Poly(acrylamide) Gels for Single-Molecule Imaging and Biophysics

3-D Localization of Single Small Fluorophores Undergoing Restricted Brownian Motion

Figure 1: Fluorescence image (25 micron x 20 micron, 1 s exposure) of single nile red molecules in a PAA gel.


Figure 2: 3-D motion determined by our TIR technique.

R. M. Dickson, S. Kummer, and W. E. Moerner

Since our initial observations of single molecules in 1989, the studies performed by many different groups have significantly furthered the understanding of single molecule behavior in low temperature glasses and crystals. In order to obtain analogous in formation from the behavior of single molecules at room temperature, we are employing near-field and far-field microscopic techniques to better understand biological systems. Although most room temperature single molecule studies have been performed in polymer hosts or on surfaces, we have developed exciting new techniques that have allowed us to study single molecules in aqueous solutions (Please see our recent Science article). Our studies have opened up an entirely new class of systems for physical and in vitro biophysical single-molecule studies. Currently we are investigating photophysical and biological properties of individual, singly-labeled proteins and their environmental interactions. By employing the exquisite local environmental sensitivity that single molecule studies enable, we are deciphering biomolecular mechanisms that are obscured in bulk biological studies.

By employing water-based polyacrylamide (PAA) gels, we have been able to restrict Brownian motion of individual dye molecules and singly-labeled proteins. The random gel matrix enhouses many water-filled cavities ("pores") through which the molecules can move. With these techniques we have directly observed the translational motion of small dye molecules in high acrylamide concentration gels and the motion of small proteins in lower concentration gels. The TIR geometry provides a strongly varying optical intensity in the axial direction, which allows determination of not only the xy position, but also the Z-position of the molecule by its overall brightness (see the lower panel in the figure below). As the acrylamide concentration is increased, the molecular motion becomes increasingly restricted; this means that large single proteins can be trapped in individual gel pores and studied for long times in aqueous environments. This represents a significant advancement over previous methods which were only able to detect the presence of single proteins in solution as the protein diffused (quite rapidly due to Brownian motion) through the focal spot of the microscope. Our techniques allow for long time study of proteins and their behavior in solution (see figure 3 below). Studies of the naturally fluorescent Green Fluorescent Protein (GFP), for example, have identified both blinking and optical switching behavior on the single molecule level (please refer to our recent article in Nature). We have many other experiments in progress and hope to extend both the gel based studies and other solution based biophysical techniques to the understanding of a wide range of biological systems.

Figure 3: 20 micron x 20 micron image of individual Cy-3 labeled BSA (Bovine Serum Albumin) proteins in a polyacrylamide gel.

Figure 4: Dr. Rob Dickson at the TIR microscope.

Recent Publications

  1. "Three-Dimensional Imaging of Single Molecules Solvated in Pores of Poly(acrylamide) Gels," by R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science, 274, 966-968 (1996).
  2. "On/Off Blinking and Switching Behavior of Single Green Fluorescent Proteins," by R.M. Dickson, A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, Nature, 388, 355 (1997).