Single Molecule Detection and Spectroscopy
 The experimental, numerical, and theoretical development of methods and
applications for single molecule detection and spectroscopy, initiated in CLA in
1989, continues to remain one of the focus areas principal research. The
experimental techniques generally involve detection of single photons of light
emitted by molecules of interest and the characterization of the physical
properties of the photons on a one-by-one basis. Interpretation of results
frequently involves fundamental physics issues in quantum and non-linear optics.
Applications in the rapidly growing biotechnology areas increasingly require
incorporation of state-of-the-art optical methods such as single molecule
spectroscopy, and the emphasis of the CLA program continues to be ultrasensitive
fluorescence detection in water-based solutions for biotechnology applications.
Over the past year, specific accomplishments have included:
- (1) Development of neural network analysis methods for spectroscopically
distinguishing different individually detected molecules. Aspects of this
work were part of the Ph.D. dissertation topic of a CLA student, who
graduated in May 2000.
- (2) Development of maximum entropy statistical methods for spectroscopic
analysis of signals from fluorescence photon counting instrumentation. Last
year CLA completed a series of research contracts on this topic for a
company interested in incorporating the methods into their existing
commercial scanning microscope instrument. This year the emphasis has been
on technology transfer of methods and findings, and more recently on
developing the results of the research into a format suitable for
presentation at conferences and in the literature.
- (3) Development of custom single molecule detection and spectroscopy
instrumentation. In prior years, CLA had developed an instrument for rapid
and efficient single molecule detection within a flow cell. This instrument
was disassembled and the components were used to build a prototype confocal
epi-illumination microscope for single molecule detection and fluorescence
correlation spectroscopy. The prototype was rebuilt twice with successive
improvements and then used to conduct many series of experiments for
characterization of single molecule interactions. Meanwhile, components for
building a new single molecule detector, including an argon/krypton ion
laser, were specified by CLA, purchased by an industry sponsor, and then
used to build yet another instrument. Following a period of technology
transfer, the instrument was transferred to the company laboratories and
optically aligned by CLA in mid December, 1999. A confocal
epi-illumination instrument for single molecule detection is in use in
the CLA labs.
- (4) Single molecule detection experiments. While most of the single
molecule interaction experiments conducted with the confocal epi-illumination
instruments described above were for the purpose of investigating novel
fluorescently-labeled bio-molecules for an industry sponsor, one series of
experiments was conducted in a collaboration with personnel at NASA
Marshall. Results were obtained to demonstrate the feasibility of using
single molecule methods to obtain new information on protein crystallization
in microgravity, and more specifically, on the interactions between protein
molecules prior to crystallization. The experiments and
aspects on the construction of the instrumentation are part of the MS thesis
of a CLA student, who is scheduled to graduate in summer 2000.
- (5) Single molecule imaging microscope. The Figure shows the
stage
of a microscope custom built by CLA over the past year using a Zeiss
axiovert frame and other
components that were evaluated and specified by CLA and purchased by an
industry sponsor. The microscope has been used to perform experiments
involving the recording of movie images of individual fluorescent molecules
in aqueous solution. The figure shows a single image
in which bright spots are due to fluorescence light collected from
individual molecules. Laser excitation is provided by total internal
reflection at the water/glass interface of a fused silica prism. Photon
imaging is provided by a state-of-the-art intensified-camera with a 5 MHz
PCI interface. Huge amounts of image data are generated in the experiments
and future work may benefit from the advanced parallel processing
computational facilities that are being developed at UTSI/CLA. The
microscope has recently been transferred to the industry sponsor. It is
planned to build a similar instrument with improvements in the year ahead.
- (6) Monte Carlo modeling of single molecule detection. CLA has
collaborated with the industry sponsor's personnel to develop a Monte Carlo
model of single molecule imaging, written in MatLab. Components of the model
are based upon earlier models developed by CLA in the C language. The new
model is being used to help design ongoing and future experiments.
Investigator: Dr. L.M. Davis
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