Biology Reference
In-Depth Information
Abstract
Fluorescence correlation spectroscopy (FCS) performed using a laser scanning con-
focal microscope is a technique with single-molecule sensitivity that is becoming
more accessible to cell biologists. In this chapter, we describe the use of FCS for
the analysis of diffusion coefficients and receptor-receptor interactions in live cells
in culture. In particular, we describe a protocol to collect fluorescence fluctuation
data from fluorescence-tagged receptors as they diffuse into an out of a small
laser-illuminated observation volume using a commercially available system such
as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the
fluctuations in fluorescence intensity provides information about the diffusion time
and number of fluorescent molecules in the observation volume. A photon-counting
histogram can be used to examine the relationship between fluorescence intensity
and the number of fluorescent molecules to estimate the average molecular bright-
ness of the sample. Since molecular brightness is directly proportional to the number
of fluorescent molecules, it can be used to monitor receptor-receptor interactions and
to decode the number of receptor monomers present in an oligomeric complex.
INTRODUCTION
Fluorescence correlation spectroscopy (FCS) is a single-molecule detection tech-
nique that measures the fluorescence fluctuations of molecules diffusing through
a well-defined volume. Introduced over 40 years ago by
Magde, Elson, and Webb
(1972)
, one of the initial applications of FCS was to analyze the interaction of
ethidium bromide with DNA in solution to measure diffusion and chemical reaction
kinetics. The application of FCS to address such questions in live cells, especially the
measurement of diffusion of cell surface proteins in biological membranes, was lim-
ited by the lack of sufficiently sensitive instrumentation, stable lasers for excitation
and a means to reduce the volume in which the measurements were made. Most of
these concerns were addressed in the early 1990s with the adoption of the confocal
microscope for FCS measurements, providing a sensitive method for monitoring
protein dynamics in living cells (reviewed in
Elson, 2013
).
The main advantage of FCS over other currently used techniques for monitoring
receptor interactions is that it provides real-time information about the two-
dimensional dynamics of single molecules diffusing within a plasma membrane with
diffraction-limited spatial and sub-microsecond temporal resolution. In addition, the
most accurate FCS measurements are made in samples with very low protein expres-
sion levels, making this technique ideal for monitoring receptors at physiological
expression levels. Confocal microscopy-based FCS experiments are performed
by focusing a laser beam into a small diffraction-limited spot (0.3
m
m) using a high
numerical aperture objective to create an observation volume on the order of
0.5
10
15
L(
Fig. 10.1
).
