CAPRYDAA, an anthracene dye analog to LAURDAN: A comparative study using cuvette and microscopy

Article abstract of DOI:10.1039/c9tb01738k

We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable by two-photon laser sources, making it easy to combine CAPRYDAA with either blue or red emission probes. In GUVs or cells, CAPRYDAA can discriminate the lipid phases and liquid-liquid phase heterogeneity. This new membrane probe shows the bathochromic properties of the PRODAN-based probes designed by Weber, overcoming the need for UV or two-photon excitation and facilitating the studies on the membrane properties using regular confocal microscopes.

Full text of DOI:10.1039/c9tb01738k

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CAPRYDAA, an anthracene dye analog to
LAURDAN: a comparative study using cuvette
and microscopy†
Cite this: J. Mater. Chem. B, 2020,
8, 88
a
Vicente Castro-Castillo,
Javier Gajardo,a Catalina Sandoval-Altamirano,a
b
c
bd
Enrico Gratton,
Susana Sanchez,
Leonel Malacrida
*
and
a
German Gunther
*
We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular
probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a
dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively.
This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe
designed to mimic the well-known membrane probe LAURDAN’s location and response in the lipid
membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption
and emission bands are red-shifted according to the polarity of the media. The photophysical behavior
of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and
in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable
physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light
(488 nm) as an excitation source. CAPRYDAA was also excitable by two-photon laser sources, making it
easy to combine CAPRYDAA with either blue or red emission probes. In GUVs or cells, CAPRYDAA can
discriminate the lipid phases and liquid–liquid phase heterogeneity. This new membrane probe shows
the bathochromic properties of the PRODAN-based probes designed by Weber, overcoming the need
for UV or two-photon excitation and facilitating the studies on the membrane properties using regular
confocal microscopes.
Received 14th August 2019,
Accepted 8th November 2019
DOI: 10.1039/c9tb01738k
rsc.li/materials-b
donor character.4 Generally, the magnitude of the spectral
changes shown by the probe is directly related to the spatial
1. Introduction
Fluorescent probes are essential tools in a wide range of biological separation between both substituent groups as well as to the
research. In particular, environment-sensitive hydrophobic basic molecular entity. The structure of the series of polarity
fluorescent probes are widely used in the study of molecular sensitive fluorescent probes, designed and synthesized by Weber
organization in the membranes.1–3 According to Weber’s definition, et al.4 in the late seventies, which undergo intramolecular
an environmental probe must contain, in its molecular structure, at photoinduced charge transfer, contains a naphthalene aromatic
least two different substituent groups or organic functionalities, moiety substituted with a dimethylamino group in position 2
both attached to an aromatic ring, with one of them having an (donor group) and an alkyl carbonyl group in position 6 (acceptor
electron acceptor character and the other with an electron group). PRODAN and LAURDAN, among others, belong to this
family of probes. LAURDAN has been widely used, both in
cuvettes and in microscopes, for the study of the synthetic
a
´
´
membranes and live cells.5–17 These studies include the char-
acterization of lipid phases in the membranes, determination of
partition coefficients,18 observation of shape changes during the
phase transition,19 observation of lipid–protein interactions,20
and discrimination of cholesterol dynamics in living cells.21 The
complex photophysics and solvatochromism properties of this
family of compounds involve the contribution of solvent dielectric
relaxation and intramolecular excited-state reactions. The separation
Universidad de Chile, Facultad de Ciencias Quımicas y Farmaceuticas,
´
´
´
Departamento de Quımica Organica y Fisicoquımica, Casilla 233, Santiago 1,
Chile. E-mail: ggunther@ciq.uchile.cl
b Laboratory for Fluorescence Dynamics, Biomedical Engineering Department,
University of California at Irvine, Irvine, CA, USA
c
´
´
´
Universidad de Concepcion, Facultad de Quımica, Departamento de Polımeros,
´
Concepcion, Chile
d
´
´
Departamento de Fisiopatologıa, Unidad de Microscopia Avanzada y Bifotonica,
´
´
Hospital de Clınicas, Facultad de Medicina, Universidad de la Republica,
Montevideo, Uruguay. E-mail: lmalacrida@hc.edu.uy
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb01738k of these two contributions allows a detailed characterization of
88 | J. Mater. Chem. B, 2020, 8, 88--99
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the processes.22 In fact, the analysis of the spectral data using possibilities for studying the membrane dynamics using the
phasor analysis allows this distinction, making phasor analysis fluorescent probes for in vitro as well as in vivo experiments and
a very useful tool to describe the complex systems.16,17,23 At this combining the opportunity to work in cuvettes and with imaging.
point, it is important to acknowledge that several researchers The major advantage of CAPRYDAA is the possibility of working
have developed other analogs of the LAURDAN/PRODAN family with single-photon excitation, thereby avoiding the need for
for imaging lipid organization in cell membranes and model 2-photon or UV illumination.
membrane systems. Among them, the fluorine-based push–pull
probes24,25 and push–pull pyrene derivatives26 show a large shift
in the emission as a function of the lipid order.27
The LAURDAN’s emission is mainly in the wavelength range
between 400 and 530 nm, with maximum excitation at 370 nm.
2. Experimental
2.1. Materials
All the reagents and solvents were purchased from Sigma-
The possibility to shift absorption/emission to longer wavelengths
Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany)
is a major goal since such a red-shift will have advantages for
and used without further purification. Dioleoylphosphatidyl-
biological imaging, including facilitating light penetration into
choline (DOPC), dipalmitoylphosphatidylcholine (DPPC), and
tissues, reducing auto-fluorescence, and opening a different wave-
cholesterol (Chol) were obtained from Avanti Polar Lipids
length window for combination with other biological fluorescent
(Alabaster, AL, USA) and used as received. Melting points were
markers. An option to achieve this purpose is to increase the
determined on a Reichert Galen III hot plate with a DUAL JTEK
number of aromatic rings in order to enlarge the distance between
substituents involved in the intramolecular charge transfer. This
methodology has been used to fabricate the anthracene derivative
Dig-Sense thermocouple thermometer and were uncorrected.
1
The H and 13C NMR spectra were recorded on Bruker Avance
spectrometers at 200 or 400 MHz and 50 or 100 MHz, respectively,
dyes. The excitation wavelengths of these derivatives are red-
using CDCl3 or DMSO-d6 as solvents unless indicated otherwise.
shifted (near-infrared in two-photon excitation),28 and they
show high quantum yields. As an example of this strategy, we
The chemical shifts were reported in terms of d (ppm), downfield
from TMS. The coupling constants ( J) were given in Hz. The
can mention the PRODAN (2-propionyl-6-dimethylaminonaph-
analytical TLC was performed on Merck silica gel 60 F254 foil-
thalene) homologs and Anthradan (6-propionyl-2-(dihexylamino)-
backed plates. The solvents used as eluent were specified in
anthracene), whose excitation and emission wavelengths shift
each case. The chromatographic purification of all the products
around 100 nm and 80 nm, respectively. Further, the batho-
was carried out on the silica gel columns, and the solvents used
chromic displacement of the absorption bands was accom-
were specified in each case.
plished when a symmetric diaryl substitution on the positions
9 and 10 of the anthracene moiety was performed. These
substituted probes (analogs of Acedan (2-acetyl-6-(dimethylamino)-
2.2 Methods
Synthesis of 2-nonanoyl-6-(dimethylamino)anthracene, CAPRYDAA.
Fig. 1 and Scheme 1 shows the molecular structure and the
synthetic path of CAPRYDAA, respectively. More details are
given in the ESI.†
naphthalene)) have been used for two-photon microscopy
imaging.29
In this report, we synthesized an anthracene derivative, with
an acyl chain of nine carbon atoms and a dimethylamino sub-
stitution at positions 2 and 6 of the anthracene ring, respectively.
The logic behind this synthetic design was to obtain a molecule
with a similar membrane location and response to the environ-
ment as that of the LAURDAN probe, but with a bathochromic
shift in the absorbance and emission bands.
We report here the synthesis, spectroscopic characterization,
and applications of 2-nonanoyl-6-(dimethylamino)anthracene,
CAPRYDAA. The photophysical behavior was measured in several
media (homogeneous, synthetic bilayers, and cells), with the
measurements being carried out both in cuvettes and under the
microscope, using one- and two-photon excitation to establish
its applicability under different experimental techniques and
conditions as well as to compare with the LAURDAN probe.
Our results show a comparable behavior of CAPRYDAA with
LAURDAN, but with the advantage of using visible light (488 nm)
for excitation. Moreover, CAPRYDAA was fully excitable with two-
photon laser sources, due to which CAPRYDAA can easily combine
with either blue or red emission probes. Further, by using
CAPRYDAA, it was possible to discriminate lipid phases in
GUVs as well as liquid phase heterogeneity in living cells.
Therefore, here, we present a novel probe that extends the
Briefly, the synthesis of 6 was carried out by using a method
reported in the literature.28 Compound 2 was synthesized in
good yield by the Sandmeyer reaction, using tert-butyl nitrite in
dry acetonitrile. The treatment of 2 with CsF in DMSO afforded
2-bromo-6-fluoro-9,10-anthraquinone, which was treated, without
purification, with aqueous dimethylamine to afford 2-bromo-6-
dimethylamino-9,10-anthraquinone (3). The subsequent sodium
borohydride reduction followed by the dehydration of 3 and
further subjection to a Rosenmund–von Braun reaction yielded
5. Finally, 6 was prepared by the nucleophilic addition of octyl-
magnesium bromide to the cyano compound followed by the
hydrolysis of the imine intermediate (more details and NMR
spectra are given in the ESI†).
Fig. 1 The molecular structure of CAPRYDAA.
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Scheme 1 Reagents and conditions: (a) tert-butylnitrite, CuBr2, CH3CN, 90 1C, 24 h; (b) (1) CsF, DMSO, N2, 170 1C, 5 h; (2) (CH3)2NH (ac) 40%, K2CO3, 70 1C,
17 h; (c) (1) NaBH4, 2-propanol, reflux, 12 h; (2) HCl (ac) 37%; (d) CuCN, DMF, N2, reflux 12 h; (e) CH3(CH2)6CH2MgBr, CuBr (cat.), THF, N2, room temperature, 1 h.
Cuvette spectroscopy measurements. The UV-vis spectra were coumarin 6, having a reference lifetime of 2.5 ns (www.iss.
recorded on an Agilent 8453 Diode-Array spectrophotometer com). For FLIM data acquisition, an ISS A320 FastFLIM box
(Agilent, USA) in the range of 250–700 nm. The fluorescence (ISS, Champaign, USA) was used to collect the lifetime decay
emission spectra were obtained by using an ISS PC1 spectro- data. The images were acquired using a 40ꢀ water immersion
fluorometer (ISS, Champaign IL) or a FluoroMax 4CP (Horiba objective 1.2 N.A. (Carl Zeiss). CAPRYDAA was excited using
Jobin Yvon, Japan) at room temperature. Further, the fluores- either a 488 nm or 900 nm excitation sources. LAURDAN was
cence lifetime measurements were carried out by using the excited using 780 nm excitation source. For the FLIM measure-
time-correlated single-photon counting (TCSPC) method on a ments, two high-efficiency GaAsP Hybrid Detectors (HPM-100-40,
PicoQuant FluoTime 200 fluorescence lifetime spectrometer Becker & Hickl GmbH) were used, and before the detector, a filter
(PicoQuant, Germany) equipped with a multichannel scaler, cube composed of two bandpass filters (440/60 nm, channel-1, and
PicoQuant’s Timeharp 250 (Pico Quant, Germany). LEDs or 500/60 nm, channel-2) and a 470 nm dichroic mirror from
lasers of appropriate wavelengths were employed as excitation Semrock was employed. We defined channel-1 and channel-2 as
sources. The fluorescence quantum yields were measured in a blue and green filters, respectively. For the hyperspectral imaging,
FluoroMax 4CP (Horiba Jobin Yvon, Japan) spectrofluorometer the lambda mode configuration of the Zeiss LSM8800 was used. A
equipped with the Quanta-Phi integrating sphere.
32-channel configuration was used with a bandwidth of 9.7 nm,
and the total range acquired was between 410 nm and 695 nm.
Partition constant measurements. The water/dodecane partition The image acquisitions in both the configurations (FLIM and
for CAPRYDAA was measured by HPLC. The solution was allowed hyperspectral) were performed with a pixel frame size of 256 ꢀ
to equilibrate for 48 h and before the measurement, the aqueous 256 and a pixel dwell time of 16.38 ms per pixel. The hyperspectral
and organic phases were centrifuged at 3000 rpm for 10 min and FLIM data were processed using the SimFCS software
(Hermle Z200A) to split the phases properly. The partition constant developed at the Laboratory for Fluorescence Dynamics (www.
was obtained from the ratios of the areas of the chromatographic lfd.uci.edu).
peaks obtained in a Waters 600 HPLC system. The HPLC was
equipped with a Waters 996 PDA pump, the column used had a
GUVs preparation. Giant unilamellar vesicles (GUVs) were
1.15 mm pore, 100 ꢀ 4.6 mm Merck Chromolith RP-18 (Merck prepared by following the electroformation protocol described
KGaA, Darmstadt, Germany), and the mobile phase and chromato- by Pott et al.30 Briefly, dioleoylphosphatidylcholine (DOPC)
graphic conditions were: isocratic acetonitrile having a flow rate or dipalmitoylphosphatidylcholine : cholesterol (1 : 1 molar,
of 1 mL minꢁ1 with subsequent detection at 315 nm.
DPPC :Chol) was prepared at a final concentration of 0.3 mM
in chloroform. CAPRYDAA or LAURDAN at 0.5% molar was
One- and two-photon lifetime and hyperspectral microscopy. added to the lipid mixture in chloroform. Then, 4 mL of the
The samples were incubated with either 1 mM of CAPRYDAA or organic mixture was applied to each platinum wire and dried
LAURDAN for 30 min at 37 1C. The fluorescence lifetime imaging under vacuum overnight. The electroformation chamber was
(FLIM) and the hyperspectral microscopy were acquired using a filled with 400 mL of 200 mM sucrose at 50 1C. To keep the
Zeiss LSM880 laser scanning microscope (Carl Zeiss, Dublin, temperature constant, we used a water circulating bath. For the
CA-USA). The instrument was equipped with a 20 MHz pulsed GUVs growth, we applied a sinusoidal potential of 2 volts and 10 Hz
supercontinuum white laser (A2, SC390, Fianium Inc, USA) for for 1.5 hours. To detach the GUVs from the wire, the frequency of
488 nm excitation and a Ti:Sapphire laser (Spectra-Physics, the sinusoidal potential was reduced to 1 Hz for 10 min, and
Newport-CA-USA) with 80 fs pulses and 80 MHz repetition then, the function generator and circulating bath were turned off.
rate for two-photon excitation. The calibration of the lifetime For the measurements, 50 mL of the GUVs dispersion were trans-
measurements was performed using a 100 nM solution of ferred to an 8-well bottom-glass imaging chamber, containing 300 mL
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ꢀ
ꢁ
Ð
2pnðl ꢁ liÞ
lmax ꢁ lmin
l
lmin
of a 200 mM glucose solution. The imaging chamber was previously
coated with 1 mg mLꢁ1 of the bovine serum albumin (BSA) solution
(to avoid vesicle burst on settling), and before transferring the GUVs,
each well was washed 10 times with 200 mM glucose solution.
Cell culture. The NIH-3T3 fibroblasts derived from mouse
(ATCCs CRL-1658t) were grown at 37 1C, with 5% CO2 in
Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific
Inc. Huntington Beach, CA-USA), supplemented with 10%
fetal bovine serum, and 5 mL of Pen-Strep. The cells (2 ꢀ
104 cells per dish) were plated 24 h before the experiments onto
fibronectin-coated 35 mm glass-bottom dishes (MatTek Corpora-
tion, MA-USA). On the day of the experiments, the cells were
incubated for 30–60 min with a final solution of 3 mM CAPRYDAA
or LAURDAN. Before imaging, the dyes were removed by replacing
it with a fresh medium. The concentration of LAURDAN
max IðlÞ sin
dl
y coordinate ¼ S
¼
(4)
Ð
ðlÞ
l
max IðlÞdl
lmin
The position for every pixel in the spectral phasor plot can
be defined by the phase angle and the modulus (M), given the
coordinates G and S (for both spectral or lifetime), in the
following way:
y = arctan(S/G)
(5)
(6)
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
M ¼ S2 þ G2
The angular position in the spectral phasor plot relates to the
center of mass of the emission spectrum, and the modulus
depends on the spectrum’s full width at half maximum
(FWHM). For instance, if the spectrum becomes broad, the
phasor cluster should move closer to the center (modulation is
decreasing). On the other hand, for the red-shifted spectra, the
position of the phasor will move counter-clockwise towards the
increasing angles from position (1,0), i.e., the phase angle will
increase. The spectral phasors share all the vector properties
with the lifetime phasors. Moreover, an in-depth description of
the properties of the spectral phasor plots is given in the ref. 16.
was determined using
a molar extinction coefficient of
20 000 Mꢁ1 cmꢁ1 at 364 nm in methanol. During the imaging,
on the Zeiss LSM880 microscope, the cells were kept at 37 1C
with 5% CO2 using the Zeiss incubator.
Data analysis and interpretation
Fluorescence lifetime imaging and phasor analysis (FLIM phasor
plot). The FLIM phasor plot was obtained using eqn (1) and (2),
as described previously.22,31 Briefly, the fluorescence decay, I(t),
was acquired at each pixel of an image, and then, the G and S
coordinates were calculated. The G and S coordinate for each
pixel was plotted in a polar plot (referred to as phasor plot or
phasor space) as follows:
3. Results and discussion
Cuvette spectroscopic characterization
The absorption and emission spectra of CAPRYDAA and LAURDAN
in solvents with increasing polarity were compared. The absorption
spectrum of CAPRYDAA (Fig. 2A) was found to undergo a shift of
around 100 nm towards the red region as compared with the
LAURDAN probe (Fig. 2D). In homogeneous media, the absorption
spectra and molar absorptivity at maxima absorption wave-
lengths, for both CAPRYDAA and LAURDAN, showed a fair
dependency on the solvent polarity. For the LAURDAN probe,
the molar absorptivity, at the absorption maxima at a longer
wavelength (around 20 000 Mꢁ1 cmꢁ1), was twice to that of
CAPRYDAA’s molar absorptivity, which was determined to be
around 9000 Mꢁ1 cmꢁ1 (Fig. 2A and D).
Ð
T0 IðtÞ cosðotÞdt
x-coordinate ¼ G
¼
(1)
Ð
ðlÞ
T0 IðtÞdt
Ð
T0 IðtÞ sinðotÞdt
y-coordinate ¼ S
¼
(2)
Ð
ðlÞ
T0 IðtÞdt
where, o is the angular modulation frequency (equal to 2pf, and f is
the repetition frequency of the laser) and T is the period of the laser
frequency. Phasors follow the rules of vector addition, orthogonality,
and reciprocity, i.e., pixels that contain a linear combination of two
independent fluorescent species will appear on the line joining the
two independent emissions in the phasor plot. A detailed descrip-
tion and discussion can be found elsewhere.32
Further, clear bathochromic shifts were observed for CAPRYDAA
(Fig. 2B and C) and LAURDAN (Fig. 2E and F). To compare the
spectral shift (Stokes shift) for CAPRYDAA and LAURDAN, the
Lippert–Mataga equation was used:33,34
Hyperspectral imaging and phasor analysis (spectral phasor
plot). The fluorescence spectrum, at each pixel, in a spectral image
was transformed into phasor coordinates, (G(l)) and (S(l)), as
described in eqn (3) and (4), respectively.16 The G(l) and S(l)
coordinates are the real and imaginary components of the Fourier
transformation. I(l) represents the intensity at every wavelength
(channel in our case), n is the number of the harmonic, and li the
initial wavelength. The x and y coordinates were plotted in the
spectral phasor plot, as previously shown by Malacrida et al.16
2
2Df ðmE ꢁ mGÞ
e ꢁ 1
n2 ꢁ 1
nA ꢁ nF
¼
;
Df ¼
ꢁ
hc
a3
2e þ 1 2n2 ꢁ 1
In this equation, mE and mG corresponds to the dipole moments
of the probe in the excited and ground state, respectively; a is a
radius of an assumed spherical Onsager cavity, n2 is the square
of the refractive index, and e is the dielectric permittivity of the
solvent. Fig. 3 shows the experimental data and fitting to the
Lippert–Mataga equation. The trend observed for both probes
was the same (similar slopes were observed in Fig. 3). Assuming
a larger volume for the cavity of CAPRYDAA, these data indicate
that the charge transfer dipole of CAPRYDAA must be smaller
than that of LAURDAN.
ꢀ
ꢁ
Ð
2pnðl ꢁ liÞ
lmax ꢁ lmin
l
lmin
max IðlÞ cos
dl
x coordinate ¼ G
¼
(3)
Ð
ðlÞ
l
max IðlÞdl
lmin
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Fig. 2 Spectroscopic characterization of CAPRYDAA and LAURDAN. (A) The absorption spectra of CAPRYDAA in solvents with increasing polarity
(hexane, benzene, chloroform, acetonitrile, and ethanol). (B) The emission spectra for CAPRYDAA in increasing solvent polarity. The excitation
wavelength used was 450 nm. (C) The spectral phasor transformation for the emission spectra of CAPRYDAA shown in (B). (D) The absorption spectra
of LAURDAN in solvent with increasing polarity (hexane, benzene, chloroform, acetonitrile, and ethanol). (E) The normalized emission fluorescence
spectra for LAURDAN in increasing solvent polarity. The excitation wavelength used was 360 nm. (F) The spectral phasor transformation for the LAURDAN
emission spectra shown in (E).
Additionally, it must be stated that both absorption and the optimal excitation by two-photon excitation was determined
emission properties as well as their dependency on the solvent to be around 900 nm, while the LAURDAN excitation maximum
for CAPRYDAA were found to be similar to that reported for was found around 780 nm.
Anthradan by Lu et al.28
Several CAPRYDAA photophysical parameters related to the
first singlet excited state in homogeneous media were measured
(Table 1). The singlet state emission decay of CAPRYDAA, in
homogenous media with different polarities, can be fitted to the
monoexponential decays, with lifetime values (t) between 5 and
12 ns (Table 1). Further, the lifetime values for LAURDAN, i.e.,
between 2.5 and 5.5 ns, were found to be similar to the values
reported in the literature.35 However, the fluorescence quantum
yield (FF) for CAPRYDAA decreased as the polarity of the solvent
medium increased (Table 1).
Fluorescence lifetime characterization (FLIM-phasor analysis)
The second comparison was the dye response to the physical
order of the lipid bilayer. The CAPRYDAA partition in water was
estimated by the partition constant measurements in a water/
dodecane mixture. A log p 4 4 was experimentally obtained,
and hence, it can be considered that there is no partition of
CAPRYDAA in the solvent. Further, the fluorescence lifetime
measurements in MLVs (multilamellar vesicles) of lipids in
different phases were used to evaluate the performance of
CAPRYDAA (Fig. 5). Three different sets of MLVs were prepared
for this comparison, and the measurements were carried out at
37 1C: MLVs of DPPC (gel phase), DOPC (fluid phase), and
DPPC : cholesterol (1 : 1, molar) were used as a liquid order model.
The vesicles were labeled with either CAPRYDAA or LAURDAN,
Additionally, the effect of temperature in the CAPRYDAA
emission from DPPC SUVs is shown in the ESI† (Fig. S9).
Microscope intensity measurements
Two-photon excitation characterization. Generally, the measure- and the lifetime measurements were performed by collecting the
ments of the LAURDAN probe in a confocal microscope require two- emission in two different channels: blue (channel-1) and green
photon excitation in order to avoid severe photobleaching that (channel-2) wavelength range of the dyes. Using this strategy, it
occurs with one-photon excitation. Thus, the first comparison was possible to isolate the polarity (blue region) from the dipolar
for the newly synthesized CAPRYDAA and LAURDAN was the two- relaxation effect of the dye in the excited state (green region).22
photon cross-sectional excitation spectra (Fig. 4). For CAPRYDAA, Additionally, the fluorescence lifetime measurements were carried
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Fig. 4 Two-photon excitation spectra for CAPRYDAA and LAURDAN: the
black line shows the CAPRYDAA absorption spectrum in DMSO. The red
line and square boxes show the two-photon excitation spectra for
CAPRYDAA in DMSO. The LAURDAN two-photon excitation spectrum in
DMSO is shown by the blue trace and circles.
different, namely being shorter in the Lb phase than in the Lo
phase (Fig. 5A); however, the two-photon excitation cannot
differentiate between these two phases (Fig. 5B, channel-1).
On the other hand, for LAURDAN the Lb phase shows the longer
lifetime in regards to the other membrane models when two-
photon excitation was used (Fig. 5C).
Fig. 3 The Lippert–Mataga plot for CAPRYDAA (red squares) and LAUR-
DAN (black circles) as a function of solvent polarity.
The lifetime data acquired in channel-2 (Fig. 5A–C; the
bottom panel) gives information regarding the dipolar relaxation,
and in the phasor representation shown in Fig. 5, this phenom-
enon is illustrated by the position of the phasors outside the
universal circle. Generally, a position outside the universal circle is
associated with a reaction in the excited state, and in this case, it is
due to the dipolar relaxation of the water molecules around the
probe. The solvent dipolar relaxation takes time, and this duration
is responsible for the increase in the phase angle, and hence, the
position outside the phasor plot is observed. For CAPRYDAA, the
dipolar relaxation was smaller for one-photon as compared with
the two-photon excitation. In Table 2, the lifetime for CAPRYDAA
and LAURDAN using phase and demodulation analysis is shown.
The difference between the tau phase (tP) and tau modulation (tM)
values in channel-1 is indicative of a complex decay. On the other
hand, the larger values for the tau phase over the tau modulation
in channel-2 were found to be related to the occurrence of a
reaction in the excited state. For instance, delays in the phase were
present when the dipolar relaxation of the solvent molecules
occurred around the probe in the excited state.
Table 1 Results summary for the photophysics properties of CAPRYDAA
in solvents with different polarity
Solvent
Hexane
FF
t/ns
l
abs/nm
l
em/nm
e/Mꢁ1 cmꢁ1
0.44
6.46
314–428
320–438
321–443
316–441
316–443
475
507
527
554
587
52 589 (314)
9437 (428)
50 566 (320)
9816 (438)
42 469 (321)
10 113 (443)
46 148 (316)
9924 (441)
42 841 (316)
10 552 (443)
Benzene
CHCl3
ACN
0.51
7.84
7.68
7.56
5.29
0.44
0.29
EtOH
0.10 (MEOH)
FF = fluorescence quantum yield. t = fluorescence lifetime. labs
absorbance wavelength. lem emission fluorescence wavelength.
e = extinction coefficient (value and absorbance wavelength).
=
=
out in a microscope, using two-photon excitation at 780 nm for
LAURDAN, and 900 nm for CAPRYDAA. For CAPRYDAA, one-
photon excitation was also used since no significant photo-
bleaching was observed.
Channel-1 (Fig. 5) gives information about the polarity of the
media surrounding the dye molecule, and therefore the physical
Spectral shift comparison
state of the membrane. As expected, the Ld phase showed a The spectral shift of the probes in different lipid environments
shorter lifetime (indicated by the red cursor in Fig. 5A–C, was analyzed using the spectral phasor approach16,36 (Fig. 6).
corresponding to channel-1). This result was consistent for both The emission spectrum of CAPRYDAA was found to be similar,
the dyes using one and/or 2-photon excitation. However, for Lb whether one-photon or two-photon excitation was used, as
and Lo phases, the FLIM-phasor analysis showed some differences shown in the spectral phasor plots (Fig. 6A and B). The spectral
between CAPRYDAA and LAURDAN. The CAPRYDAA probe phasors for CAPRYDAA in the Ld phase showed the largest
showed differences in the lifetime values when one or two- phase and smallest modulation, as evidenced by the more red-
photon excitation was used (Fig. 5A and B). When one photon shifted spectra and higher emission heterogeneity (broader
excitation was used, the lifetime values of CAPRYDAA were spectrum) (Fig. 6A and B). Further, the blue-shifted spectra
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Fig. 5 CAPRYDAA and LAURDAN lifetime comparison in multilamellar vesicles by FLIM-phasor plot. (A) The FLIM-phasor plot for CAPRYDAA in MLVs
with Lo, Ld, and Lb phases using 20 MHz pulsed supercontinuum laser (488 nm excitation) and collecting the emission decay in two channels: channel-1
(polarity) and channel-2 (dipolar relaxation). The arrows in the inset illustrated the shift of the phasor position from channel-1 to channel-2. (B) The
phasor plot distribution for CAPRYDAA in MLVs with Lo, Ld, Lb phases using 900 nm excitation at 80 MHz. (C) The phasor plot distribution for LAURDAN in
MLVs with Lo, Ld, and Lb phases using 780 nm excitation at 80 MHz. Lo was DPPC : cholesterol 1/1 molar, Ld was DOPC, and Lb was DPPC.
Table 2 Fluorescence lifetime of CAPRYDAA and LAURDAN in MLVs
Channel-1
Channel-2
Lipid mixture
tP
tM
tP
tM
CAPRYDAA 900 nm
DPPC
DOPC
7.07 ꢂ 0.05
4.37 ꢂ 0.12
9.60 ꢂ 0.05
11.33 ꢂ 0.05
5.50 ꢂ 0.00
11.73 ꢂ 0.17
34.00 ꢂ 2.405
13.67 ꢂ 0.05
9.50 ꢂ 0.08
13.33 ꢂ 0.12
a
—
DPPC : Chol (1 : 1)
29.37 ꢂ 4.84
LAURDAN 780 nm
DPPC
DOPC
5.27 ꢂ 0.05
2.37 ꢂ 0.05
5.23 ꢂ 0.05
5.37 ꢂ 0.05
2.47 ꢂ 0.05
5.43 ꢂ 0.12
8.10 ꢂ 0.22
5.93 ꢂ 0.05
8.07 ꢂ 0.17
5.93 ꢂ 0.12
3.87 ꢂ 0.05
5.93 ꢂ 0.12
DPPC : Chol (1 : 1)
tP, tau phase. tM, tau modulation. n = 3 for each sample.a Phase larger than 90 degree.
were found for CAPRYDAA in the membranes in the Lo phase. Lo, Ld, or coexistence of the Lo/Ld phases (Fig. 7). Using the cursor
Finally, the gel phase (Lb) had similar modulation as Lo but selection on the spectral phasor plot (Fig. 7A and B), we were able
with an increased phase angle, indicative of a blue-shifted to segment the liquid order from the liquid disorder membrane
spectrum. For LAURDAN, a larger spectral shift was obtained, either in the single lipid phases or in the coexistence of Lo/Ld
but the most blue-shifted spectrum was identified for DPPC phases.
(gel phase, see Fig. 6C). The liquid order membrane showed a
slightly increased phase angle, but with a significant decrease
in modulation (Fig. 6C).
Spatial resolution in live cells
After the GUV experiments, we moved to in vivo cell imaging. We
used NIH-3T3 cell, and the CAPRYDAA fluorescence was acquired
by using the lifetime and hyperspectral imaging (Fig. 8 and 9,
Spatial resolution in model systems
To test the spatial resolution of CAPRYDAA under hyperspectral respectively). Using intensity or hand-drawn image segmentation,
imaging, i.e., its ability to discriminate between the membrane we explore the dye response in different subcellular environments.
phases, we prepared giant unilamellar vesicles (GUVs) containing The lipid droplets were assigned to the high-intensity pixels due to
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Fig. 6 Spectral phasor characterization for single and two-photon excitation of CAPRYDAA versus LAURDAN in multilamellar vesicles. (A) Spectral
phasor characterization of CAPRYDAA in MLVs with different lipid phases (Lb-DPPC green cursor; Lo-DPPC:cholestrol 1/1 molar pink cursor; Ld-DOPC
red cursor) using single photon excitation (488 nm). (B) Spectral phasor characterization of CAPRYDAA in MLVs with different lipid phases using two-
photon excitation (900 nm). (C) Spectral phasor of LAURDAN in MLVs with different lipid phases using two-photon excitation (780 nm). All the
experiments were done at 37 1C. At the right of each figure is an enlargement of the ROI represented as the dashed box.
the accumulation of the dye in the cell interiors and significant the interior plus plasma membrane or the plasma membranes,
increment in the quantum yield (Fig. 8A and B). The spectral isolated by the hand-drawn masks, were analyzed (Fig. 8C,
shift was color coded using the three-cursor selection as G, D and H). The histogram of the spectral shift was plotted
described previously by Ranjit et al.37 (Fig. 8E and H). Then, as a fraction of the green cursor, representing the fraction of the
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Fig. 7 CAPRYDAA fluorescence in GUVs with different phases. (A) The spectral phasor plot for CAPRYDAA in Ld, Lo : Ld, and Lo membranes. (B) Zoom of
(A). (C) The representative images of GUVs with Ld, Lo : Ld, and Lo phases, respectively.
Lo membrane (Fig. 8J). Using this approach, we can quantita-
tively evaluate the differences within the cellular membranes,
4. Final remarks
since the lipid droplets correspond to the bluer environment. At Biological membranes are a supramolecular assembly, classically
this point, it is important to mention the biochemical and associated with the subcellular compartmentalization; however,
structural differences between the membrane and the lipid dro- membranes are sensitive to the thermodynamic considerations,
plets. Further, concerning the solvent polarity, the dye present in and thus, their physical order and dynamics can be modified. An
the membrane senses the polarity at the phospholipid/water important methodology to study the membrane dynamics and
interphase, but in the lipid droplet, the dye will report from the organization is the combination of fluorescent dyes with micro-
homogenous apolar environment of the core. This capability to scopy. In this study, environment-sensitive probes were designed
sense the two environments has also been demonstrated in other with the final goal of understanding the macromolecular
dyes, like push–pull fluorenes25 and pyrenes.27 CAPRYDAA fluctuations and conformation. LAURDAN has been widely
efficiently discriminates between the plasma membrane and described as a probe located at the membrane interphase and
the intracellular membranes. In Fig. S10 in the ESI,† we present due to the charge transfer upon excitation, the increase in the
the ‘‘classical’’ generalized polarization5 (GP = Ch1 ꢁ Ch2/Ch1 + dipole moment was used to test the environment. Weber has
Ch2) from the hyperspectral images using the two bands centered at originally described the fundamentals between the polarity and
536 nm (channel-1) and 614 nm (channel-2).
the dipolar relaxation. The polarity was modified due to the
Using the two FLIM channels, we can independently study apparent dielectric constant at the membrane interphase,
the polarity and the dipolar relaxation of CAPRYDAA (Fig. 9). while the dipolar relaxation was found to be related to the
Additionally, the ‘‘classical’’ FLIM image generated by the tau re-orientation of the solvent dipoles around the dye during the
phase histogram is shown in Fig. S11 of the ESI.† In the blue excited-state lifetime. LAURDAN is perhaps one of the most
channel, we can see a trajectory associated with different widely used membrane dyes in the literature, and up until now,
fractions of Lo and Ld membranes in the cell (Fig. 9A–C). The it is the probe with the best-understood photophysics behavior.
dipolar relaxation of water around the dye at the membrane An important limitation of the use of LAURDAN is the need for
interphase was assessed using the green FLIM channel two-photon excitation with microscopy in addition to the
(Fig. 9D–F). An increased dipolar relaxation was found at the restriction to the blue spectral emission. Even upon using
intracellular interior, while the plasma membrane presented one-photon excitation of 405 nm, it is important to consider
lower dipolar relaxation when compared with the intracellular that such red excitation selects a subpopulation of LAURDAN’s
membranes (Fig. 9F).
ground state, and thus the UV illumination may be detrimental
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Fig. 8 Hyperspectral study of CAPRYDAA fluorescence: (A) intensity image for CAPRYDAA fluorescence using two-photon excitation. (B) Pseudo-color
image generated by the spectral phasor analysis of the CAPRYDAA spectrum shift represented as a fraction of green cursor in the spectral phasor plot of
figure (C). The color scale is representative of the membrane order, where blue means ordered membrane and red means fluid membrane. (C) The
spectral phasor plot of CAPRYDAA fluorescence. The cloud of pixels was colored using the scale shown between the red and green cursors. The red and
green cursors are associated with the fluid to ordered fraction, respectively. (D) The histogram of CAPRYDAA spectral shift in different cellular
membranes, obtained by masking the subcellular regions (inset figures at the left). The fraction of green is related to the fraction of membrane order.
to live cells. Therefore, novel probes with more red-shifted excitation when exploiting the linear combination properties of the phasor
spectra and capabilities for either one- or two-photon excitation will plots since the dye can be used in combination with the red
prove to be very useful in overcoming some of these drawbacks.
CAPRYDAA was designed based on the LAURDAN logic and
fluorescent proteins to track particular subcellular structures.
CAPRYDAA was also sensitive to the dipolar relaxation; this
considering the availability of new spectroscopy and image property of LAURDAN is unique and is related to the delay in
analysis tools. The rationale for the design of CAPRYDAA was the emission due to the reorganization of the water molecules
related to the spectral-shifted absorption of anthracene with around the dye in the excited state. Water has a rotational time
respect to naphthalene, but keeping the electron donor and of the order of the probe lifetime, and for instance, any change
acceptor groups as a key point in the photophysics of the dye. in the number of water molecules or their dynamics can be
This new probe demonstrated spectral shifts similar to sensed as a dipolar relaxation. The physical state of the membrane
LAURDAN with increasing solvent polarity but was found to is responsible for the final number of water molecules, and
undergo red-shift, making it easier to excite by the single- thus, the dipolar relaxation can also be related to the membrane
photon methods. This point must be emphasized, considering dynamics and organization. Interestingly, CAPRYDAA can be
the limitation on the use of 405 nm for the LAURDAN excita- studied either by spectral or lifetime shifts, showing a tight
tion. Nowadays, some variants of LAURDAN, like C-LAURDAN correlation between both. The use of the phasor plots (for
or M-LAURDAN, claim better photostability than LAURDAN lifetime or spectra) enhances the bathochromic properties of
under UV excitation. Nevertheless, the major limitation of this CAPRYDAA in the synthetic lipid vesicles. In GUVs, it was
kind of excitation is the deleterious effect on cells and the possible to identify the liquid/liquid phase separation and the
preferential ground state selection due to the red-edge excitation. characteristic domain formation in a ternary lipid mixture,
CAPRYDAA was also excitable by two-photon excitation at 900 nm, which is the representative of lipid rafts. Finally, when CAPRYDAA
which facilitates the combination with other fluorophores showing was tested in vivo with live cells, it was possible to isolate the
blue or red excitation spectra. This characteristic can be very useful organization of the subcellular membrane by the spectral phasor
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Fig. 9 FLIM characterization of CAPRYDAA fluorescence in cells: (A, B and D) the FLIM-phasor analysis of CAPRYDAA using a blue filter. The blue filter is
used to characterize the polar part of the CAPRYDAA emission. Using the color scale shown in (A) we generated (C). Red and green cursor represent the
‘‘pure’’ fluid and ordered phases, respectively. In between it is possible to see the cloud of pixels with different fraction of the two components. (D–F) The
FLIM-phasor analysis of CAPRYDAA fluorescence using a red filter. The red filter is used to identify the occurrence of dipolar relaxation. The dipolar
relaxation is a reaction in the excited-state and for instance should produce a delay in the phase, which pull out the universal circle the pixels that
undergoes to dipolar relaxation, see (D). To monitor the change in the dipolar relaxation, we use a three cursor analysis as previously described by
Malacrida et al.23 And the dipolar relaxation axis, the DR-axis was colored using the color scale shown in (D). In the dipolar relaxation (F) red means higher
dipolar relaxation. It is important to notice that almost all the pixels are outside the universal circle.
analysis and image segmentation. This spectral shift is very useful LAURDAN is located right behind the carbonyl group at the
in addressing the membrane dynamics in the in vivo experiments, membrane interphase. On the other hand, it looks as if CAPRYDAA
as well as the dynamic range showed by the phasor plot can be was positioned a little deeper in the bilayer, which can also be
applied to different kinds of experiments. With the FLIM data, a explained by the smaller phase-shift observed in the green channel.
map of the polarity or the dipolar relaxation in the cell can be An important aspect of the probe to highlight is its lack of distribu-
generated, which is valuable to understand the supramolecular tion to the water molecules during the water/dodecane experiment,
heterogeneity of live cells. These two properties (polarity and which besides its low emission from the water, is fundamental for
dipolar relaxation) provide complementary information, in which studying the membranes avoiding an emission component from
the first one was related to the apparent dielectric constant at the the bulk phase. This characteristic is shared with LAURDAN and
membrane interphase and the second one to the relaxation of is an important advantage of the probe over other soluble
water molecules around the probe during its excited-state lifetime. LAURDAN versions, which always present a water component.
It is interesting to discuss the difference in the dipolar relaxation
Finally, CAPRYDAA shows excellent labeling in the in vivo
observed by LAURDAN and CAPRYDAA, namely, it seems that experiments, where a hundred percent of the cells were strongly
LAURDAN has a larger dipolar relaxation shift than CAPRYDAA. stained after 30 minutes of incubation, with the staining being
However, it is important to consider the difference of the laser homogeneous from cell to cell and inside the cell (data not
sources (one or two-photon), and another important point is the shown). This point is an important property of CAPRYDAA to
actual position of each dye in the membrane. It is known that highlight, considering there is no preferential affinity for different
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12 S. A. Sanchez, L. A. Bagatolli, E. Gratton and T. L. Hazlett,
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is one of the fundamental use.
To summarize, we introduced a new molecular probe for
membrane dynamics and supramolecular organization with an
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standard’’ in the field, i.e., LAURDAN.
15 C. C. De Vequi-Suplicy, C. R. Benatti and M. T. Lamy,
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Author contribution
JG and VCC synthetized the probe; GG, LM and SS designed the
experiments; CSA, GG, SS and LM performed the experiments;
CSA, SS, GG and LM analyzed the data; SS, GG and LM wrote
the manuscript; EG, SS, GG and LM revised and discussed the
manuscript.
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Conflicts of interest
The authors declare no competing interests.
Acknowledgements
We would like to dedicate this paper to the memory of Dr Vicente
Castro-Castillo who passed away during the revision stage of this
manuscript. Vicente was fundamental to the development of
CAPRYDAA and he was a wonderful scientist and person. He will
be greatly missed by all who knew him. The authors would like to
thanks to MilkaStakic for the cells preparation and Prof. David
Jameson for his valuable suggestion during the manuscript
preparation. This work was partially supported by grants Grants
NIH P41-GM103540 and NIH P50-GM076516. LM is supported for
the Universidad de la Repu´blica-Uruguay as a full time professor.
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