Ca2+ as a Second Messenger: New
Reporters for Calcium (Cameleons
The intracellular Ca2+
ion concentration has been
found to be associated with a wide variety of cellular
processes (Carafoli, 2003). These include diverse
events such as secretion, fertilization, cleavage, nuclear
envelope breakdown, and apoptosis. Several diseases,
including types of muscular dystrophy, diabetes, and
leukemia, involve proteins that directly respond to or
. Indeed, it may be more difficult to find
cellular processes that do not involve Ca2+
that do. From decades of research we have learned that
the process of Ca2+
signaling consists, in general terms,
of molecules for Ca2+
signal production, spatial and
temporal shaping, sensors, and targets that elicit
changes in biological function. Hence, this has
prompted the development of sensitive signaling techniques
to measure and image submicromolar levels of
] and decode the dynamic Ca2+
the propagation of the signal.
transients have traditionally been measured
using synthetic fluorescent chelators (such as Fura-2
and Quin2) or recombinant aequorin (Grynkiewicz et al.
, 1985: Montero et al.
, 1995). Synthetic molecules
provide a bright fluorescent signal, but these dyes
are not easy to load and gradually leak out of cells at
physiological temperatures. Cellular targeting is also
not specific and some chemical indicators have been
shown not to accumulate well in certain organelles.
Aequorin is targeted easily but it requires incorporation
of the cofactor coelenterazine, is irreversibly consumed
, and is very difficult to image due to
low bioluminescence. By comparison, green fluorescent
protein (GFP) and calmodulin (CaM)-based
"cameleon" probes have been developed and retain
several of the benefits of the aforementioned indicators,
yet also provide significant improvements for in
vivo imaging (Miyawaki, 2003; Zhang et al.
Truong and Ikura, 2001).
The use of cameleon indicators is gradually becoming
more common within the Ca2+
and the literature is rich with examples of various
applications. For instance, fusion of cameleons to specific
signal sequences has successfully sorted them to
nuclei, endoplasmic reticulum, caveolae, and secretory
granule membranes (Isshiki et al.
, 2002; Demaurex
and Frieden, 2003; Emmanouilidou et al.
, 1999). In
addition to their use in detecting rapid stimulusinduced
] transients, genetic studies in which
cameleons were stably expressed in Arabidopsis
stomatal guard cells (Allen et al.
, 1999), nematode
pharyngeal muscle (Kerr et al.
, 2000), or larval thermoresponsive
neurons of Drosophila
(Liu et al.
show that the sensors are also applicable to long-term
monitoring of Ca2+
concentration. This has been
demonstrated further for murine cells where the circadian
rhythm of cytosolic but not nuclear Ca2+
suprachiasmatic neurons was demonstrated
(Ikeda et al.
It is hoped that this article provides some explicit
and practical information relevant to the laboratory
use of cameleon fluorescence resonance energy transfer
(FRET) indicators. Our aim is that it will benefit and
be of interest to colleagues both unfamiliar or experienced
in using fluorescent Ca2+
II. MATERIALS AND
A. Expression and Purification of Cameleons
Enhanced cyan fluorescent protein (ECFP) and
enhanced yellow fluorescent protein (EYFP) expression
constructus (Clontech Cat. No. 6075-1 and 6004-1,
respectively); calmodulin cDNA (M. Ikura); pRSETB
prokaryotic expression vector (Invitrogen Cat. No.
V351-20); Luria broth (LB) media; isopropyl-β-D
(IPTG, Fermentas Cat. No.
R0391); complete protease inhibitor cocktail tablets
(Roche Cat. No. 1697498); Ni-NTA agarose (Qiagen
Cat. No. 1018240); Escherichia coli
BL21 (DE3) strain
(Stratagene Cat. No. 200133); sonicator; EGTA buffer:
KCl, 50 mM
HEPES (pH 7.4), and 10 mM
buffer: 100 mM
KCl, 50 mM
7.4), 10 mM
EGTA, and 10 mM
B. In Vitro Fluorescence Quantitation
Shimadzu spectrofluorometer RF5301; 10-mm pathlength
C. In Vitro Imaging of Cameleons
pcDNA3 eukaryotic transient expression vector
(Invitrogen Cat. No. V790-20); uncoated; γ-irradiated,
35-mm tissue culture dishes with glass bottom No. 0
(MatTek Cat. No. P35G-0-10-C); Dulbecco's modified
Eagle medium (DMEM) supplemented with 10% dialyzed
fetal bovine serum (FBS, Invitrogen Cat. No.
26400044), Hanks' balanced salts solution (HBSS) with
(Invitrogen Cat. No. 14170120); 37°C CO2
HeLa cells or appropriate eukaryotic strain; Lipofectamine
(Invitrogen Cat. No. 18324012) and PLUS
(Invitrogen Cat. No. 11514015) reagents; histamine,
ionomycin, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'
-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'
tetrakis(acetoxymethyl ester) (BAPTA-AM) (Sigma
Cat. No. H7125, I0634, E0396, and A1076, respectively);
Olympus IX70 inverted epifluorescence microscope;
Olympus Xenon lamp; MicroMax 1300YHS CCD
camera and Sutter Lambda 10-2 filter changers
controlled by Metafluor 4.5r2 software (Universal
Imaging); ECFP-EYFP FRET filter set (Omega Optical);
440AF21 excitation filter (ECFP excitation), 455DRLP
dichroic mirror, 480AF30 emission filter (ECFP emission),
and 535AF26 emission filter (EYFP emission);
neutral density (ND) filter set (Omega Optical); UApo
40xOil Iris/340 objective (Olympus); U-MNIBA bandpass
mirror cube unit (Olympus).
A. Engineering Cameleon Constructs
The history of cameleon engineering is reflected in
its nomenclature (Table I). Optimization of Ca2+
pH dependency, maturation time, and other
parameters is by no means complete. However, we
present construction of a general cameleon designed
in our laboratory, YC6.1, to serve as a reference point
for future work (Truong et al.
, 2001). Molecular biology
techniques for manipulating recombinant DNA are not
given as they can be obtained from common reference
|FIGURE 1 Schematic depiction of YC6.1 FRET in response to
[Ca2+]. See text for details.
In these constructs, ECFP and EYFP function as a
donor-acceptor pair for nonradiative, intramolecular
FRET. During FRET, excitation of the donor (cyan)
leads to emission from the acceptor (yellow), provided
that the molecules are close enough (within 80 Å) and
in a parallel orientation. In this way, on binding Ca2+
the CaM wraps around its adjacent CKKp target
peptide and ECFP and EYFP are brought closer to each
other and FRET increases (Fig. 1).
B. Overexpression and Purification
- A successful construction of GFP-fused protein
indicators is facilitated greatly by careful inspection of
available three-dimensional structure information of
the protein or protein domain used for sensing functions.
Typically, such structural analysis can be done
using SwissPDBviewer (Windows) and MODELLER
(Unix), which allow the measurement of atomic distances
and the molecular modeling of fusion proteins,
respectively. This was also the case for designing
YC6.1. We found that by virtue of the hairpin-like
complex structure of CKKp, the peptide can be
inserted into the domain linker region (residues 78-81)
- Insert a CaM-binding peptide derived from
CaM-dependent protein kinase kinase (CKKp;
residues 438-463) between the terminal EF hand Ca2+-
binding domains (N-CaM and C-CaM) within the
CaM linker domain (between CaM residues 79 and 80)
and connect via two Gly-Gly linkers.
- Add ECFP- and EYFP-encoding open reading
frames to termini of the (N-CaM)-GG-CKKp-GG-(CCaM)
module by recombinant DNA methods. Expression
vectors for many GFP family members are
available through Clontech. Cameleon constructs can
benefit from truncation of the last 11 C-terminal amino
acids of ECFP (the minimal region to form GFP) to reduce the relative tumbling of the fluorophores. Additionally,
linkers introduced between CaM domains
and the target peptide can be optimized for complex
- Sequence construct to ensure polymerase chain
reaction errors are not present. Note that oligonucleotides
designed to the 5' or 3' end of ECFP open
reading frames will also recognize the counterpart
sequences in EYFP due to high sequence identity.
- Subclone this cameleon domain into either
pRSETB plasmid, for prokaryotic protein expression
sufficient for biochemical and biophysical characterization,
or pcDNA3.1 for mammalian expression and in
vivo Ca2+-imaging experiments. If optimal expression
is not crucial, we also found that the pTriEx3
(Novagen) vector is convenient for expression in both
prokaryotic and eukaryotic systems.
- In addition to this cytoplasmic version of
cameleon YC6.1, nucleus- and endoplasmic reticulumtargeted
versions (YC6.1nu and YC6.2er, respectively)
can be constructed by the addition of appropriate
signal sequences to termini.
- From a single colony of newly transformed E. coli strain BL21(DE3), grow liquid cultures at 37°C in
LB medium containing 100µg/ml ampicillin.
- At OD600, induce cultures with 0.5mM IPTG at
- Harvest cells by centrifugation at 6500rpm for
20min at 4°C.
- Resuspend cell pellets in 1/20 culture volume of
lysis buffer [50mM HEPES (pH 7.4), 10% glycerol,
100 mM KCl, 1 mM CaCI2, and 1 mM phenylmethyl
sulfonyl fluoride (PMSF)], sonicate, and centrifuge
at 15,000rpm for 30min to remove debris.
- Incubate the supernatant with nickel chelate agarose
for 1 h at 4°C and wash with of 50mM HEPES
(pH 7.4), 100mM KCl, and 5 mM imidazole.
- Elute YC6.1 with 300mM imidazole in the aforementioned
buffer. Proteolytic cleavage to remove
the (His)6 tag is not necessary as it does not interfere
with YC6.1 fluorescent properties.
- Optional: The eluant can be then purified further
on a Superdex 75 HR 10/30 FPLC column using
20 mM HEPES, pH 7.5, 150 mM KCl, 5% glycerol,
5 mM dithiothreitol, and 1 mM PMSE
- Use the fraction that has the highest FRET ratio for
- Dialyze the sample against 2 liter of 50 mM HEPES
(pH 7.4), 100 mM KCl at 4°C.
- Optional: Glycerol can be added to the sample at a
final concentration of 20%, and aliquot, flash freeze
in liquid N2, and store the YC6.1 at -70°C.
If necessary, it is possible to perform the characterization
using a mammalian cell lysate. For harvesting,
cells should be transfected in several 100-mmdiameter
culture dishes, washed thoroughly to remove
traces of phenol red and serum, and lysed in a hypotonic
lysis buffer [50 mM
HEPES (pH 7.4) 100 mM
, and 0.5% Triton X-100]. Following
removal of cellular debris by centrifugation, dialyze
the supernatant in 2 liter of buffer [50 mM
7.4) and 100mM
KCl]. Finally, the sample can be used
for characterization as described.
C. In Vitro Cameleon Fluorescence
- Record fluorescence spectra on a Shimadzu
spectrofluorometer RF5301 using a 10-mm path-length
quartz cuvette at room temperature.
- Dilute cameleon chimeric proteins in 50mM HEPES (pH 7.4), 100mM KCl, and 20µM EGTA to a
final concentration of 60nM. Although many dilution
factors are acceptable, intermolecular ECFP-EYFP
FRET will occur at higher concentrations, thereby
inflating the emission signal falsely.
- Excitate at 433 nm and monitor the fluorescence
emission between 450 and 570nm with excitation and
emission slit widths of 5 nm (Table II).
- Record the fluorescence emission spectra of
buffer (background) and cameleon protein solutions.
- Subtract the background spectrum for buffer
alone from the cameleon sample to find spectra of the
cameleon in the absence of Ca2+.
- Determine the fluorescence emission ratio (R) by
dividing the integration of fluorescence intensities of
the FRET acceptor (for EYFP, between 520 and 536 nm)
by that of the FRET donor (for ECP, between 470 and
- Determine Rmin from this spectrum. A key parameter
for a Ca2+ indicator is its dynamic range in
response to [Ca2+]. The dynamic range of a cameleon
is defined as the division of the maximum ratio, Rmax,
by the minimum ratio, Rmin.
- In the presence of 1 mM CaCl2, repeat to find
spectra of the cameleon in the presence of saturating
amounts of Ca2+. Determine Rmax from this spectrum.
-binding curve is used to assess the effective
range of [Ca2+
] measurement. Ca2+
/EGTA buffers are used as standards because
even trace Ca2+
contaminants can significantly distort
values at low [Ca2+
] (Bers et al.
Miyawaki et al.
D. Live Cell Cameleon Fluorescence Imaging
- Prepare EGTA buffer [100 mM KCl, 50 mM HEPES (pH 7.4), 10mM EGTA] and CaCl2 buffer
[100mM KCl, 50mM HEPES (pH 7.4), 10mM EGTA,
10 mM CaCl2]. pH should be held constant.
- Use a 1-ml cuvette and dilute the sample in the
EGTA buffer. Record the fluorescence emission spectrum
from 450 to 570nm at 433-nm excitation. Determine
the emission ratio.
- To obtain the Ca2+-binding curve, add successive
fractions of the CaCl2 solution to the sample and determine
the emission ratio. Given that the experiment is
performed in 20°C with these EGTA and CaCl2 buffers,
the free calcium can be calculated by solving the quadratic
equation: [Ca2+]free2 + (10,000,060.5- [Ca2+]total) *
[Ca2+]free - 60.5 * [Ca2+]total = 0.
- To produce the Ca2+-binding curve, plot the
[Ca2+]free versus emission ratio change (percentage of
maximum). Initial cameleons show biphasic Ca2+ dependency, whereas YC6.1 has a monophasic
- Extract the apparent dissociation constant (K'd)
and Hill coefficient (n) from the fitted curves.
- The emission ratio can then be transformed to
[Ca2+] according to
|[Ca2+ ] - K'd [(R - Rmin)/(Rmax - R)]1/n
This section describes a Ca2+
using HeLa cells; however, with minor modifications
the method can be applied to other cellular and physiological
- Plate HeLa cells on 35-mm-diameter glassbottom
dishes with DMEM-10% FBS media.
- Incubate the cells at 37°C (5% CO2) until cells are
- Transfect cells with the mammalian expression
plasmid containing your cameleon using Lipofectamine and PLUS reagents (Invitrogen) according to the
- Remove the transfection mixture after 5-18 h and
replace with fresh 1.5 ml of DMEM-10% FBS media.
- Incubate the cells at 37°C (5% CO2) for 24h.
The cells are ready to perform the Ca2+-imaging
- All data acquisition should be performed in a
dark room to reduce background light.
- Wash with 1 ml of HBSS (+CaCl2) and add 1 ml of
fresh HBSS (+CaCl2).
- Put the cells on the stage of the microscope. Cells
are viable and healthy for at least 60 minutes at
ambient conditions. A CO2 box and temperature
controller are required for long-term/extended time
- The MetaFluor software controls the shutters,
filter exchangers, and camera during data acquisition.
- Screen for EYFP fluorescence (which is brighter
and distinguished more easily than ECFP fluorescence)
using the eyepiece to find transfected cells
expressing the cameleon construct. Turn the filter
turret to the U-WNIBA band-pass mirror cube and use
ND filter in the range of 0.1-10% to reduce photobleaching
depending on the intensity of fluorescence
emission (Table III).
- Using the 40× oil objective, center the microscope
viewing area on a cell that has a healthy morphology
and displays a strong cytosolic fluorescence.
- Acquire single images on the computer screen
using MetaFluor while adjusting the focus until you
have the sharpest screen image. Focus through the
eyepiece and the CCD usually vary slightly.
- Turn the filter turret to the cube with the
455DRLP dichroic mirror. This allows visible light
shorter and longer than ~455 nm to be reflected as excitation
to the sample and collected as emission from the
sample, respectively. Consult spectra from Omega Optical for relatively wavelength transmission
- To monitor the peak emissions of ECFP and
EYFP as a result of ECFP peak excitation over time, set
the data acquisition conditions as follows: time interval
to every 10 s and exposure time to 200 ms for ECFP
and EYFP. Longer exposures improve resolution at the
expense of bleaching the fluorescent signal. MetaFluor
will display the emission ratio over time.
- Draw a region of interest on the field of view of
the CCD. Usually, this region will outline the whole
cell, but one can specify only a portion of a cell if
desired (e.g., the nucleus). It is important that the stage
or the cell does not move during the observation
period as the region initially drawn may drift from
the region of interest. Additionally, the intensity in the
region (the signal) should be at least five times the
intensity of the background.
- The emission intensities of both ECFP and EYFP
will decrease over the course of the experiment due to
some unavoidable photobleaching; however, the effect
on the emission ratio should be negligible. To reduce
photobleaching, decrease exposure time and excitation
light intensity. Also, binning can sum the signal from
multiple pixels on the CCD camera so that less light is
required while keeping a good signal-to-noise ratio.
- MetaFluor will record the fluorescence emission
intensities of the ECFP and EYFP, together with their
emission ratios in the regions over time.
- When the emission ratio reaches a steady state,
add 50µl of 2mM histamine to the culture dish for a
final concentration of 100µM. Be careful not to move
the culture dish in this process. The histamine binds to
cell receptors on the plasma membrane that set off a
signaling cascade, resulting in the release of Ca2+ from
the endoplasmic reticulum through the inositol-l,4,5-
triphosphate receptor. This should cause a conformational
change in the cameleon that can be observed by a rise in emission intensity of EYFP and a decline in
ECFP intensity. Therefore, the emission ratio should
increase. The EYFP/ECFP emission ratio should return
to steady-state levels when the effect of the histamine
wanes. Figure 2 shows a representative example of
MetaFluor software as it is collecting data.
- In order to correlate the emission ratio to
[Ca2+]cytosolic, it is necessary to determine Rmin and Rmax so that emission ratios can be mapped to the Ca2+-
binding curve. Add 50 µl of 20 µM ionomycin for a final
concentration of 1 µM. Ionomycin open pores on the
plasma membrane to allow permeability to Ca2+ ions. Because the medium is saturated with CaCl2, the ratio
will rise to Rmax. To determine Rmin, add 50µl of 100mM EGTA and 600µM BAPTA-AM for a final concentration
of 5mM and 30µM, respectively. The emission
ratio should drop to Rmin.
|FIGURE 2 Example of Ca2+ imaging experiment using MetaFluor software. The region of observation is
highlighted green in the 440e535 panel. The 440e535 panel and graph plot the change in EYFP fluorescence
as a result of ECFP excitation; the 440e480 panel and graph plot the change in ECFP fluorescence as a result
of ECFP excitation; the EYFP/ECFP panel and graph are the ratio of 440e535 and 440e480 panels and graphs.
In this experiment, the graphs display a sharp rise in [Ca2+]c from the initial stimulation with histamine
followed by a slow decline in [CA2+]c to baseline levels. The second stimulation with histamine causes a
significantly more rapid return to baseline levels.
IV. OTHER APPROACHES
Several other Ca2+
probes are also being developed,
including the so-called "camgaroos" and "pericams." Camgaroos take an alternative approach to designing
sensors based on CaM and GFP family
members (Baird et al.
, 1999). While ECFP and EYFP in
cameleons are appended to the amino and carboxyl
termini of CaM and Ca2+
binding is detected by FRET,
camgaroo indicator proteins take advantage of the
robust structure and profound fluorescence sensitivity
of GFP to altered pKa
values and chromophore orientation.
Circular permutations and insertion of whole
CaM in place of Tyr-145 within EEYFP thereby render
this indicator responsive to Ca2+
binding. As a result,
both excitation and emission spectra of camgaroo
simply increase in amplitude by up to seven-fold upon
saturation with Ca2+
, without any significant shift in
peak wavelength. This Ca2+
enhancement is substantially larger than other published
genetically encoded fluorescent indicators.
However, camgaroos are limited by pH sensitivity
inherent in the current mechanism of modulating fluorescence
via changes in pKa
of the chromophore and
have to date only been preliminarily subjected to systematic
Pericams, in which EYFP is circularly fused to
CaM and the M13 myosin light chain kinase peptide,
improve approximately 10-fold upon the low affinity
of camgaroo for Ca2+
= 7 µM
) and are thereby better
capable of sensing low physiological changes in
] (Nagai et al.
, 2001). Taken together,
these strategies offer alternatives complementary to
cameleons for creating genetically encoded, physiological
V. OTHER CONSIDERATIONS
A. Ca2+ Ion Sensitivity
Although cameleon probes vary in their Ca2+
most are suitable for monitoring [Ca2+
] between 0.5
and 100 µM
. This poses a problem for examining the relatively
] found in the endoplasmic reticulum
of resting cells (approximately 500 µM
). However, CaM
mutagenesis studies have shown that substitution of
a conserved glutamic acid residue at the 12th position
of each Ca2+
-binding loop abolishes its Ca2+
ability (Zhu et al.
, 1998). The effect of combinations of
these mutations on cameleon Ca2+
range is currently
being examined further in our laboratory.
GFP variants have been developed in which chromophore
oxidative maturation (and thereby become fluorescent) occurs more quickly and efficiently at
37°C. It would be advantageous to utilize efficiently
folding versions, such as the recently developed
"Venus" form of EYFP (F46L/F64L/M153T/V163A/
S175G). These EYFP mutations confer an eight-fold
increase of fluorescence intensity when expressed in
mammalian cells (Nagai et al.
, 2002; Rekas et al.
This will enable assay of cells 24 h postrecovery transfection,
if so desired.
C. pH Sensitivity
The hydrogen bond network within the 13 barrel of
the chromophore is sensitive to external pH. Hence, in
order to analyze Ca2+
levels in acidic organelles (such
as secretory vesicles), the FRET donor/acceptor pair
must be engineered so that it is pH resistant. Two
mutations within EEYFP (V68L and Q69K) have
been shown to decrease its pKa
to 6.1 (Miyawaki et al.
1999). The pH sensitivity was improved further via a
Q69M or "citrine" mutation (pKa
5.7; Griesbeck et al.
2001). Another approach would be to change the
donor/acceptor pair to the pH-insensitive sapphirered
cameleon probe (SapRC2). Although this construct
has a tendency to aggregate and form homotetramers,
the recent engineering of a monomeric mRFP1 offers
an interesting alternative (Campbell et al.
GFP family proteins have been observed to form
obligate dimers and may thereby generate falsepositive
FRET signals. However, this aggregation
problem need not preclude their use in biological
systems, even when present in higher local concentrations.
Nonoligomerizing mutants of EYFP have been
suggested from its crystal structure (Wachter et al.
1998; Rekas et al.
, 2002), but these have yet to be
validated experimentally. Alternatively, using a
monomeric version of the evolutionary distinct RFP
(mRFP1) in tandem with a EYFP donor would also
serve to eliminate this issue (Campbell et al.
E. Influences on Biological Systems
It is very important to consider potential competition
of the FRET indicator for native CaM or CaMdependent
enzymes. Previous comparisons of the
effect of recombinant CaM and cameleon chimeras on
prototypical CaM-dependent enzymes have revealed
that the primary effect of cameleons is on buffering
] and not interfering with CaM-mediated signaling
(Miyawaki et al.
, 1999). This could be due to the
CaM component of the cameleon being inhibited by the adjoining CKKp efficiently occupying its substratebinding
site. Also, as the YC6.1 CKKp is embedded
within the cameleon polypeptide, it is not likely to
interact with endogenous CaM proteins.
F. Interpretation of Live Cell FRET Data
There are two commonly used simple and practical
approaches. The first, measurement of donor emission
quenching and acceptor emission enhancement by
using three filter sets and then mathematical processing
to determine emission/FRET ratios, is described in
Section III. The alternative approach is by detection of
donor dequenching following acceptor bleaching. We
find bleaching with a minimum of 200ms, compared
to 1 ms for control excitation, is sufficient.
G. Additional Ways to Do Ratio Imaging
FRET requires rapid intensity measurements at different
wavelengths. Switching time (in the millisecond
range) may be an important parameter for some applications.
New imaging systems have become available
in the marketplace, notably TILLvisION (T.I.L.L.
Photonics) and AquaCosmos (Hamamatsu), which
allow for excellent time resolution for fluorescenceintensity
H. FRET Using Red Fluorescence Protein
For YC6.1 applications, the ECFP-EYFP FRET filter
set is sufficient. However, if you are using RFP for
FRET, the following dichoric mirrors and filters from
Omega Optical (or equivalents) will be needed: 450-
520-590TBDR for the dichoric mirror; 440DF21 for
ECFP excitation; 510DF23 for EYFP excitation;
575DF26 for RFP excitation; 480AF30 for ECFP emission;
535AF26 for EYFP emission; and 600ALP for RFP
emission. This filter set allows you to excite or acquire
emission from ECFP, EYFP, and RFP individually,
albeit with a tradeoff in efficiency.
I. Imaging Systems
Using a confocal microscope is the best way to
increase spatial resolution for FRET experiments.
Confocal YC6.1 measurements can be performed with
single-photon excitation using the 458-nm line of an
argon laser, but much more efficient excitation of ECFP
is attained with the 442-nm line of a HeCd laser. Twophoton
excitation microscopy, in addition to providing
optical sections of a specimen as with confocal
microscopy, offers certain advantages. Its applicability
to cameleons has been demonstrated using video-rate scanning instrumentation (Fan et al.
, 1999). This latter
imaging approach may not be readily available to most
J. Preparation of Ca2+/EGTA Buffers
The accuracy of the Ca2+
-binding curve depends
on accurate preparation of the Ca2+
/HEEDTA systems below 10-5
M free Ca2+
above (Bers et al.
, 1994). The purity of
EGTA, temperature, and pH are all practical issues.
We are grateful to Atsushi Miyawaki for his help in
setting up a FRET microscope system in our laboratory,
as well as for much advice on the use of GFP
variants. This work was supported by grants from the
Cancer Research Society Inc. and the Institute for
Cancer Research of the Canadian Institutes of Health
Research (CIHR). K.P.H. is a recipient of a NCIC
Research Fellowship, K.T. holds a CIHR scholarship,
and M.I. is a CIHR senior investigator.
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