Biophysics:
to investigate biological
systems at the single-molecule level. With a new
thrust in biophysics, the Quantum Physics Division aims
to investigate critically important biological systems at
the single-molecule level, drawing upon our measurement
expertise and experience with atomic and quantum systems.
INTENDED OUTCOME AND
BACKGROUND
As JILA and the Quantum Physics Division look to the future, it is
clear that an important scientific revolution is presently taking place in the
area of biophysics. Accordingly, we are evolving a part of our research program
in this direction to help NIST contribute to and be a meaningful part of this
scientific future.
Our strengths - namely, our ability to build institutional bridges to
university departments that are already strong in this area, a superlative
infrastructure that serves to extend the quickness of our eyes and the reach of
our hands, our experience in manipulating and measuring similarly sized atomic
and quantum-mechanical systems, and a reputation that allows us to attract and
successfully hire the best and brightest of today's young scientists - all
suggest that we can hope to become a significant contributor to this scientific
revolution. We have therefore embarked on a program in biophysics that is to be
carried out in close collaboration with the University of Colorado, in
particular with the Department of Molecular, Cellular, and Developmental
Biology, and with the Biochemistry Division of the Chemistry Department.
We expect that bridging JILA to additional departments will enhance the very
productive, interdisciplinary character of JILA. Most importantly, we expect to
be able to find a niche where, by capitalizing on our existing expertise, we
will be able to bring this program to the same very high stature as we have for
our other programs.
This year, three research projects were initiated, with NIST and other-agency
support. They build on our fundamental laser skills and chemical-physics
experience, and contribute as well to the rapidly evolving NIST research
programs in nanotechnology and single-molecule biophysics.
Accomplishments
Fluorescence Microscopy Studies of Biomolecular Conformational
Dynamics
This biometrology project is based on ultrasensitive time-, color-, and
polarization-resolved fluorescence detection of single biomolecules
(specifically, dye-labeled DNA and RNA oligomers) in a high-numerical-aperture
confocal microscope.
The operation of this apparatus is as follows. A pulse train from a mode-locked
laser (532 nm, doubled Nd:YAG, 80 MHz repetition rate) is focused
into an aqueous sample with a water-immersion microscope objective
(NA = 1.3), thereby illuminating  0.1 femtoliter of solution. For
sufficiently dilute samples of labeled DNA/RNA, this corresponds to less than
single-molecule occupancy in the detection region, which permits laser-induced
fluorescence from single molecules to be unambiguously monitored.
The resulting weak fluorescence is collimated, separated from the
108
fold stronger, incident laser with high-rejection dichroic filters, sorted by
both polarization and color, and finally imaged on single-photon-counting
avalanche photodiodes. The individual photon events are then efficiently sorted
by color and polarization and stored as a function of time-after the incident
laser pulse. The fluorescence dynamics of the biopolymers are then extracted
via time-correlated, single-photon counting. This permits measurement of
fluorescence decay rates of the labeled DNA/RNA species, or
fluorescence-resonant-energy-transfer (FRET), between donor and acceptor dyes
on a single DNA strand.
In a complementary thrust, we are developing methods for immobilizing
dye-labeled biomolecules in aqueous gels to allow us to measure fluorescence a
nd to image single molecules by raster-scanning of the laser over the sample
with a precision, PZT (piezoelectric transducer), servo-controlled stage.
The combination of FRET, immobilized labeled biomolecules, and ultrasensitive
single-molecule detection methods offers new opportunities for directly probing
the extremely important conformational dynamics of biomolecules in real time,
e.g., folding and unfolding. This class of information is of crucial relevance
to issues concerning RNA-based enzymes, so-called ribozymes.
Single-Biomolecule Electrophoresis
A second new biometrology project is single-molecule electrophoresis. The
apparatus for these studies is based on wide-field microscopy through a thin
gel-electrophoresis cell, in which weak electric fields (1 V/cm to
5 V/cm) are used to coax single DNA molecules through a 20 µm × 20 µm field of view.
The DNA molecular motion can be studied by labeling with highly fluorescent
dyes that are illuminated with cw 488 nm laser excitation and detected by
imaging on an intensified, charged coupled device (CCD) camera. The sensitivity
of the method is sufficient to image at a 10 Hz frame-repetition rate.
This permits monitoring of single DNA electrophoretic dynamics in real time.
These sorts of studies permit detailed tests of "reptation" models of
electrophoresis and can begin to address issues in improving separation
efficiency in the limit of high biomolecular strand lengths. Also of interest
are kinetics/dynamics of protein-DNA/RNA binding, which are relevant to cell
regulatory processes and would now be amenable to detection at the
single-molecule level.
Single-Molecule Measurement with Nanometer Resolution
The biochemical cycle of mechano-enzymes generates a force and a displacement
that can be measured at the single-molecule level. The third new project aims
to determine how motor proteins transduce chemical energy into physical motion.
This research focuses on developing assays and precision instrumentation to
measure the properties of single DNA-based molecular motors. The enabling
technology is optical tweezers, a focused laser beam that can manipulate
micrometer-sized beads in solution, allowing measurements of position and force
in the nanometer and piconewton ranges.
© Geoffrey
Wheeler
Figure 3. Using a modified microscope, Tom Perkins measures the motion
generated by a single enzyme moving along DNA. |
Measurement of steps and stall forces provide fundamental information on the
mechanics of motion. For many enzymes, different proposed mechanisms of motion
predict different step sizes. Steps have been seen for myosin along actin
(5.5 nm) and for kinesin along microtubules (8 nm).
Enzymatic motion along the DNA is measured by anchoring the enzyme to a surface
and monitoring the position of an optically trapped bead attached to the DNA's
distal end. To date, unitary steps of DNA-based molecular motors have been too
small to resolve. Their presumed step sizes are 1.4 nm or smaller, but
such steps may be attenuated by the compliance of DNA.
Building on our demonstration that 0.3 nm steps of a stuck bead can be
resolved, we are building a microscope for measuring 1 nm motion along DNA.
The electronics for this microscope are a high bandwidth (250 kHz)
quadrant photodetector connected to a differential normalizing amplifier. These
detectors do not exhibit wavelength-dependent reduction in bandwidth as seen in
earlier designs. By incorporating PZT mirrors instead of acousto-optic
deflectors, we eliminate the nanometer-sized, artifactual steps observed to
arise from standing waves inside the acousto-optic crystal.
First strategic focus |
Second strategic focus |
Third strategic focus
"Technical Activities 2002" - Table of Contents |
