Work in our lab makes use of advanced methods in fluorescence spectroscopy and imaging. Central to these methods is a technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), which very accurately measures structure and dynamics in biological membranes.
An inherent limitation to biological fluorescence imaging is the optical diffraction limit, which is larger than the size of typical protein assemblies in membranes. PIE-FCCS is a method that uses time resolved fluctuations in the signal to gain single molecule sensitivity. Experiments are conducted on a customized multi-platform microscope (photo above, schematic below). Two series of laser pulses are interleaved in time so that individual photons can be uniquely assigned to an excitation laser by single photon counting detectors and electronics. The microscope is also equipped with TIRF excitation and and EM-CCD for single molecule tracking experiments.
We are interested in further development of time-resolved fluorescence methods including super-resolution correlation methods. As an example of our tinkering, Here’s an animated GIF of the laser launch that directs light into the microscope.
Comar, W. D., Schubert, S. M., Jastrzebska, B., Palczewski, K. and Smith, A. W. (2014) Mobility and clustering of the opsin G protein-coupled receptor in live cells with time-resolved fluorescence spectroscopy. Journal of the American Chemical Society 136, 8342.
Endres, N. F.*, Das, R.*, Smith, A. W.*, Arkhipov, A., Kovacs, E., Huang, Y., Pelton, J.G., Shan, Y., Shaw, D.E., Wemmer, D.E., Groves, J.T., Kuriyan, J.(2013) Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor. Cell 152, 543-546. *Equal Contribution