Calcium rubies: A family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging

Article abstract of DOI:10.1021/ja304018d

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca²⁺) indicators as new tools for biological Ca²⁺ imaging. The specificity of this Ca²⁺-indicator family is its side arm, attach

Full text of DOI:10.1021/ja304018d

Article
pubs.acs.org/JACS
Calcium Rubies: A Family of Red-Emitting Functionalizable Indicators
Suitable for Two-Photon Ca2+ Imaging
Mayeul Collot,†,‡,§ Christina Loukou,†,‡,§ Aleksey V. Yakovlev,◆,∥,⊥,○ Christian D. Wilms,¶,□
Dongdong Li,∇,#,△ Alexis Evrard,∇,#,△ Alsu Zamaleeva,◆,∥,⊥ Laurent Bourdieu,◆,∥,⊥
Jean-Franco
̧
is Leger,◆,∥,⊥ Nicole Ropert,∇,#,△ Jens Eilers,¶ Martin Oheim,∇,#,△ Anne Feltz,◆,∥,⊥
́
and Jean-Maurice Mallet*,†,‡,§
†UPMC Universite
‡CNRS UMR 7203, Paris F-75005, France
§Laboratory of Biomolecules (LBM), Paris F-75005, France
◆Institut de Biologie (IBENS), Ecole Normale Super
ieure (ENS), Paris F-75005, France
́ ́
Paris 06, Ecole Normale Superieure (ENS), Paris, F-75005 France
́
∥
INSERM U1024, Paris F-75005, France
⊥CNRS UMR 8197, Paris F-75005, France
¶Carl-Ludwig-Institute for Physiology, Leipzig University, D-04103 Leipzig, Germany
∇INSERM U603, Paris F-75006, France
#CNRS UMR 8154, Paris F-75006, France
△Laboratory of Neurophysiology and New Microscopies, Universite
France
́ ́
Paris Descartes, PRES Sorbonne Paris Cite, Paris F-75006,
S
* Supporting Information
ABSTRACT: We designed Calcium Rubies, a family of
functionalizable BAPTA-based red-fluorescent calcium (Ca2+)
indicators as new tools for biological Ca2+ imaging. The
specificity of this Ca2+-indicator family is its side arm, attached
on the ethylene glycol bridge that allows coupling the indicator
to various groups while leaving open the possibility of aromatic
substitutions on the BAPTA core for tuning the Ca2+-binding
affinity. Using this possibility we now synthesize and
characterize three different CaRubies with affinities between
3 and 22 μM. Their long excitation and emission wavelengths
(peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca2+ uncaging or
optogenetic stimulation with Ca2+ imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of
Ca2+ transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca2+-translocating
channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence
lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca2+ chelation. Since only the
fluorescence quantum yield but not the absorbance of the CaRubies is Ca2+-dependent, calibrated two-photon fluorescence
excitation measurements of absolute Ca2+ concentrations are feasible.
of most mammalian cells to >100 μM at the peak of Ca2+
1. INTRODUCTION
microdomains. Thus, depending on the specific Ca2+ signal
investigated, Ca2+ indicators with different affinity for Ca2+
binding (KD,Ca) will be required as fluorescent reporters. Its fast
on-rate for Ca2+ binding and high selectivity for Ca2+ over Mg2+
has made BAPTA3 (1,2-bis(o-aminophenoxy)ethane-
N,N,N′,N′-tetraacetic acid) the most popular Ca2+ chelator
used in the synthesis of chemical Ca2+ indicators. A broad range
Fluorescent Ca2+ indicators are indispensable tools for studying
spatiotemporal fluctuations of intracellular free Ca2+ concen-
tration ([Ca2+]i).1,2 Ca2+ is an ubiquitous second messenger
involved in numerous intracellular signaling cascades. Biological
Ca2+ signals gain their specificity from operating at different
temporal, spatial, and concentration scales. Temporally, Ca2+
transients cover the submillisecond to hour scale. Confined
Ca2+ microdomains coexist with large-scale fluctuations which
propagate through multicellular networks that extend over
hundreds of micrometers. Cellular Ca2+ signals cover
concentrations from near ∼100 nM for the basal free [Ca2+]i
Received: May 20, 2012
Published: July 20, 2012
© 2012 American Chemical Society
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of indicators has been synthesized by linking or integrating
BAPTA to various chromophores.4
are reported, allowing calibrated [Ca2+]i measurements with
2PEF time-correlated single-photon counting (TCSPC).19
Most Ca2+ indicators, and certainly those that work best,
combine BAPTA with fluorescein derivatives and hence emit
yellow/green fluorescence.5,6 However, the increasing use of
cells transfected with fluorescent proteins (FPs),5 of FP-
expressing transgenic mice for targeting identified subpopula-
tions of cells, together with the advent of optical techniques for
purposes other than imaging require the development of new
genetically encoded2,7−9 and chemical Ca2+ probes.2,10−12
Although monomeric red FPs are available, green or yellow
FP tags are the most common. Introduced in 2012, GECO-R13
is the only red-emitting genetically encoded Ca2+-sensor
whereas other FP-based Ca2+ indicators remain limited to
green hue. The demand for longer-wavelength and higher
signal-to-noise chemical Ca2+ indicators is accentuated by the
recent trend toward all-optical manipulation and recording.
Photopharmacology, photochemical uncaging, and optoge-
netics14,15 all use near-ultraviolet or short visible wavelengths
that further restrain the part of the visible spectrum available for
Ca2+ imaging. Taken together, to be valuable for biological Ca2+
imaging, new Ca2+ probes should be bright, operate in spectral
2. RESULTS AND DISCUSSION
The three fluorescent Ca2+ indicators described in this article
consist of a BAPTA Ca2+-chelating moiety with a -Cl, -F, or
Me-substituted on one of the aromatic rings. The functionaliz-
able azido side arm is introduced at an early stage of the
synthesis whereas the X-rhodamine is synthesized thereafter,
leading to the functional Ca2+ indicator, which we refer to as
CaRuby-Cl, -F, or -Me depending on the substitution. The
detailed synthesis of these molecules is reported in the
Supporting Information.
In a solution containing 100 mM KCl, 30 mM MOPS (3-(N-
morpholino)propanesulfonic acid), and 10 mM NTA (nitrilo-
triacetic acid) (at pH 7.2), CaRuby-Cl, -F. or -Me had apparent
K
D,Cas of 3, 6, and 22 μM (Figure 1a and Table 1). For all
probes fluorimetric titration curves indicated a 1:1 Ca2+
binding, suggesting that the presence of the linker arm does
not modify the stoichiometry of Ca2+ binding. All three
CaRubies had absorbance and fluorescence peaks near 586 and
604 nm, respectively (Scheme 1, inset), and fluorescence
quantum yields φ were around 0.55 when bound to Ca2+
(Table 1 and Figure S1 of the Supporting Information).
CaRubies were virtually nonfluorescent in a nominally Ca2+-free
solution containing 10 mM ethylene glycol tetraacetic acid
(EGTA). Relative peak fluorescence (dF/F0, measured at 604
nm) increased more than 50-fold upon Ca2+ binding and
resulted from a Ca2+-dependent change in φ, rather than
absorbance.16
windows outside the yellow/green, and have a tunable KD,Ca
.
In this context, we introduced the red-emitting low-affinity
(KD,Ca ∼ 20 μM) indicator CaRuby-Cl,16 a chloride-substituted
BAPTA incorporated into an extended rhodamine (X-Rhod-
amine) having an extra side arm for conjugation to dextrans,17
nanoparticle surfaces,18 or other molecular targets. The
attachment of the linker arm on the ethylene glycol bridge
leaves us with the possibility for aromatic substitutions on the
BAPTA (Scheme 1) that did not change the rhodamine
Ca2+ indicators are exposed to ionic conditions that
potentially interfere with Ca2+ chelation. In the cytoplasm,
Mg2+ is the most prevalent divalent cation. We therefore first
measured CaRuby fluorescence in a pH- and Ca2+-buffered
solution containing 50 μM free Mg2+ and 50 μM free Ca2+ (free
ion concentrations in the presence of pH and Ca2+ buffers were
calculated as described in the Supporting Information of ref 16)
and found no difference to the fluorescence measured in 50 μM
free Ca2+ alone (Figure 1b).
Scheme 1. Generic Structure of the CaRubies
For testing how the presence of other divalent cations
affected the fluorescence, we established a fluorescence baseline
with copper quenching20 and used an ion-displacement assay:
CaRuby-Me was strongly fluorescent in solutions containing 50
μM Ca2+, Ba2+, or Cd2+, whereas the paramagnetic transition
metal ions (Co2+, Cu2+, Ni2+) acted as quenchers. Adding 50
μM Ca2+ to these solutions did not measurably alter CaRuby-
Me fluorescence, suggesting that Ca2+ was unable to displace
already complexed Co2+, Cu2+, Mn2+, Ni2+, or Zn2+ (Figure 1c).
The corresponding and similar data for CaRuby-F are shown in
Figure S2 of the Supporting Information. Finally, we
investigated the pH-dependence of CaRuby fluorescence and
found intensity changes below 20% over the physiologically
relevant range from 6.8 to 7.6, with a pKa around 6.2 for all
three CaRubies (Figure 1d). Taken together, our new Ca2+
indicators combine the spectral properties of an X-rhodamine
with the high selectivity for Ca2+ over Mg2+ and low pH
sensitivity of a BAPTA-based indicator.3
spectrum (inset). We now systematically exploit these two
properties to enlarge the CaRuby family and provide red-
fluorescent indicators covering the micromolar range of
affinities for Ca2+ binding (KD,Ca).
This paper is organized as follows. After their physicochem-
ical characterization, we validate the chloride-, fluoride-, and
methyl-substituted CaRubies for biological Ca2+ imaging by the
detection of light-evoked near-membrane Ca2+ transients in
cultured astrocytes expressing CatCh (calcium translocating
channelrhodopsin), a Ca2+-translocating channelrhodopsin,
using total internal reflection fluorescence microscopy
(TIRFM). We next characterize, using infrared fs-pulsed
excitation, the utility of the CaRuby family for two-photon
excitation fluorescence (2PEF) microscopy, both in the
intensity- and fluorescence-lifetime domain. Fluorescence
lifetimes of the Ca2+-free and -bound forms of all three dyes
The major asset of our CaRubies over existing rhodamine-
based Ca2+ probes21 is the combination of their tunable affinity
for Ca2+ binding and the functionalizable azido arm. The azido
group enables different kinds of coupling reactions (Figure 2).
We illustrate this capacity by coupling an alkyne or active ester
via the Huisgen 1,3-dipolar cycloaddition (click chemistry) or
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Figure 1. Physico-chemical characterization of CaRubies. (a) Fluorimetric titration against Ca2+ in a buffer containing (in mM) 100 KCl and 30
MOPS (pH 7.2). Lines are Hill fits with all data points from three independent titrations for each dye. See Table 1 for KD,Ca values. (b) Fluorescence
of CaRuby-Cl, -F, and -Me, in the presence of 50 μM free Ca2+ alone, gray, and in the presence of 50 μM free Ca2+ and 50 μM free Mg2+, white. (c)
Normalized fluorescence of CaRuby-Me (relative to that in the presence of 50 μM free Ca2+) in solutions containing 50 μM of either Ca2+, Ba2+,
Cd2+, Co2+, Cu2+, Mn2+, Ni2+, or Zn2+, gray, and upon addition of 50 μM Ca2+, white. (d) pH dependence of of CaRuby-Me and -F fluorescence in a
buffer containing (in mM) 100 KCl, 30 MOPS, 10 NTA, and 0.024 free Ca2+. pKa values from Hill fits were 6.27
0.06 and 6.18
0.14,
respectively. In the physiologically relevant range between pH 6.8 and 7.6, fluorescence varied from 0.87 to 1.05 (unity at pH 7.2). Symbols and
through lines show mean 1 SD from three independent titrations and fit.
a
Table 1. Physicochemical Properties of the CaRuby Dyes
CaRuby
KD,Ca (μM)
dF/F0
φCaF
φCaB
pKa
-Me
-F
3.4 0.47
6.2 1.03
21.6 2.7
62.5 5.2
49.7 5.8
51.9 1.4
0.004 0.02
0.007 0.02
0.007 0.02
0.58 0.06
0.57 0.05
0.55 0.05
6.27 0.06
6.18 0.14
6.22 0.07
-Cl
a
Mean 1 SD values; n = 3 independent titrations; φ: quantum yield in Ca free and 2 mM Ca2+ (CaF; CaB); others, see text).
the reduction of the azido group, respectively. These reactions
were very efficient and are suitable for biological conjugation
reactions. As an example, we linked CaRuby-Me with up to
76% yield to different molecular-weight poly(ethylene glycols)
(PEGs). Conjugation had no adverse effect on Ca2+ chelation
nor on the dynamic range for Ca2+ sensing (Figure S3,
Supporting Information). Compared to their arm-free ana-
logues, X-rhod-5F and X-rhod-1 (Figure S4, Supporting
Information) CaRubies display a 3- to 4-times higher Kd,Ca
presumably due to a restricted ethylene glycol C−C bond free
rotation. However, their unique linker arm should make our
CaRubies a versatile building block for subcellularly targeted
Ca2+probes, allowing their coupling to antibodies and bio-
orthogonal tags or linking it to dextrans, nanoparticles, latex
beads, or other substrates.17,18,22
HEK293, or BON cells in culture, even when present overnight
in the extracellular fluid.17 A notable exception were cultured
mouse cortical astrocytes that readily internalized the acid
forms of all three CaRuby dyes following short incubations at
room temperature (5 μM, 10 min, 20−22 °C). We observed a
similar spontaneous uptake into astrocyte cultures with the
structurally related rhodamines sulforhodamine 101 (SR101),
R101, and SR-B. Depending on the dye, the subcellular
distribution differed (Figure S5, Supporting Information). The
mechanism of this astrocyte-specific uptake and compartmen-
talization is unknown but could result from a combined effect
of charge and partitioning into lipids,23,24 or the presence of an
astrocyte-specific permeation or transport pathway.
In astrocytes expressing a light-gated Ca2+-translocating
channelrhodopsin coupled to yellow FP (CatCh-YFP)25,26
and labeled with CaRuby-Me (Figure 3a), short flashes of blue
light (458-nm, 5s, 13.6 mW/mm2) elicited robust and
reproducible near-membrane Ca2+ transients detectable with
TIRFM (Figure 3b). The peak dF/F0 values observed in
response to this stereotyped light-evoked Ca2+ influx scaled in
an affinity-dependent manner, following the sequence -Me > -F
> -Cl (Figure 3c). The amplitude and time-course of light-
Fluorescent Ca2+ indicators are commonly prepared as
membrane impermeant salts for cytoplasmic loading by
infusion from a patch pipette or as membrane-permeable
esters that allow the simultaneous bulk loading of hundreds of
cells.1 We found earlier that CaRuby-Cl with a molecular
weight of ∼1000 Da and a size of ∼1.5 nm was membrane-
impermeable when applied to chromaffin cells, neurons,
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Figure 2. Coupling reactions involving CaRubies: click chemistry and amine-acid coupling (Asc: ascorbate; Suc: succinimide).
evoked Ca2+ transients could be further shaped by varying the
duration of the light pulse (Figure 3d). Astrocytes responded to
local ATP application (300 μM) with fast, spatially confined
and high-amplitude Ca2+ transients detected with CaRuby-Me
(dF/F0 = 13.4 4.6%; mean fwhm of 84.9 40.2 ms; n = 24
events from N = 4 cells; Figure 3e) that were occluded by the
global, homogeneous fluorescence increase seen in parallel
experiments with the green-fluorescent high-affinity Ca2+
indicator Fluo-4, AM (KD,Ca 345 nM). In response to the
same stimulus, the later reported slower and lasting Ca2+
Figure 5) which is close to those of enhanced cyan fluorescent
protein (ECFP) (860 nm),30 EGFP (920−960 nm),30−32 EYFP
(950 nm),30,32 and channelrhodopsin (920 nm).33,34 In vivo, 20
min after a topical application of CaRuby-Me (10 μL, 100 μM)
in a small cranial window above the rat cortex, we observed a
perivascular labeling using 2PEF excitation at 900 nm (Figure
5). Perivascular staining has been reported recently for the
structurally unrelated fluorophore AlexaFluor 633.35
Estimating absolute [Ca2+]i levels from fluorimetric 2PEF
measurements is made difficult by unknown dye concentrations
and the attenuation of both the excitation power and 2PEF
collection efficiency36 with imaging depth. Pseudoratios with a
second Ca2+-insensitive reference dye37,38 correct for some
distortions but do not give access to absolute [Ca2+]i levels.
Advantageously, fs-pulsed excitation lends itself in a natural way
to fluorescence lifetime measurements provided the decays for
the Ca2+-bound and free forms of the indicator are measurably
different.19 In Ca2+-buffered solutions containing between 1 nM
and 100 μM free Ca2+, CaRuby-Me displayed a double-
exponential fluorescence decay that was Ca2+-dependent
(Figure 6a). The amplitudes α of the fractional decay
components were used to derive the amount of Ca2+-free and
-bound dye, respectively (Figure 6b). The KD,Ca and a
correction factor S (see ref 19) were obtained by fitting the
Hill equation with the normalized pre-exponential coefficients
of the fast and slow decay components and were in good
agreement with the KD,Ca values obtained from steady-state
fluorescence measurements, Table 2. Taken together, their
steady-state and time-resolved fluorescence properties make
CaRuby dyes excellent candidates for quantitative 2PEF
imaging of [Ca2+]i in intact preparations.
transients having a mean amplitude of 49.7
7.5% and
fwhm of 11.8 3.8 s (19 ROIs from 4 cells; Figure 3f).
The sulfonated X-rhodamines SR101/TexasRed are known
to display a cell-type specific tropism for astrocytes when
applied extracellularly to brain slices or as a drop on the cortical
surface of the intact brain after craniotomy.27,28 Similar results
have been obtained after i.v. injection of different rhodamines.29
In view of their spontaneous internalization into cultured
astrocytes, we therefore tested whether CaRubies selectively
labeled cortical astrocytes in situ. However, incubation (1 μM,
15 min) of acute brain slices from adult Aldhl1L1-EGFP mice
that express EGFP under the control of an astrocyte-specific
promoter revealed no astrocytic tropism of any of the three
CaRubies whereas parallel experiments using SR101 confirmed
the well-described astrocytic staining. Only at the surface of the
brain slices was observed some sporadic labeling, suggesting a
nonspecific uptake into cells damaged during the slicing
procedure (Figure 4). Whether this difference between
rhodamines concerning their astrocytic uptake is due to the
bigger size of the CaRubies or resulting from a molecular
recognition of the sulfonated X-rhodamines is beyond the
scope of this paper.
3. CONCLUSION
Their long emission wavelengths make our CaRubies
attractive probes for Ca2+ imaging in scattering media using
two-photon excitation fluorescence (2PEF) detection. To
determine the optimal excitation wavelength, we scanned the
entire tuning range of the Ti:Sapphire laser. Femtosecond- (fs-)
pulsed excitation in the 750 to 1000 nm range revealed 2PEF
excitation peaks near 915 nm for all three CaRubies (inset in
We have enlarged the CaRuby family to provide three
functionalizable red-emitting medium- to low-affinity Ca2+
indicators with physicochemical properties making them useful
in a wide array of multicolor biological applications. The
common and defining feature of our CaRubies is the linker arm
allowing diverse couplings of the dyes. Unlike a linker anchored
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Figure 3. Multiband Ca2+ imaging and optogenetic stimulation with CaRuby. (a) Dual-color excitation (488/568 nm) and dual-color emission
TIRFM images of an astrocyte expressing the light-gated Ca2+-translocating CatCh-YFP (yellow fluorescent protein) loaded with CaRuby-Me (5
μM, 10 min). Scale bar, 10 μm. (b) CaRuby-Me, -F, and -Cl were used to monitor light-evoked Ca2+ rises (458 nm, 5 s, 13.6 mW/mm2). Thin light
traces show individual astrocytes, thick darker lines are population mean +1SD. (n = 9 trials in N = 3 cells for CaRuby-Me and -F, 4 trials in 2 cells
for CaRuby-Cl). (c) Comparison of the peak amplitude (dF/F0 = 8.7 2.0%, 6.7 1.7%, and 4.3 1.6% for CaRuby-Me, -F, and -Cl, respectively)
and the duration (full width at half-maximum, fwhm) of the Ca2+ responses scaled as expected from the lower Ca2+ affinity of CaRuby-Cl (KD,Ca, 21.6
μM) compared to CaRuby-Me (KD,Ca, 3.4 μM) and CaRuby-F (KD,Ca, 6.2 μM). *, p < 0.05; **, p < 0.01. (d) In comparison with the responses
evoked with 5 s light pulses, CaRuby-Me and CaRuby-Cl reported significantly lower Ca2+ rises with 2 s light pulses (5.1 1.3%, 8 trials in 3 cells vs
8.7 2.0%, 9 trials in 3 cells; 4.0 0.6%, 6 trials in 2 cells vs 6.7 1.7%, 9 trials in 3 cells, respectively; p < 0.01). Each trace represents the
stimulation of a single astrocyte. (e) Local application of 300 μM ATP (adenosine triphosphate) from a puff pipet results in spatially confined Ca2+
transients in astrocytes detected with CaRuby-Me. Top panels show TIRFM images of an astrocyte acquired before and after stimulation. Circles
identify seven regions of interest (ROIs, 2 μm diameter). Green traces show the evolution with time (imaged at 20 Hz) of dF/F0 responses in the
ROIs identified by number. Scale bar, 5 μm. (f) Same experiment with the high-affinity green-fluorescent Ca2+ indicator Fluo-4, AM that reported a
homogeneous elevation throughout the entire astrocyte with no detectable Ca2+ microdomains. White circles identify five ROIs with the
corresponding fluorescence responses shown as gray traces.
to the BAPTA aromatic ring,22 its localization on the ethylene
glycol bridge permits aromatic substitutions and hence a fine-
such experiments that put strong constraints on the available
wavelength windows.
tuning of the KD,Ca
.
Compared with the red-emitting bioluminescence resonance
energy transfer (BRET)-based tandem-dimer tomato-aequorin9
or the genetically encoded red-emitting Ca2+-probe R-GECO,13
the CaRubies build on the large and established toolbox of
organic synthesis and can be prepared in a variety of different
forms, as salts, as membrane-permeable esters, be attached to
With their long-wavelength emission and nonlinear ex-
citation properties compatible with popular FPs, our CaRubies
are excellent probes for Ca2+ imaging in otherwise challenging
experiments relying on several excitation or emission bands.
The combined light-stimulation and Ca2+-imaging experiments
using CatCh-YFP expressing cells validate the new indicators in
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Figure 4. Comparison of sulforhodamine 101 (SR101) and CaRuby-
Me labeling in acute coronal slices of adult transgenic Aldh1L1-EGFP
mice. (a) Example of infrared (>900 nm) differential-interference
contrast (DIC) and 488/568-nm dual-color epifluorescence raw
images of cortical astrocytes loaded with SR101 (1 μM, 15 min)
and expressing enhanced green fluorescent protein (EGFP) under the
control of the astrocytic-specific marker Aldh1L1 (aldehyde
dehydrogenase 1 family, member L1).39 The pseudocolor overlay
(merge) reveals a significant overlap between SR101 and EGFP
fluorescence (yellow) as expected from earlier studies.27−29 Similar
results were obtained in the hippocampus (n = 3 slices of N = 2
animals, data not shown). (b) DIC, CaRuby-Me-, and EGFP-
fluorescence images revealed distinct distributions of EGFP and
spontaneously internalized CaRuby-Me in the cortex of Aldh1L1
transgenic mice. Similar nonoverlapping distributions were observed in
various regions of n = 5 slices from N = 3 mice (not shown). The
uptake of CaRuby-Me was prominent near the slice surface, suggesting
that the dye might accumulate in cells damaged during slicing. Scale
bar, 100 μm for all panels. Note the absence of cross-excitation and
fluorescence bleed-though, respectively, allowing the simultaneous
imaging of EGFP and CaRuby fluorescence.
Figure 5. Two-photon excitation at 900 nm of CaRuby-Me at ∼70 μm
depth in the cortex of an anesthetized 3 months rat in vivo, 20 min
after topical application of CaRuby-Me revealed perivascular labeling
(white arrows). Blood vessels remained unstained (white stars). A
deeper, large vessel (2 stars) is seen as a shadow. Scale bar, 50 μm.
Inset: Normalized 2PEF spectra of CaRuby-Me (green), CaRuby-F
(black), and CaRuby-Cl (red) bound to Ca2+ 2 mM (solid) and, for
CaRuby-Cl, in nominally Ca2+-free solution containing 5 mM EGTA
(dashed). Spectra show a peak of 912 nm for CaRuby-Cl and 917 nm
for CaRuby-F and -Me, respectively. For the less fluorescent Ca2+-free
(apo-) form of the dye (dashed) the higher excitation intensity
required introduced a contamination at the edges of the scanned
region (black arrows). No peak shift was detected upon Ca2+ binding.
large MW dextrans, or linked to specific bio-orthogonal tags
penicillin (5 U/mL), and streptomycin (5 μg/mL) at 37 °C in a
humidified 5% CO2 atmosphere. Astrocytes kept for one more week in
secondary culture and transfected with CatCh-YFP (see below) were
incubated with CaRuby-Me, -F, or -Cl salt (5 μM, 10 min) at room
temperature (RT, 20−22 °C) and perfused 30 min in dye-free medium
to allow for complete wash-off of membrane-bound CaRuby-Me.
During experiments, cells were continuously perfused at 0.5−1 mL/
min with extracellular saline containing (in mM): 140 NaCl, 5.5 KCl,
1.8 CaCl2, 1 MgCl2, 20 glucose, 10 HEPES (pH 7.3, adjusted with
NaOH).
4.1.3. Plasmids. Cultured astrocytes were transfected with CatCh-
YFP25 using lipofectamine 2000 and following standard protocols. We
used 1.4 μg DNA plasmid for each coverslip. Cells were maintained in
culture for 1−2 days after transfection to allow the expression and
proper targeting of CatCh to the cell surface, detectable by TIRF
imaging (see below) of the near-membrane space.
4.2. CaRuby Uptake in Acute Brain Slices. Adult Tg(Fthfd-
EGFP)1 Gsat/Mmcd) mice expressing EGFP under the control of
formyltetrahydrofolate dehydrogenase (Aldh1L1 or Fthfd)42 were
obtained from the Mutant Mouse Regional Resource Centers
(MMRRC), Jackson Laboratory (Bar Harbor, MN), and backcrossed
on an NMRI background. Mice were anesthetized by intraperitoneal
injection of pentobarbital (20 mg/kg) and decapitated. Their brains
were quickly removed and placed in ice-cold (2−4 °C) oxygenated
(5% O2, 95% CO2) standard artificial cerebrospinal fluid (ACSF)
containing, in mM: 126 NaCl, 2.85 KCl, 1.25 KH2PO4, 1.5 MgSO4, 2
CaCl2, 26 NaHCO3, 5 sodium pyruvate, and 10 glucose. Coronal slices
(400 μm thick) were cut on a vibratome (VT1000S, Leica, Wetzlar,
Germany) and first maintained during 1 h at 33 °C for recovery, then
at RT in oxygenated ACSF. Subsequently, slices were either incubated
permitting their subcellular targeting.22
4. EXPERIMENTAL SECTION
For general analytical procedures and synthetic protocols used to
prepare the CaRubies and their PEGylated derivatives, please see the
Supporting Information.
4.1. Reagents and Materials. 4.1.1. Chemicals. The following
chemicals were utilized in this investigation: 1 M CaCl2 (Fluka
21115); DMEM (Invitrogen 31885); ethanol (Merck cat. no.
100983); Fluo-4, AM (Invitrogen cat. no. F-14201); 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich; cat.
no. H3375); KCl (Prolabo cat. no. 215GC); lipofectamine 2000
(Invitrogen; cat. no. 11668-027); methanol (Merck cat. no. 106009);
MOPS (Sigma-Aldrich cat. no. M-1254); NTA (Sigma-Aldrich cat. no.
N-9877); rhodamine 101 (Sigma-Aldrich; cat. no. 83694); sulforhod-
amine 101 (Sigma-Aldrich cat. no. S-7635); sulforhodamine-B (Sigma-
Aldrich; cat. no. R6626); water (Fluka cat. no. 95305); X-rhod-1, AM
(Invitrogen cat. no. X-14219).
4.1.2. Cells. Experiments followed the European Union and
institutional guidelines for the care and use of laboratory animals
(Council directive 86/609EEC). Cortical astrocytes were prepared
from P0-1 (P0 being the day of birth) NMRI mice (Janvier , St Genest
sur Ile, Mayenne, France) as previously described.40,41 Briefly, the
neocortex was dissected and mechanically dissociated. Cells were
plated and maintained in Petri dishes for one week to reach confluence
before their transfer onto coverslips (#1.5, BK-7, 25 mm diameter,
Marienfeld Superior, Menzel ThermoFisher, Braunschweig, Germany).
Secondary cultures were maintained in Dulbecco’s modified Eagle's
medium (DMEM) supplemented with 5% fetal bovine serum,
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φSR101(s/sSR101)(nwater/nEtOH
)
as a correction44 for the different
2
refractive indices (n = 1.33 and 1.36 for water and ethanol,
respectively, at 535 nm. Here, s is the slope of a plot of the integrated
fluorescence vs absorbance (Figure S1, Supporting Information). To
avoid self-absorption, we worked with solutions having OD 0.01−0.1.
The sequence of measurements was altered between titrations to
exclude bleaching or evaporation effects.
4.3.2. 2PEF Excitation Spectra. 2PEF was measured at RT upon
excitation with a wavelength-tunable fs-pulsed Ti:Sapphire (MaTai
HP, SpectraPhysics-Newport, Santa Clara, CA). The excitation
wavelength was scanned at equal excitation power from 750 to 1000
nm and the laser beam focused with a water immersion objective
(XLUMPFLN 20XW, NA1.0, Olympus, Hamburg, Germany) into a
cuvette containing, in mM, 100 KCl, 30 MOPS at pH 7.2. 2PEF was
epi-collected, extracted with a 750DCSPXR dichroic filter and ET605/
70 band-pass filter (both from Chroma, Bellows Falls, VT) and was
detected on a photon-counting GaAsP PMT module (Hamamatsu,
H7421-40MOD). The mean excitation power (maintained constant at
5 mW at all wavelengths) was monitored on a power meter
(SpectraPhysics Newport, 407A; 1 s integration time) placed
immediately after the sample, on the opposite side of the objective.
The laser beam was attenuated using a combination of a rotatable half-
wave plate and linear polarizer.
4.3.3. Time-Correlated 2PEF Lifetime Measurements. Fluores-
cence lifetime (FL) curves were acquired as described previously19,45
using a time-resolved spectrometer (FluoTime 100, PicoQuant; Berlin,
Germany) and a time-correlated single-photon counting (TCSPC)
computer board (SPC-730, Becker & Hickl; Berlin, Germany). The
excitation source was a mode-locked Ti:sapphire laser (Tsunami,
Spectra Physics Newport, Santa Clara, CA). A fast photodiode (TDA
200, PicoQuant, Berlin, Germany) synchronized the photon counting
to the excitation pulses (880 nm, 80 MHz). A 650 nm short-pass filter
(Chroma, Bellows Falls, VT) and a polarizer set to the magic-angle
orientation were placed in the emission path. The instrument response
function (IRF) was obtained by recording the hyper-Rayleigh
scattering from a colloidal gold suspension (G-1652, Sigma, Seelze,
Germany). Stock solutions (1 mM) of the dyes were prepared in
methanol. Subsequently, the dyes were diluted 1:100 in buffers
containing 10 mM EGTA or 10 mM CaEGTA (Calcium Calibration
Buffer Kit #1, Invitrogen), used for reciprocal dilution series
(Invitrogen) adjusted to cover the Ca2+ range of the dyes. The
recorded fluorescence decay curves of each calibration were subjected
to a double-exponential iterative reconvolution fit (FluoFit software;
PicoQuant, v.4.53) in which the decay time constants were set as
global parameters. The fast time constant (τf) was assigned to the
Ca2+-free (apo-) form of the dye under study, and the slow one (τb)
was assigned to its Ca2+-bound form. The fractional amplitudes of the
decay components resulting from the global fit (αf and αb) were the
Ca2+-dependent parameters in the calibration equations. Ca2+-
insensitive offsets in the decay components, attributable to dye
impurities45or unresolved lifetime components46 were negligible and
ignored in the calibration. Specifically, offsets for the slow component
were absent with all dyes, offsets for the fast component were <5% for
CaRuby-F, <3% for CaRuby-Cl and <1% for CaRuby-Me and, in all
cases, not significantly different from an accordant distribution around
0%. The S parameter was extracted from the calibration curves as
Figure 6. (a) Normalized 2PEF lifetimes of CaRuby-Me at different
[Ca2+] between 1 nM and 100 μM, color-coded as in part b; the black
trace is the instrument response function. (b) Normalized amplitudes
of the pre-exponential coefficients α of the fast and slow decay
components (closed and open triangles, respectively) of a double-
exponential global reconvolution fit to the data shown in part a. Solid
lines are fits with the αf and αs values, yielding a KD,Ca of 3.4 0.3 μM
(dashed vertical line) indistinguishable from that obtained by steady-
state fluorimetric titration (3.4
0.47 μM). See Table 2 for
fluorescence lifetime data for CaRuby-Cl and -F.
a
Table 2. Fluorescence Lifetimes of CaRuby Dyes
CaRuby
tf (ps)
217
ts (ns)
S-factor
KD,Ca (μM)
-Me
-F
3
2
9
3.66 0.12
3.74 0.02
3.61 0.04
0.14 0.01
0.09 0.02
0.12 0.01
3.4 0.3
(9.4 2.5)
20.6 4.4
232
160
-Cl
a
Mean and SD values; n = 3 measurements for each dye.
at 33 °C in CaRuby-Me (1 μM, 15 min) from a stock dissolved in
methanol (1:2000 final) or S101 (1 μM in water, 15 min). Parallel
controls with SR101 dissolved 1:2000 in methanol excluded a possible
solvent effect. During experiments that were performed at room
temperature (RT, 20−23 °C) slices were constantly perfused with
ACSF at 3 mL/min. Cells located near the slice surface were imaged
with 1PEF epifluorescence as described below.
4.3. Fluorimetry, Spectroscopy, and Lifetime Measure-
ments. 4.3.1. Evaluation of Divalent-Cation and pH Sensitivity.
Methods used here have been published elsewhere.16,17,20 Briefly,
stock solutions of the three CaRubies were prepared at 1 mM in
DMSO and diluted to final concentrations in a reference buffer
containing in mM: 100 KCl, 30 MOPS, pH 7.2. Absorbance spectra
were recorded on a double-beam UV/vis spectrophotometer (Varian
Cary 300, Agilent, Massy-Palaiseau, France). From the same samples,
we recorded fluorescence emission spectra in the 544 to 700nm range
on a spectrofluorometer (Jasco, France) housing a Xe-arc lamp.
Excitation and emission slit widths were both set to 2.5 nm. All
measurements were carried out at RT in quartz cuvettes having 1 cm
described earlier.45 Ca2+ concentrations were estimated as Ca2+
=
KDSαb/αf (cf. equation in ref [45).
4.4. Microscopy. 4.4.1. Total Internal Reflection Fluorescence
Microscopy and Photoactivation of CatCh-YFP in Cultured
Astrocytes. Dual-color excitation dual-color emission TIRF micros-
copy and CatCh photoactivation experiments were performed on a
custom microscope described in detail elsewhere.26 Briefly, the 488
and 568 nm lines of an Ar+/Kr+-mixed gas laser (CVI MellesGriot,
Voisins Le Bretonneaux, France) were isolated with an acousto-optical
tunable filter (AA.Opto-Electronic, Orsay, France), focused in the
back-focal plane and directed through the periphery of a high-NA oil-
immersion objective (PlanApo TIRFM x60/NA1.45, Olympus,
Hamburg, Germany). As a consequence, a collimated beam emerges
from the objective at an angle exceeding the critical angle for total
optical path length (QS-115, Hellma, Mullheim, Germany).
̈
Fluorescence quantum yield φ was evaluated relative to that of
SR101, φ = 1.0 in absolute ethanol,43 by integrating the spectral
emission of the respective CaRubies and SR101 and applying φ =
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internal reflection. The penetration depth (1/e2 intensity) of the
evanescent wave was ∼200 nm. Fluorescence was extracted with a
custom 488/568 nm dual-band dichroic (AHF Analysentechnik,
4.5. Data Analysis and Statistics. Image and spectral data were
exported and analyzed with MetaMorph (Molecular Devices,
Sunnyvale, CA) and IGOR Pro (Wavemetrics, Lake Oswego, OR).
Data are shown as mean 1 SD, unless otherwise stated. N is the
number of independent experiments (e.g., animals, titrations), and n is
the number of cells or of regions interest (ROIs). The significance of
the difference between samples was evaluated by ANOVA and t-test
with MATLAB (The MathWorks, Natick, MA). p values less than 0.05
were considered significant.
Tubingen, Germany) and home-built dual-viewer device housing a
̈
HQ600LP secondary dichroic, HQ535/50 band-pass (green) and
HQ600LP long-pass filter (red) (all from Chroma, Bellows Falls, VT)
and detected on an electron-multiplying charge-coupled device camera
(QuantEM512SC, Photometrics, Tucson, AZ, 16 μm pixel size). All
setup components were controlled with Metamorph (Molecular
Devices, Sunnyvale, CA). The 488 nm TIRF excitation beam, 488/
568 dual-band dichoric and DF535/50 emission filter were used to
search for CatCh-YFP expressing cells, before illumination was
switched to 568 nm 1 Hz TIRF imaging for monitoring the CaRuby
fluorescence. For the photoactivation of CatCh we used brief (0.5−5
s) flashes of 458 9 nm epi-illumination (13.6 mW/mm2) from a
tunable polychromatic light source (Poly II, TILL Photonics,
ASSOCIATED CONTENT
■
S
* Supporting Information
Six supplementary figures and their legends. Details of synthesis
and purification of CaRuby-Cl, CaRuby-F, and CaRuby-Me are
given. This material is available free of charge via the Internet at
http://pubs.acs.org. Complete NMR and mass spectra files are
available from the authors upon request.
Grafelfing, Germany). Ca2+ imaging was stopped during photo-
̈
activation. Time-lapse fluorescence data was corrected with the
prestimulus baseline to account for dye leakage or photobleaching.
TIRFM was also used to monitor ATP-evoked [Ca2+]i changes in
the near-membrane region of cultured astrocytes loaded with the Ca2+
indicator Fluo-4, AM (KD,Ca ∼345 nM). Cells were incubated in dye-
containing extracellular solutions (30 min, RT) and then washed for at
least 30 min prior to Ca2+ imaging. ATP (300 μM) was applied locally
via a double-channel perfusion system, one channel delivering the
control buffer and the other the ATP-containing solution. To start the
stimulation, the control channel was stopped while switching on the
ATP channel at the same time, and the reversed procedure was carried
out to terminate the stimulation. The different solutions were
delivered through plastic tubings (0.8 mm ID, Tygon, Charny,
France) to a multichannel holder (AutoMate Scientific, Berkeley, CA)
connected to a small (250 μm ID) silica pipette (WPI, Saratosa, FL,
USA) positioned ∼200 μm away from the cell. Images were streamed
at 20 Hz.
AUTHOR INFORMATION
■
Corresponding Author
jean-maurice.mallet@ens.fr
Present Addresses
○Biology Faculty, Kazan Federal University, Kazan, Russia.
□Wolfson Institute, University College London, London, U.K.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank K. Her
́
ault for astrocyte culture, Prof. Dr. E. Bamberg
(MPI fur Biophysik, Frankfurt) for the CatCh-YFP plasmid,
̈
4.4.2. Epifluorescence 1PEF Imaging of Acute Brain Slices. Acute
mouse brain slices were imaged on a custom upright microscope fitted
for alternate 1PEF wide-field epifluorescence and 2PEF scanning
microscopy.47 For 1PEF epifluorescence, we used a Hg-arc lamp,
attenuated and filtered with HQ470/40x and HQ560/80x excitation
band-pass filters for EGFP and for CaRuby/SR101, respectively.
Fluorescence was extracted with 500DCXR and 600DCXR dichroics,
DF535/35 and FF01-654/75-25 emission band-pass filters, respec-
tively, and captured on a Marconi Quantix57 CCD detector
(Photometrics, Tucson, AZ, 13 μm pixel size). All filters were from
Chroma Technology (Bellow Falls, VT) or Semrock (Rochester, NY).
For the images shown in Figure 4, the effective pixel size was 630 nm
in the sample plane (XLMPlanFluor, x20/NA0.95, Olympus,
Hamburg, Germany).
4.4.3. 2PEF in Vivo Imaging. Two 3 months old (body weight 250
g) male Sprague−Dawley rats were anaesthetized with isoflurane (1−
1.5%) in O2/NO2 (20%/80%), placed on a heating blanket and
maintained in a stereotaxic frame using a custom-made mouth piece
Heartbeat and breathing were continuously monitored. A 3 mm
diameter craniotomy centered at 2.5 mm from the bregma and 5.5 mm
laterally was performed above the right hemisphere, and the dura
mater was removed. Throughout the surgery the brain was bathed in
external saline (in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25
NaH2PO4, 2CaCl2, 1 MgCl2, and 20 glucose).
and Drs. E. Audinat and J. S. Kehoe for comments on early
versions of the manuscript. This work was supported by the
French Agence National de la Recherche (ANR P3N,
nanoFRET2 grant to A.F., J.M.M., and M.O.) and the European
Union (FP6 STRP AUTOSCREEN grant and FP7 ERA-NET
Neuron nanosyn grant to M.O.).
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