Robert Youker

By Robert T. Youker

The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.

Part of the book: Photon Counting

By Robert T. Youker and Danielle Voet

G-protein-coupled receptors (GPCRs) are seven transmembrane receptors that form the largest superfamily of signaling proteins, and the family members function in a diverse array of metabolic pathways including cardiac function, immune response, neurotransmission, smell, taste, cell differentiation and growth, and vision. It is becoming clear that alteration in the quaternary structure of the GPCR receptor can have a profound impact on signaling capabilities. Biochemical, biophysical, physiological, x-ray crystallographic, and computational methods have been used over the last 40–50 years to study the structure and function of GPCRs. Evidence from these studies confirm that GPCRs can be organized as monomers, dimers, and higher-order oligomers. However, many times, these methods require extraction of the receptor from its native environment and high levels of expression and only provide a snapshot of information. A need arose for techniques that could measure the assembly and disassembly of receptors at few-to-single molecule resolution in their native environment at fast time scales. In the last 20 years, fluorescence fluctuation techniques have filled this need and provided new insight into the dynamics of GPCR organization in the absence and presence of ligands, agonists, and antagonists. In this book chapter, we provide a brief introduction to GPCR structure and function [Section 1]. An overview of the theoretical basis for fluorescence fluctuations techniques (FFTs) and how FFTs can be used to study the oligomeric structure of GPCRs in live and fixed cells is explained [Section 2]. We discuss the advantages and limitations of FFTs [Section 3], and finally, we summarize select case studies on GPCR structure and function revealed by FFT experiments [Section 4].

Part of the book: Fluorescence Methods for Investigation of Living Cells and Microorganisms