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
94720-7300; 510-642-5515; http://www.advancedlab.org, tcolton@berkeley.edu
*Supported by a donation from Stanford Research Systems
I. Introduction
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
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.
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.
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 - 100 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.
VII. Intracellular Transport in Onion Cells
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.
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.
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.
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 812 hours/week in lab).
• 35-55 students per semester.
IV. Simulating Brownian Motion
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.
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.
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.