Ca2 dependent Clconductance This oscillation is rapidly induced with bath application of the agonist Fig 1 Oscillations in the membrane conductance in an oocyte We show the membrane currents of a native xenopus oocyte observed under voltage clamp in response to the onset of perfusion with serum arrow Data of undergraduate student Brenda Bloodgood 1 lysophosphatidic acid a component of normal serum Further oocytes can transformed with the injection of mRNA so that they express a desired channel such as the shaker K current Thus oocytes provide a means for students to learn electrophysiology and modern manipulation techniques Compared with approaches using invertebrate ganglia which have the virtue of rich electrophysiological responses across different cell types the oocyte preparation is technically simpler and adds the possibility of genetic manipulation The laboratory sequence includes Project 1 Set up of electrophysiology rig and basic tests of equipment with a model cell Current and voltage clamp for measurements of passive membrane parameters Voltage clamp in the presence of serum to determine and characterize oscillatory membrane currents see Fig 1 Manipulation of the membrane currents with external ion exchange and buffering of Ca2 intracellular via intracellular injection of EGTA and BABTA Project 2 Preparation of mRNA from cDNA to be supplied by established research laboratories using commercially available viral kits Intracellular injection and expression of mRNA Voltage clamp of voltage dependent channels e g shaker A type K current see Fig 2 or the rapidly inactivating Na current Fig 2 Voltage clamp of A type K current in an oocyte Voltage clamp records from a xenopus oocyte subsequent to the injection and expression of the mRNA for the shaker A type K current Note the onset of the rapidly inactivating outward current that occurs commensurate with the increase in amplitude of the command voltage steps Data of undergraduate students Jennifer Coates and Gary Tedeschi Over time the electrophysiology sequence will be extended to single channel currents and their statistics This will cement the connection between noise and thermodynamic equilibrium and ion kinetics Biophotonics This module explores the use of light and optics for imaging cellular function and for spectroscopy and manipulation of bioactive agents Since much of the instrumentation involved is quite flexible e g optical benches can be reconfigured we incorporated a substantial degree of flexibility in this module Some of the experiments depend on a readily available source of cultured neurons albeit small numbers of plates these are now a common feature of many research laboratories Project 1 Imaging of Ca2 intracellular during Ca2 IP3 oscillations in oocytes This project builds on the electrophysiology module We consider a confocal measurement using a pinhole and a modified conventional microscopes and optical sectioning using a vibrating 2 grating technique This project also introduces the modeling of reaction diffusion systems as is appropriate for Ca2 waves Project 2 Imaging the dynamics of subcellular organization The availability of neuronal cultures with protein markers e g EGFP labeled actin will enable study the dynamics of cellular outgrowth using time lapse fluorescent microscopy Project 3 Optical tweezers for understanding the fluctuation dissipation theorem central to any system in equilibrium This can be addressed by measuring the force of a trap on polystyrene balls in terms of the relaxation of the ball upon a perturbation of its position versus a measurement of the fluctuations in the position of the ball found from an ensemble of snapshots of the position within the trap see Fig 3 Fig 3 Optical trapping A one micron diameter bead that is trapped at the focus of 1 3 NA objective 100 mW 832 nm wavelength The field of view is 25 m m Out of focus particles are also visible Data of undergraduate student Peter Rickgauer Project 4 Optical tweezers for the manipulation of subcellular organelles One means that we tested involved the manipulation of neuronal processes in cultured hippocampal cells that engulfed polystyrene balls Over time and with the continued drop in price of lasers and optical components it will be possible to set up a scanning system for optical sectioning of cells This represents a natural evolution of the optical tweezers whose realization includes a slow scan system Spectroscopic analysis This module spans the use of nonionizing radiation both light and radio waves for the analysis of macromolecules and small 0 1 m m assemblies This module serves to illustrate basic optical concepts magnetic