Single Molecule Spectroscopy
Starting with single molecules provides the ultimate level of reductionism needed to take on such a complex system. We can seek a full understanding of each component, starting with neurotransmitter receptors and scaffolding, and work our way towards higher levels of complexity. Our solution is to watch it all happen in real time with video microscopy.
In the Bowen Lab
The Bowen lab uses single molecule methods to study the structure, localization, interactions and activity of proteins. The lab is equipped with two Total Internal Reflection Fluorescence (TIRF) microscope systems capable of single molecule detection. We have seven different lasers giving us eight colors of excitation and photoactivation capability. We use synthetic dyes and fluorescent proteins to image recombinant proteins as well as fixed and live cells. We exploring the use of Image-based stabilization to permit long-term imaging and increase resolution. The optical systems are under constant revision in house to incorporate the latest advances and technology.
Magnification has been with humankind for more than a millennium and, by the close of the 20th century, we had seen light emitted from single molecules. Diffraction limited optical microscopy, when combined with laser excitation and electron-multiplied CCD cameras, allows us to follow individual molecular trajectories to “watch” individual biological molecules doing their job. This advance has revealed a new level of detail in molecular understanding of protein structure and enzyme activity. A population of chemically identical molecules does not always behave the same or work in synchrony. This heterogeneity can only be resolved by probing each molecule, which can reveal details of mechanism obscured by classical methods that measure large ensembles, containing billions or more molecules, at once.
Single molecules can be detected at optical resolution, which is limited by the wavelength of light and the physical properties of the microscope system. Unfortunately, the size of synapse is close to the size of optical resolution so everything becomes blurred. This is made worse when molecules are packed closely together, so their images start to overlap. In the previous decade, several scientists realized that we could determine the location of a single molecule to a precision greater than optical resolution by finding the center of the blurred spot. A further advance was the realization that by turning on molecules one by one, we can reconstruct a super-resolution image of the sample.