Biology Reference
In-Depth Information
within the center of the observation volume is critical for accurate molecular bright-
ness determination of fluorescence-tagged membrane receptors. Positioning of the
plasma membrane in the center of the observation volume is best achieved by scan-
ning the sample along the
z
-axis while simultaneously monitoring the photon counts
per molecule. The
z
position corresponding to the maximal photon counts per mol-
ecule is selected for FCS recording. FCS measurements generally are made on the
upper plasma membrane, directly above the cell nucleus. We have discovered sev-
eral factors that influence the shape of the cell and thus the ability to achieve optimal
membrane position within the observation volume including the cell type, the recep-
tor being expressed, and the choice of substrate for cell adhesion. Control samples
should most closely approximate the conditions used for the receptor being studied.
SUMMARY
During the past four decades, fluorescence fluctuation spectroscopy has evolved into
a highly sensitive method for monitoring protein dynamics in living cells. Laser
scanning confocal microscopes fully equipped with sensitive photon-counting detec-
tors and FCS analysis software have greatly expanded the application of FCS to ad-
dress current issues in cell biology research at the single-molecule level. In this
chapter, we reviewed a basic method for monitoring receptor-receptor interactions
using FCS and molecular brightness analysis. Once a technique used predominantly
by biophysicists and chemists, commercial availability of sensitive instrumentation
and step-by-step protocols for performing FCS experiments are making the FCS
method more accessible to researchers in general.
Acknowledgments
Funding for this work is provided by NIMH R21MH086796 to KHD.
References
