Single Molecule Biophysics in Live Cells

Fluorescence imaging is the most ubiquitous technique used for biological imaging, especially on the cellular level. Recently, single molecule fluorescence methods have been shown to provide valuable information to biological studies of relevance. The removal of ensemble averaging from hundreds or thousands of fluorescent molecules provides additional information on nanoscale biological functionality that simply cannot be garnered in other ways. Fluorescence methods can be applied in non-invasive physiologically relevant environments so that little to no extrapolation must be made in interpretation of results. Organic fluorophores and quantum dots have both been shown to provide fluorescence blinking (intermittency) that is often indicative of local (nanometer) environments and reaction pathways. This blinking behavior is only observable with single molecule or single quantum dot methods since the statistical variation in total fluorescence that arises from the blinking of tens or hundreds of fluorophores is not measureable.

We have recently developed a simple, unique laser scanning confocal microscopy system that allows us to scan the optical beam using a unique scanning system that is much easier to implement than conventional mirror systems available in commercial systems. It also removes the need for sample scanning, which is often not desirable for scanning live biological systems. In addition, we will discuss the use of quantum dots in biological single molecule systems. In particular, we will discuss unique uses for the blinking behavior from quantum dots that allow the addition of new contrast mechanisms for single molecule biological imaging. We will present the application of these methods to myosin V motion with 4 nm accuracy and single molecule imaging in live cells.