Biomedical Engineering Reference
In-Depth Information
based “cameleon”, which is the genetically encoded calcium-sensitive fluorescence
protein, was reported to visualize odorant-evoked intracellular calcium concentra-
tion changes [
74
,
75
]. Cameleons for measuring the level of intracellular Ca
2+
ions
are genetically engineered proteins that can detect free Ca
2+
and are comprised of
tandem fusions of a blue or cyan mutant of the green fluorescent protein (GFP),
calmodulin, calmodulin-binding M13 fragment of myosin light chain kinase. If
Ca
2+
binds to the calmodulin of these cameleons, the distance between the two GFP
variants shortens and FRET follows. The light emitted from the first GFP variant
(458 nm) excites the other GFP variant (480 nm), and the second variant emits a
photon with an even longer wavelength (520 nm). Hence, the FRET between the
GFP variants can be used to monitor the localized Ca
2+
signals in olfactory cells
upon ligand stimulation [
75
].
BRET studies are conducted commonly during the heterologous expression of
GPCRs in mammalian cells [
76
]. The BRET method is based on the FRET occur-
ring in some marine species (i.e.
Renilla reniformis
) and leading to a non-radiative
energy transfer between an energy donor and an energy acceptor [
70
]. The ho-
modimerization of the hOR17-40 protein and its involvement in receptor activa-
tion upon odorant ligand binding was addressed by BRET approaches [
71
] (see
Fig.
11.4
). This study demonstrated that the BRET signal increases upon odorant
ligand stimulation, which supports a conformational change of the OR. Modu-
lation of the BRET signal upon stimulation with increasing amounts of helional
displays a bell-shaped curve and a two-state model (active and inactive states) of
OR addressed by different ligand concentrations. Most BRET studies in GPCR
rely on the detection of the consequences of GPCR activation by monitoring the
conformational change of the receptor dimer [
77
-
79
]. Also, a cAMP sensor using
YFP-Epac (known as a cAMP-regulated guanine nucleotide exchange factor)-Luc,
a quantitative cAMP BRET-sensor assay able to monitor he modulation of cAMP
levels in cells, has been developed and used to follow G protein activation by GP-
CRs [
80
] (Fig.
11.6
).
11.2.4
Total Internal Reflection Based Sensor
Total internal reflection fluorescent microscopy (TIRFM) provides high signal
to noise ratio by collecting signals form a thin region of a biological specimen [
82
].
In total internal reflection, one can expect there will be evanescent wave propa-
gating into the second medium at the boundary according to Maxwell's equation.
Assuming harmonic oscillation of an electric field of the incident light, the transmit-
ted electric field, denoted
E
T
can be obtained through the combination of Maxwell
equation and Snell's law as shown in equation, derived from the Helmholtz equa-
tion with dispersive, homogeneous, isotropic conditions.
ω
ε
2
2
∇+ =
E
E
0
2
c
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