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Single Molecule Micro-Spectroscopy

Single Molecule Probes of Biological and Polymer Systems

In recent years the development of techniques to measure and manipulate single organic or biological molecules is facilitating a new understanding of molecular interactions and dynamics in biophysics, cell and molecular biology, and polymer science. In Biophysics and molecular biology, the ability to identify, track, and measure the conformation and interaction of e.g., single proteins or RNA molecules, is leading to new insights in protein folding and RNA interactions and functionality, and is helping to validate models of protein folding or molecular dynamics. The extension of single molecule techniques to track and measure individual biomolecules in living cells has the potential to revolutionize our understanding of cell signaling. In polymer science, visualizing the motion of individual molecules aids enormously in understanding flow, phase transitions, and other polymer dynamics.

Single molecule measurements as applied to polymer or biomolecular systems tend to fall into three categories; optical measurements (typically utilizing single molecule fluorescence or nonlinear spectroscopies), electronic measurements (e.g., single ion-channel measurements), and force measurements using optical or magnetic tweezers or microcantilevers. Techniques for manipulating single molecules might involve magnetic tweezers, optical tweezers, micro or nanofluidcs, and bioMEMs devices.

Our program specializes in the use of optical techniques for physical and spectroscopic measurements on single molecules in both biophysical and polymeric systems.
  1. Studies of rotational dynamics of single molecules in Polymer thin films.

  2. Characterization of a Guanosine-Analogue, 3-MI, For Use as a Single Molecule Probe.

  3. Quantitative Single Molecular Pair FRET.
1. Studies of rotational dynamics of single molecules in Polymer thin films.
(K.D. Weston and L.S. Goldner)

We use polarization modulation confocal microscopy to measure the absorption dipole orientation and reorientation dynamics of individual dye molecules physisorbed to glass and embedded in thin, spin-cast polymer films under ambient conditions (Ref. 3). Surprisingly, discrete jumps in absorption dipole orientation were observed for a significant fraction of dye molecules in all samples tested. A sub-population of dye molecules that is stationary on the time scale of these experiments (32 s) is observed and persists even at high excitation power. Figure 1 shows an image acquired while modulating the polarization of the scanned excitation light, giving rise to apparent stripes that run through stationary molecules and making it very easy to identify molecules that change their orientation. A dependence of the reorientation dynamics on film thickness was identified: DiIC18 molecules reorient with higher frequency and a broader distribution of jump rates in progressively thinner polymer films.

Figure 1
Figure 1. Demonstration of the technique used to quickly assess the location and fraction of rotationally mobile molecules in a sample. The beginning of each scan line (horizontal) is synchronized with the initiation of polarization rotation. The bright and dark stripes correspond to excitation light parallel to or perpendicular to the absorption dipole orientation of a molecule. Discontinuity in the stripes indicates a molecule has reoriented during imaging. In (a) a 300 pixel × 600 pixel (10 µm × 20 µm) image is shown. In (b)-(g), expanded views of 6 different molecules taken from the image of (a) are shown. The molecule in (b) is stationary while the molecules in (c)-(g) are rotationally active.

2. Characterization of a Guanosine-Analogue, 3-MI, For Use as a Single Molecule Probe.
(J.E. Sanabia, L.S. Goldner, P.-A. Lacaze, M.E. Hawkins, J.T. Krug)

We characterized the guanosine-analog 3-MI [3-Methyl-8-(2-deoxy-β-Dibofuranosyl)isoxanthopterin], a pteridine widely used in studies of DNA binding and dynamics, using UV fluorescence fluctuation correlation spectroscopy (FCS) measurements (see Figure 2). The photon count rate per molecule, for both monomeric 3-MI and a 3-MI-containing oligonucleotide, and a comparison with a bright laser dye, are shown in Fig. 2, right side. For the monomer, we find a photon count rate per molecule above 4 kHz and a signal to background ratio of 5. For incorporated 3-MI, both parameters are a factor of 4 smaller. We investigated triplet and photobleaching behavior of 3-MI and the possibilities of using this analog in single molecule studies of DNA dynamics. The conclusion was that this is a good dye for use in burst experiments but unless a way can be found to minimize photobleaching, it will probably not work in a single molecule tracking experiment since it photobleaches too fast. Comparisons are made to the behavior of stilbene 3, a brilliant laser dye.

Figure 2. Left        Figure 2. Right
Figure 2. Left Side: Top: Guanosine. Bottom: 3-MI. Right Side: The count rate per molecule, η, is plotted as a function of the input intensity. Diamonds: stilbene 3. Squares: 3-MI monomer. Asterisks: 36 mer oligo containing 3-MI.

3. Quantitative Single Molecular Pair FRET.
(G.M. Lowman, J. Tang, P.B. Yim, E.S. Dejong, L.S. Goldner)

A large portion of our work involves developing a quantitative understanding of fluorescence resonance energy transfer probability in single donor-acceptor pairs. This involves developing quantitative measurement protocols as well as models to help understand the various mechanisms that become important in single molecule measurements. The Förster distance, R0, which is defined to be the distance at which the probability of energy transfer is 1/2, is well known to be a function of both the spectral properties of the fluorophores (overlap integral and donor quantum yield) and their relative orientation, although the latter is often ignored through the assumption of freely-rotating dyes.

Figure 3. Left      Figure 3. Right

Figure 3. Left: Model of 8mer RNA with Cy dyes attached at the 3’ ends. Right: (A) Donor (green and symbols) and acceptor (red) data from a Cy3-Cy5 pair attached to the 8mer as shown on the left. (B) FRET ratio calculated from the data and background. (C) Proximity ratio calculated from the data and background. See Ref. 1 for details regarding how the data were acquired and analyzed.

We have developed measurement methods for accurate measurement of FRET from a surface-tethered, donor-and-acceptor-labeled A-form RNA duplex. We compare single molecule FRET data with polarization anisotropy data and structural molecular models, based on NMR and X-ray crystallography, which include both the angular relationship and distance information of the dye pair. This analysis shows that for surface-tethered RNA duplexes the dyes assume neither a freely-rotating nor a base-stacked conformation along the RNA chain and so the usual assumption of free-rotation is not possible. This makes using FRET as a "molecular ruler" a difficult proposition, since in fact for these RNA systems it is more of a "molecular protractor."

Our long-term goal is the development of quantitative single molecule FRET as a complement to NMR for elucidating RNA secondary structure.

Resources

Several confocal and TIRF microscopes with cooled ccd cameras and photon counting detectors; Atomic Force microscopes and a molecular puller; optical tweezers; femtosecond tunable pulsed lasers; access to complementary scanning microscopies such as SEM, AFM, and NSOM.

Representative Publications

  1. P.B. Yim, X. Zhang, E.S. DeJong, J.M. Carroll, J.P. Marino, and L.S. Goldner,
    "Single RNA Kissing Complexes Studied by Fluorescence Resonance Energy Transfer," (2.3 MB preprint Get PFD file)
    in Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications III, ed. by D.V. Nicolau, J. Enderlein, R.C. Leif, D.L. Farkas, and R. Raghavachari,
    Proc. SPIE 5699 (2005), 450-460.


  2. J.E. Sanabia, L.S. Goldner, P.-A. Lacaze, and M.E. Hawkins,
    "On the Feasibility of Single Molecule Detection of the Guanosine-Analogue 3-MI," (195 kB Get PFD file)
    J. Phys. Chem. B 108(39), 15293-15300 (2004).


  3. K.D. Weston and L.S. Goldner,
    "Orientation imaging and reorientation dynamics of single dye molecules," (837 kB Get PFD file)
    J. Phys. Chem B 105(17) 3453-3462 May 3 (2001).

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For technical information or questions, call:
Lori S. Goldner
Phone: (301) 975-3792
Fax: (301) 840-8551
Email: lori.goldner@nist.gov

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Online: November 1999   -   Last updated: January 2006