Our understanding of the structure-photochemistry
relationships of green fluorescent protein (GFP)
(Tsien, 1998) has enabled the development of genetic
calcium probes based on a circularly permuted GFP
(cpGFP) in which the amino and carboxyl portions
have been interchanged and reconnected by a short
spacer between the original termini (Baird et al.
The resulting new amino and carboxyl termini of the
cpGFP have been fused to calmodulin and its target
peptide M13, generating a chimeric protein named
pericam (Nagai et al.
, 2001). This new protein was fluorescent,
and its spectral properties changed reversibly
concentration, probably due to the interaction
between calmodulin and M13, which alters the
environment surrounding the chromophore. Three
types of pericam have been obtained by mutating
several amino acids adjacent to the chromophore. Of
these, "flash pericam" becomes fluorescent with
, whereas "inverse pericam" dims.
However, "ratiometric pericam" has an excitation
wavelength that changes in a Ca2+
thereby enabling dual-excitation ratiometric Ca2+
imaging. Ratiometric dyes permit quantitative Ca2+
measurements by minimizing the effects of several
artifacts that are unrelated to changes in the concentration
of free Ca2+
]), such as uneven loading or
partitioning of dye within the cell or varying cell thickness.
This article presents an outline of an imaging
experiment using HeLa cells expressing ratiometric
pericam to measure receptor-stimulated changes in
) (Protocol 1).
In contrast to cameleons (Miyawaki et al.
which are fluorescence resonance energy transferbased
indicators, pericams can be easily targeted
into the mitochondria matrix using an upstream targeting
sequence encoding subunit IV of cytochrome c
oxidase. Ratiometric pericam has been used successfully
to monitor changes in [Ca2+
] in mitochondria
) (Nagai et al.
, 2001). In most dual-excitation
imaging experiments, the excitation wavelength is
alternated using a rotating wheel containing two bandpass
filters. However, it is also possible to use a highspeed
grating monochromator to increase the rate at
which the ratio measurement is conducted to approximately
10 Hz. The latter instrumentation enables the
measurement in spontaneously contracting cardiac
myocytes of beat-to-beat changes in [Ca2+
to the demonstration that [Ca2+
synchronously with cytosolic [Ca2+
] during beating
(Robert et al.
, 2001). This article discusses factors to be
considered when using such a monochromator with
Although the aforementioned measurements are
typically performed using conventional microscopy,
the monitoring of changes in [Ca2+
] is often severely
limited by the poor spatiotemporal resolution of such
wide-field techniques. To obtain a more reliable representation
of changes in subcellular [Ca2+
], it is necessary
to increase the z
-axis resolution and the speed of
production and collection of the ratios of the excitation
peaks. This article provides a detailed protocol
describing a modified laser-scanning confocal microscopic
(LSCM) system for ratiometric pericam (Protocol
2). In our experiment (Shimozono et al.
, 2002), fast
exchange between two laser beams was achieved using acousto-optic tunable filters (AOTFs). Samples
were scanned on each line sequentially by a violet laser
diode (408nm) and a diode-pumped solid-state laser
(488 nm). In this way, the ratios of the excitation peaks
can be obtained at a frequency of up to 200 Hz.
HeLa cells (ATCC # CCL-2.2)
Culture medium: Add fetal bovine serum (Gibco BRL,
Life Technologies) to Dulbecco's modified Eagle's
medium (DMEM) (Sigma Aldrich) to a final concentration
of 10% (v/v)
Hank's balanced salt solution (HBSS): Dissolve CaCl2
(0.14g), KCl (0.40g), KH2
O (0.10g), MgSO2·
O (0.10g), NaCl (8.00g),
(0.048g), and D-glucose
(1.00 g) in 800ml of distilled H2
O, adjust the pH to
7.4, and then adjust the volume to 1 liter.
Histamine solution: Dissolve 1.841 mg of histamine
dihydrochloride (Sigma Aldrich, H7125) in 1 ml of
HBSS to make a 10 mM
Ratiometric pericam cDNA
Ratiometric pericam mitochondrial cDNA
35-mm glass-bottom cell culture plates (Matsunamiglass,
400DF15, XF1006, Omega
485DF15, XF1042, Omega
535AF45, XF3084, Omega
505DRLP, XF2010, Omega
505DRLP-XR, XF2031, Omega
500ALP, XF3092, Omega
A. Conventional Microscopy for Time.Lapse
Dual-excitation imaging with ratiometric pericam
uses two excitation filters (485DF15 and 400DF15),
which are alternated by a filter changer (Lambda 10-2,
Sutter Instruments, Novato, CA), a 505DRLP-XR
dichroic mirror, and a 535AF45 emission filter. The
excitation and emission spectra of ratiometric pericam in the presence and absence of Ca2+
with passbands of
the emission filter and two excitation filters are shown
in Fig. 1A. Also, the transmittance of the dichroic
mirror (505DRLP-XR) is superimposed. The dichroic
mirror has an eXtended Reflection region below
505nm; it was designed originally for the measurement
(fura-2) and pH (BCECF). In this situation,
the broad reflection of a dichroic mirror is
imperative. A more common long-pass dichroic mirror
(505DRLP) cannot be used; superimposition of its
transmission spectrum (Fig. 1B) makes one notice a
complex transmission band at short wavelengths,
which prevents reflection of the 400-nm light. The
author strongly recommends that researchers make
graphs of spectra plotted as percentage transmittance
for the interference filters (excitation filters, emission
filters, and dichroic mirrors) that are actually used,
together with excitation and emission spectra of the
relevant fluorescent dyes. The transmittance curves for
the filters (normal incidence) and dichroic mirrors (45° incidence) can be measured easily using a conventional
spectrophotometer. This measurement also provides
a chance for researchers to check the quality of
their interference filters, which may deteriorate with
B. A Modified LSCM System for Ratiometric
|FIGURE 1 (A) Fluorescence excitation and emission spectra of
ratiometric pericam in the presence (solid line) and absence (broken
line) of Ca2+. Transmittance spectra for filters (400DF15, 485DF15,
and 535AF45) and the dichroic mirror (505DRLPXR) are shown with
solid and dotted lines, respectively. (B) The transmittance spectrum
for a 505DRLP dichroic mirror.
Although the following procedure assumes familiarity
with LSCM, and with the Olympus LSCM in particular,
the procedure can be adapted easily to other
LSCM types. The microscope and lasers we use are
described in Fig. 2. The beam from a diode-pumped
solid-state laser (Sapphire 488-20, COHERENT)
directly enters an AOTF (AOTF1; AOTE8C, AA Opto-
Electronic). The light is relayed through an optical fiber
(FV5-FUR, Olympus) to a confocal microscope scan
head and passes through a dichroic mirror (DM1; DM420SP, Olympus). A laser diode (NLHV3000E,
NICHIA) is mounted on a heat sink with temperature
control, which consists of an LD mount and heat sink
[F125-4A (Suruga Seiki)], LD current driver [LDX-3525
(ILX Lightwave)], and LD temperature controller
[LDT5525 (ILX Lightwave)]. The output of the laser
diode is collimated (made parallel) using multiple
lenses, and its power is stabilized by a feedbackregulated
device. The beam is directed into another
AOTF (AOTF2; AOTE4C UV, AA Opto-Electronic).
The diffracted light is sent through another fiber-optic
line (FV5-FUR-UV, Olympus) to the scan head and is
then reflected on DM1. The two AOTFs are controlled
electronically, allowing the reciprocal choice of one of
the two laser lines. The chosen laser beam is reflected
on a second dichroic mirror (DM2; FV5-DM442,
Olympus). The scan head is coupled to an inverted
microscope (IX70, Olympus) through its right-side
port. The objective lens used is a PlanApo 60× N.A. 1.00 WLSM (Olympus). The fluorescent light is descanned,
passed through DM2, a pinhole, and an emission
filter (BA505-525 (Omega)), and detected by a
photomultiplier. Fluorescence signals with excitation
wavelengths of 408 and 488nm are distributed into
two channels during analog-to-digital conversion. The
pair of galvanometer mirrors, the digitized detector
output, and the AOTF controller are orchestrated. The
excitation and emission spectra of ratiometric pericam
in the presence and absence of Ca2+ , together with the
passband of the emission filter (BA505-525) and the
two laser lines, are shown in Fig. 3.
|FIGURE 2 Schematic diagram of the laser-scanning confocal microscopy system for fast dual-excitation
ratiometric imaging. DPSS, diode pumped solid-state laser; LD, laser diode; PD, photodiode; BS, beam splitter;
DM, dichroic mirror; EM, emission filter; PMT, photomultiplier tube; ADC, analog-to-digital converter.
|FIGURE 3 Fluorescence excitation and emission spectra of ratiometric
pericam in the presence (solid line) and absence (broken line)
of Ca2+. The wavelengths of the two laser lines, 408 nm (laser diode)
and 488nm (diode-pumped solid state), are indicated by vertical
lines. The wavelengths that pass through the emission filter
BA505-525 are shown by a box. Modified with permission from T.
Nagai, A. Sawano, E. S. Park, and A. Miyawaki, Proc. Natl. Acad.
Sci. U.S.A. 98, 3197 (2001). Copyright (2001) National Academy of
The procedure for time-lapse [Ca2+
HeLa cells is as follows.
- Plate HeLa cells or cells of interest onto a 35-ram
glass-bottom dish with culture medium.
- Transfect the cells with 1Bg per dish of
cDNA encoding ratiometric pericam mitochondria
using Lipofectin according to the manufacturer's
- Between 2 and 10 days after cDNA transfection,
image HeLa cells on an inverted microscope (IX7) with
a cooled CCD camera (MicroMax or Cool Snap HQ,
Roper Scientific, Tucson, AZ). Expose cells to reagents
in HBSS containing 1.26 mM CaCl2. Image acquisition
and processing are controlled by a personal computer
connected to a camera and a filter wheel (Lambda 10-2, Sutter Instruments, San Rafael, CA) using the
program MetaFluor (Universal Imaging, West Chester,
PA). The excitation filter wheel in front of the xenon
lamp (Lambda 10-2, Sutter Instruments, San Rafael,
CA) is also under computer control. Excitation light
from a 75-W xenon lamp is passed through a 400DF15
(400 ± 7.5) or 485DF15 (485 ± 7.5) excitation filter. The
light is reflected onto the sample using a 505-nm longpass
(505DRLP-XR) dichroic mirror with an extended
reflection. The emitted light is collected with a 40X
(numerical aperture: 1.35) objective and passed
through a 535 ± 22.5-nm band-pass filter (535AF45).
Interference filters are from Omega Optical or Chroma
Technologies (Brattleboro, VT).
- Define several factors for image acquisition,
including (i) excitation power, which depends on the
type of light source and neutral density filter, (ii)
numerical aperture of the objective, (iii) time of exposure
to the light, (iv) image acquisition interval, and
(v) binning. The last three factors should be considered
in terms of whether temporal or spatial resolution is
- Choose moderately bright cells. Select regions of
interest so that pixel intensities are averaged spatially.
- At the end of an experiment, convert fluorescence
signals into values of [Ca2+]. Rmax and Rmin can be
obtained as follows. To saturate the intracellular indicator
with Ca2+, increase the extracellular [Ca2+] to
10-20mM in the presence of 1-5 µM ionomycin. Wait
until the fluorescence intensity reaches a plateau.
Then, to deplete the Ca2+ indicator, wash the cells
with Ca2+-free medium (1 µM ionomycin, 1 mM EGTA,
and 5 mM MgCI2 in nominally Ca2+-free HBSS). The in
situ calibration for [Ca2+] uses the equation [Ca2+] = K'd[(R - Rmin)/(Rmax- R)](1/n), where K'd is the apparent
dissociation constant corresponding to the Ca2+ concentration
at which R is midway between Rmax and Rmin and n is the Hill coefficient. The Ca2+ titration curve of ratiometric pericam can be fitted using a
single K'd of 1.7 µM and a single Hill coefficient of 1.1
(Fig. 4). A typical time course of [Ca2+]i reported by
ratiometric pericam is shown in Fig. 5.
|FIGURE 4 The Ca 2+ titration curve of ratiometric pericam. Modified
with permission from T. Nagai, A. Sawano, E. S. Park, and A.
Miyawaki, Proc. Natl. Acad. Sci. U.S.A. 98, 3197 (2001).
(2001) National Academy of Sciences, U.S.A.
|FIGURE 5 Typical [Ca2+] i transients and oscillations induced by receptor stimulation in HeLa cells expressing
ratiometric pericam. The sampling interval was 3-5s. (Top) Excitation ratios of 480 and 410 nm. The righthand
ordinate indicates [Ca2+]i in µM with Rmax and Rmin indicated by an arrow and arrowhead, respectively.
(Bottom) Excitations of 480nm (black line, left-hand scale) and 410nm (gray line, right-hand scale). Modified
with permission from T. Nagai, A. Sawano, E. S. Park, and A. Miyawaki, Proc. Natl. Acad. Sci. U.S.A. 98, 3197
(2001). Copyright (2001) National Academy of Sciences, U.S.A.
The following is a procedure for confocal imaging
in HeLa cells using the modified LSCM
A. pH and Photochromism as Practical
- Plate HeLa cells or cells of interest onto a 35-mm
glass-bottom dish with culture medium.
- Transfect the cells with 1µg per dish of cDNA
encoding ratiometric pericam mitochondria using
Lipofectin according to the manufacturer's instructions.
- Incubate the cells in a humidified atmosphere at
37°C under 5% CO2 for 1 to 3 days.
- Replace the culture medium with HBSS.
- Observe the cells on an inverted microscope
(IX70, Olympus) for green fluorescence of the indicator
(ratiometric pericam-mt) using a 490-nm excitation
light generated using 490DF10.
- Choose moderately bright cells in which the fluorescence
is well distributed in the mitochondria.
- Switch on the lasers, AOTF controller, scanning
head, and the computer. The lasers need 30min to
warm up and stabilize.
- Start the scanning process with only excitation
from the 408-nm laser diode. Set the scan mode
to "Normal (unidirectional)" and scan speed to
"Fast." While looking at the acquired fluorescence
images, adjust the laser intensity to the minimum
level that allows easy identification of individual cells, adjust the sensitivity of the photomultiplier
tube (PMT) for an optimal signal-to-noise ratio, and
adjust the size of the pinhole for acceptable depth of the
image. There is a trade-off between the scan speed and
the pixel size: faster scanning speeds decrease resolution
because fewer pixels of information are collected.
- Adjust the intensity of the 488-nm laser by
setting the microscope to "Normal (unidirectional)"
scan mode and "Fast" scan speed. While looking at the
acquired fluorescence images, adjust the laser intensity
to a minimum level that allows easy identification of
individual cells and adjust the sensitivity of the PMT
for an optimal signal-to-noise ratio and the size of the
pinhole for acceptable depth of the image. Establishing
the microscope settings described in steps 8 and 9
is a process that must be iterated to produce adequate
- Start the control software for line-sequential
dual-excitation ratio analysis. The "Normal" scan
mode and "Fast" scan speed, with adequate pixel size,
enable rapid exchange between the two lasers on a line
every 5 to 20ms.
- Determine the desired scanning field (image
size) and reduce the height of the image so that 5 to
10 frames can be obtained per second.
- Start to acquire images every 100 to 200ms for
1 to 5min.
- During image acquisition, add histamine solution
to a final concentration of 10µM for receptor
- Determine the image ratio by dividing the
images acquired with excitation at 488nm by those
acquired at 408 nm.
Ratiometric pericam is sensitive to changes in pH in
both the presence and the absence of Ca2+
(Fig. 6). pHrelated
artifacts were not an issue in experiments that
used HeLa cells because agonist-induced [Ca2+
did not induce any intracellular pH changes
detectable by the pH indicator, BCECF (data not
shown). However, ratiometric pericam expressed in
dissociated hippocampal neurons was perturbed by
acidification following depolarization or glutamate
stimulation (data not shown).
|FIGURE 6 pH dependency of the 495/410-nm excitation ratio in
the presence () and absence (O) of Ca2+. Modified with permission
from T. Nagai, A. Sawano, E. S. Park, and A. Miyawaki, Proc. Natl.
Acad. Sci. U.S.A. 98, 3197 (2001). Copyright (2001) National
Academy of Sciences, U.S.A.
Pericams are derived from yellow fluorescent
protein (YFP). If YFP is excited too strongly, its fluorescence
will be reduced. This apparent bleaching
is actually photochromism because the fluorescence
recovers to some extent spontaneously and can be
restored further by UV illumination (Dickson et al.
1997). Intense excitation of ratiometric pericam also
causes photochromism, which results in a decrease in
the 490/410-nm excitation ratio independent of Ca2+
change. The extent of photochromism is dependent on
excitation power, numerical aperture of the objective,
and exposure time. Therefore, it is necessary to optimize
these factors for each cell sample in order to
minimize photochromism while preserving a high
signal-to-noise ratio. A good solution is to bin pixels at
the cost of spatial resolution. The increased signal-tonoise
ratio permits a decrease in intensity of the
excitation light with a neutral-density filter and the
observation of [Ca2+
oscillations without significant
photochromism of the indicators.
B. Use of a High-Speed Grating
Instead of a filter wheel containing 400DF15 and
485DF15 excitation filters, a fast wavelength exchanger
based on a grating monochromator (U7773-XX and
U7794-16, Hamamatsu Photonics) can be used with
a fast-acquisition CCD camera (HiSCA, C6790-81,
Hamamatsu Photonics). The spectrum of the excitation
light when the wavelength was set at 410 or 490nm is
shown in Fig. 7. Because light acquired with the setting
at 490nm spills over into the observing optical path,
it is preferable to eliminate the unwanted light by
putting a short-pass filter like 500SP (Fig. 7, broken
line) in front of the microscope.
C. Calcium Transients in Motile
|FIGURE 7 Spectrum of the 410- or 490-nm light selected by the
monochromator (U7773-XX and U7794-16, Hamamatsu Photonics)
(solid line) and transmittance of a 500SP short-pass filter (Omega,
3rd Milenium) (broken line). (Inset) The crossing of the two curves
for 490-nm light and 500SP on an expanded scale.
When changes in [Ca2+
are monitored by alternating
the excitation wavelength automatically with
wide-field conventional microscopy, the rate of acquisition
of the excitation ratio is about 10 Hz, which is
identical to the frame rate. Despite this rapid rate of
data collection, the [Ca2+
measurements are often
affected adversely by the active motion of mitochondria,
especially at warmer temperatures. Using our
LSCM technique, we attempted to increase the speed
of the alternation of excitation wavelengths so that it
was faster than the movement of mitochondria. With
this method, the frame rate was 5 Hz and the rate of
ratio acquisition was 200 Hz. Although the frame rate
did not allow us to fully follow the rapid movement of mitochondria, the fast rate of ratio acquisition minimized
the time lag between the two measurements
used to calculate the ratiometric signal. We feel that
this method of imaging [Ca2+
effectively corrects for
mitochondrial movement in all three possible both laterally
and into and out of the optical section. After the
application of histamine, spots of [Ca2+
within a single mitochondrion were identifiable (Fig.
8A) and the global increase in [Ca2+
was found to
occur relatively slowly (Fig. 8B).
|FIGURE 8 Confocal and dual-excitation imaging of [Ca2+]m using ratiometric pericam mitochondia.
(A) Ratio images before and after application of 10µM histamine. Scale bar: 5µm. (B) Time course of
averaged fluorescence signals from the white box in A with excitation at 488 (green) and 408 nm (violet) (top)
and their ratio (bottom).
The arrowhead indicates the time when histamine was applied.
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