Biomedical Engineering Reference
In-Depth Information
Fig. 11.7
A typical FCS set
up on an inverted laser-scan-
ning microscope. In a fixed
focal volume, diffusion and
binding of labeled proteins in
aqueous environment can be
monitored with FCS. To mea-
sure the molecular events in
the membrane, scanning FCS
is employed and the focal
volume is scanned verti-
cally through the membrane.
(Reprinted from Ref. [
89
]
with permission from NPG.)
semiconductor based photodetector, avalanche photodiode (APD). Information
regarding molecular kinetics including can be acquired through FCS analysis in-
cluding diffusion coefficient of analytes (denoted as
τ
d
), average concentration
of analytes (denoted as N), and kinetic factor by external perturbation (denoted
as
τ
f
) as shown in equation 1. In GPCR applications, fluorescently labeling the
molecule of interest is the very first step to utilize this powerful technique. The
detailed information can be found in a review paper by Jakobs et al. [
90
]. Also
the FCS application for single-cell pharmacology is well described by Briddon
et al. [
91
]. Other detailed methods for examining GPCRs including FCS itself,
were well introduced in the topic by Poyner et al. [
92
]. Examples of GPCR ap-
plications are numerous. First, Herrick-Davis et al. determined the diffusion coef-
ficient and oligomeric size of GPCR by using an FCS and photon counting his-
togram (PCH) and concluded that dimeric 5-HT
2C
receptors freely diffuse within
the plasma membrane [
93
]. Gao et al. [
94
] utilized FCS to identify SP-bound
NK1R-containing NLPs for measuring their dissociation constant (~ 83 nM) in
aqueous solution. For general FCS applications in observing intracellular events,
two review papers are very helpful to understand the practical and theoretical ap-
plications of FCS to cell biology [
95
,
96
].
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