What is single molecule analysis?
Single molecule analysis in biology has been in use in a variety of areas. A few examples include electron microscopy, patch clamp methods in membrane channel studies, and methods involving optical tweezers in studies of motor proteins. Use of the fluorescence microscope for single molecule detection became possible with new developments in optical systems and the availability of high sensitivity cameras. Although only a limited number of studies have been published using fluorescence microscopes to detect single fluorophores for bio-molecular interaction studies to date, recent developments in total internal reflection fluorescence microscopy appear particularly suited for this purpose. Briefly, the scheme is as follows: Each molecule to be observed is fluorescently labeled. Different molecules can be labeled with distinct fluorophores if necessary. One of the components of the reaction to be studied (typically unlabeled) is immobilized at a very low density on the surface of a cover glass in contact with the reaction mixture containing the labeled components. This surface is illuminated by an excitation beam at a shallow angle, such that the beam experiences total internal reflection without penetrating into the reaction mixture. Under these conditions, the fluorophores situated within a fraction of the wavelength from the reflecting surface will be excited by the evanescent field generated by the illumination, but the bulk of fluorophore away from the surface will not be excited. Thus, practically the only labeled molecules observed would be limited to those that are bound to the surface for a substantial period of time. If the density of the surface immobilized molecules is low enough compared to the optical resolution of the microscope, each binding event of the labeled molecule in solution to a single surface immobilized molecule will be observed as a fluorescent spot at a fixed point within a field of view. The real time on-off events can be recorded using a highly intensified video camera or digital CCD camera. By using dichroic beam splitters and multiple cameras, one can in principle simultaneously observe the behavior of differentially labeled molecules in the reaction.
Advantages of the single molecule analysis and why it's suited
for our project.
The experimental approach briefly outlined above offers unique advantages over conventional biochemical kinetic analysis methods for the type of complex macromolecular assembly processes we are interested in characterizing in our project. The end products of the assembly processes involve multiple components, and are very stable, indicating involvement of the practically irreversible step(s). The assembly steps may not be ordered. Thus, there are likely to be multiple kinetic paths for assembly and the kinetic parameter for each unique step would be distinct from each other. In addition, there may be a significant contribution of non-productive irreversible steps in the process, possibly depending on the experimentally convenient reaction conditions available. There are simply too many independent variables to narrow down the possible kinetic models by a set of conventional experiments. To make matters even more difficult, the apparent overall rate of the assembly process of these reactions tends to be very sensitive to minor variations of reaction conditions, making quantitative kinetic experiments difficult, if not impossible. One of the major difficulties associated with the conventional approach derives from the fact that all the measured parameters represent population averages, and if there is heterogeneity in the population, in many cases it is very difficult to deconvolute the mixture. The single molecule approaches have the potential to make dissection of a mixture of multiple reaction paths relatively simple. Therefore, if successful, the information obtained through this approach will have a great complementary value to that obtained by more conventional experiments.
What needs to be developed ?
Instrumentation:
The microscope system: We have designed and are in the process
of fabricating the first prototype system intended for this purpose. The system
comprises two different wavelength laser illumination sources, through-the-objective-lens
type total internal reflection illumination optics with a modified nose piece,
standard inverted microscope stand, dichroic beam splitter and focusing optics,
and two intensified digital CCD cameras with adjustable rapid frame rate. This
system is a modification and improvement (hopefully) over the design developed
in the Yanagida lab in Osaka Japan, where several variations of this type of
instrument have been developed and used for the study of motor proteins and
for the analysis of protein-DNA interactions. Because of the added requirements
for our research, we will be testing several new design features with our instrumentation.
Another item that must be custom designed and fabricated is the flow through
sample cell with sample mixing capabilities. Because of the nature of our reactions
of interest, a sample cell for a steady state reaction mixture is not very useful.
Thus, we will be fabricating and testing sample cells for our microscope system
with different types of sample mixing capabilities.
Materials: We will be developing and testing the fluorescence labeled reaction
components. We can readily prepare fluorescence labeled DNA substrates. We will
test several ways of making fluorescence labeled protein samples of our interest.
One way is to make GFP fusion proteins. The other way is to mutate out all (or
all except one at selected positions) surface-exposed cys-residues from the
protein and add single cys at one of several surface-exposed positions. Cys-reactive
fluorescent reagents will be used to label the protein. A number of modified
proteins will be made and tested for their activity. If these methods fail to
yield labeled active proteins, other structure specific chemical reactions will
be tested.
Last updated, August 12th, 2002
