Methods

The Rizzo Lab uses a variety of techniques to quantify cellular processes and develop new reagents.

FRET Microscopy

The Anisotropy Förster Resonance Energy Transfer (AFRET) method can be used to detect protein-protein interactions between two fluorescent protein conjugated targets. The large size of the fluorescent proteins slows their rotational diffusion. Thus, when excited by polarized light, the emitted photons have virtually the same orientation, or polarization, as the excitation source. Such probes are said to have a high anisotropy. Energy transfer to a second probe has the effect of depolarizing the fluorescence by introducing photons coming from geometries random respective to the original excitation source. We can take advantage of this phenomenon to detect FRET between two interacting proteins by using fluorescence polarization microscopy. Excitation light is filtered through a linear polarizer, and parallel and perpendicular fluorescence polarizations are separated using a Photometrics Dual-View separation device.

TIRF Microscopy

Total-Internal Reflection Fluorescence (TIRF) microscopy is used to selectively illuminate structures at or near the plasma membrane. Reflection of laser light off a glass coverslip generates a small evanescent wave, capable of exciting fluorescent molecules within ~100 nm of the coverslip. We use this method to quantify insulin secretory granule fusion in pancreatic β cells. TIRF equipment was obtained through funds made available by the American Recovery and Reinvestment Act of 2009 and administered through a NIDDK grant program (3R01DK077140-02S1).

Structure-Based Molecular Design

Novel genetically-encoded optical biosensors designed in the lab are informed by protein structures. We used the X-ray crystal structure of Cerulean cyan fluorescent protein to inform optimization of the second and third generation Cerulean proteins.