Biology Reference
In-Depth Information
CHAPTER
Interactions
20
Kenichi G.N. Suzuki
*
,{
, Rinshi S. Kasai
*
,{
, Takahiro K. Fujiwara
*
, and
Akihiro Kusumi
*
,{
*
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan
{
Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
{
National Centre for Biological Sciences (NCBS)/Institute for Stem Cell Biology and Regenerative
Medicine (inStem), Bangalore, India
Single-Molecule Imaging
of Receptor-Receptor
CHAPTER OUTLINE
Introduction ............................................................................................................ 374
20.1 Receptor Expression and Fluorescent Labeling .................................................376
20.1.1 Expression of Receptors in Cell Plasma Membranes at Low Density 378
20.1.2 High-efficiency Fluorescent Labeling of Receptors ........................ 378
20.2 Single-Molecule Imaging.................................................................................380
20.3 Data Analysis..................................................................................................382
20.3.1 Determination of Receptor Monomer, Dimer, and Oligomer
Fractions in Single-Color Experiments .......................................... 382
20.3.2 Determination of Receptor Dimer Lifetimes in Single-Color
Experiments............................................................................... 384
20.3.3 Colocalization Lifetimes of Receptor Dimers in Dual-Color
Experiments............................................................................... 385
20.3.4 Outline of the Theory for Estimating Colocalization Lifetimes ......... 386
Acknowledgments ................................................................................................... 387
References ............................................................................................................. 387
Abstract
Single-molecule imaging is a powerful tool for the study of dynamic molecular in-
teractions in living cell plasma membranes. Herein, we describe a single-molecule
imaging microscopy technique that can be used to measure lifetimes and densities of
receptor dimers and oligomers. This method can be performed using a total internal
reflection fluorescent microscope equipped with one or two high-sensitivity cam-
eras. For dual-color observation, two images obtained synchronously in different
