Biomedical Engineering Reference
In-Depth Information
a
b
c
0.8
Cameleon
0.4
0
M13
CaM
0
50
100 150 200 250
time (se
c
)
Ca
2+
GCaMP
23°C
M13
CaM
17°C
17°C
YFP image CFP image
or
RFP image GCaMP image
0
50
100 150 200 250
Ca
2+
time (sec)
Fig. 1.3
(
a
) Two of major GECIs: Cameleon and GCaMP. Both contain fl uorescent protein(s),
calcium-binding sites (
black dots
) of calmodulin, and calmodulin-binding site M13. (
b
)
Simultaneous dual fl uorescent imaging is usually required for calcium imaging. Cell-specifi c
expression of the indicators enables to detect single neuronal signal. Thermosensory neuron AFD
is pictured here. Scale bar indicates 100
m. (
c
) An example of temperature-responding activity of
AFD thermosensory neuron. In this case, YFP/CFP increase with temperature increase
μ
several improved variants and further the improvement of such calcium indicators is
still actively promoted because of its utility. We shortly describe about these two
GECIs for
C. elegans
studies.
1.4.1
Principles of GECIs
Different principles are implemented in Cameleons and GCaMPs. Cameleons consist
of two fl uorescent proteins, calcium-binding domains of calmodulin, and calmodulin-
binding peptide called M13 (Fig.
1.3a
, Miyawaki et al.
1997
). Four Ca
2+
binding sites
of calmodulin cause conformational change by interacting with M13, and thus, rela-
tive orientation between the two fl uorescent proteins such as cyan fl uorescent protein
(CFP) and yellow fl uorescent protein (YFP) changes. This relative orientation of the
fl uorescent proteins affects to energy transfer from CFP to YFP called Förster
Resonance Energy Transfer (FRET). Eventually, monitoring intensity of YFP/CFP
(Fig.
1.3b
; sometimes differently calculated) or fl uorescence lifetime refl ects concen-
tration of Ca
2+
in the site where a probe expressed. In the case of GCaMPs, circularly
permutated GFP changes intensity of fl uorescence emission depending on the Ca
2+
bindings of the calmodulin domain (Fig.
1.3a
, Nakai et al.
2001
). Improved GCaMP
called GCaMP3 provides high sensitivity and fast response for Ca
2+
concentration
(Tian et al.
2009
); it is becoming quickly popular in the studies of
C. elegans.
To
compensate the artifact caused by the animal movement, microscopic focal plane
change, and photobleaching, another fl uorescent protein with different emission
wavelength such as red fl uorescent protein (RFP) is often used (Fig.
1.3b
).
