III. The Course Physics 111 Advanced Lab 3rd or 4th-year course, follows one-semester lab course in electronics and computerized data acquisition. Students choose 4 experiments to perform from a list of 18, sign up for time on the apparatus, which is permanently set up. Lab open and staffed 4 hours/day (a student spends hours/week in lab) students per semester. A Biophysics Experiment for the Advanced Physics Laboratory * Thomas Colton, Steven Wasserman, Jan Liphardt, University of California Berkeley, Physics Department, 366 LeConte Hall, Berkeley, CA ; ; *Supported by a donation from Stanford Research Systems Students learn some theory of Brownian motion and basics of programming in Matlab through a simulation exercise including the following tasks: Simulate random motion of particles in one and two dimensions, calculate squared displacements and a diffusion coefficient (D) from the simulated data, and compare with a theoretical calculation of D. Model the effect of bulk flow on the simulated particle motion to discriminate graphically between random motion and bulk flow. Investigate the distributions and statistics of random displacements and squared displacements to learn how to determine appropriate sample sizes. Calculate autocorrelations and cross correlations of particle displacement. IV. Simulating Brownian Motion VI. Tracking Nanoparticles Data Acquisition. A CCD camera mounted on the microscope interfaces via Firewire to a computer. Software written in C# and.NET controls camera settings and imports movies of particle motion into the computer. Data Analysis. A Matlab program filters the images, identifies particles within a frame, correlates particles from frame to frame, calculates particle trajectories, displacements, and many other useful statistics, and displays several data plots for the movie. Students are encouraged but not required to delve into the Matlab code to modify some settings and algorithms. VI. Investigating Brownian Motion Students design and conduct an experiment to determine the relative effects of solute molecular weight, viscosity of the medium, and particle size on the characteristics of Brownian motion, including the diffusion coefficient. Students are provided with the following options: Beads: gold nm, polystyrene – 500 nm, 1  m, 2  m Glycerin - m.w. 92 Polyvinylpyrollidone (PVP) - m.w. 380,000 Viscosities can be varied by dilution with water. Experimental designs frequently include 16 or more treatments, with replication resulting in well over 100 movies processed and analyzed. Data analysis and interpretation are complex and allow considerable creativity. VIII. Discussion The use of artificial beads and onion cells as study systems for nanoparticle transport provides a good biophysics experience without requiring the facilities and expertise necessary to maintain microorganisms and tissue cultures more commonly used in this field. This experiment is expected to take students 6 afternoons of lab work (approximately 24 hours) to complete, not including reading, final analysis, and lab report. It is a challenge for physics students to prepare slides and operate a research-grade microscope in Kohler and darkfield illumination. We want students to develop and use programming skills in this experiment. Some are well-prepared, successfully manipulating algorithms and data in Matlab. Others are not, so rely on provided programs and complete their data analysis in Excel. We are currently developing a modular program for this lab in C# and.NET that is amenable to student programming in Microsoft Visual Studio. I. Introduction Why track particles? A key task in contemporary biophysics is to track organic fluorophores or quantum dots in vitro or inside living cells. This method is used to characterize the motions of molecular machines (e.g. the hand-over-hand walking of kinesin). determine the spatial distribution of proteins/mRNA inside cells. localize and characterize distinct lipid environments in the cell’s membrane by tracking single fluorophores as they diffuse on the membrane. If the motion of the probe is unhindered, then the spatial trajectory of the molecule will be described accurately by 2-D Brownian motion. If the probe’s motion is constrained by interactions with a membrane protein, then the probe’s trajectory will deviate from a random walk. Motivation: Many of our physics undergraduates go on to study biophysics, but our curriculum gives them little opportunity (or time!) to explore this dynamic new field. To meet this need, we are developing new biophysics experiments for our Advanced Lab Course. This first one focuses on tracking the motion of nanoparticles II. Objectives 1. Introduce skills and techniques useful in biophysics, including light microscopy and the theory and practice of tracking nanoparticles to infer mechanisms of motion. 2. Improve students’ ability to design and conduct experiments and analyze data. 3. Encourage students to write or edit code to acquire and manipulate data, rather than relying on provided programs and user interfaces. VII. Intracellular Transport in Onion Cells Students observe the motion of nanoparticles in a living onion cell. Cytoplasmic streaming is a bulk flow of granules accomplished by motor proteins dragging pieces of endoplasmic reticulum along actin filaments. Individual granules are also transported more directly by attached motor proteins ratcheting themselves along microtubule tracks within the cytoplasm. The motion of various granules (organelles) can be tracked and compared to the Brownian motion investigated earlier. V. Microscopy Wet specimens (living cells or suspensions of synthetic beads) are viewed on a Zeiss Axiovert 200 inverted microscope with either Kohler or darkfield illumination.