Improved push-pull-push E-Bodipy fluorophores for two-photon cell-imaging

Article abstract of DOI:10.1039/b911587k

Quadrupolar Bodipy dyes exhibiting TPA activity and high brightness with an emission at 660 nm were synthesized and internalized in HeLa cells, whereupon FLIM experiments were conducted.

Full text of DOI:10.1039/b911587k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Improved push-pull-push E-Bodipy fluorophores for two-photon cell-imaging
Pascal Didier,a Gilles Ulrich,b Yves Me´lya and Raymond Ziessel*b
Received 12th June 2009, Accepted 9th July 2009
First published as an Advance Article on the web 23rd July 2009
DOI: 10.1039/b911587k
Quadrupolar Bodipy dyes exhibiting TPA activity and high
brightness with an emission at 660 nm were synthesized and
internalized in HeLa cells, whereupon FLIM experiments
were conducted.
Optical imaging is one of the most attractive techniques commonly
used in biomedical research.1 Recent progress has raised new
demands for dyes with improved properties including good
solubility, biocompatibility, chemo- and photo-stability, one and
two photon excitation, high brightness when internalized into
cells, and minimal cytotoxicity.2 Internalization in cells can be
realized by using a vector that facilitates cell entry and may
promote selective localization in a given organelle.3 Alternatively,
the chemical structure of the dye can be modified to favour cell
penetration.4 We recently developed a new class of diethynyl
dipyrromethene-boron dyes (E-Bodipy) that can be modified at
Scheme 1
living cell.12 As expected from the design of the dyes, the presence
of a strong electron donor (styrylanisole push moieties) and the
electron accepting abilities of the Bodipy (pull moieties) resulted
in a pronounced intramolecular charge transfer (ICT) observed
at 370 nm in both dyes (Fig. 1). This type of transition greatly
enhances the two photon absorption cross-section (TPACS).13
will, and present improved stability without detriment to the
optical properties.5 To extend the development of stable dyes
emitting in the 600 to 800 nm range, a window widely recognized as
being attractive for bioanalysis and in vivo imaging,6 we have now
developed new dyes emitting around 660 nm as well as being stable
(ethynyl substitution at the boron), cell permeable, and having a
pronounced internal quadrupole (push-pull-push), a prerequisite
for two photon absorption spectroscopy and imaging.7 We report
here the design, the one- and two-photon photophysical properties
and the two-photon imaging applications of these improved
E-Bodipy fluorophores.
The target dyes 1 and 2 were prepared in two steps according
to Scheme 1: (i) a Knoevenagel condensation of the 3,5-methyl
groups with 4-methoxybenzaldehyde in basic conditions8 to
provide the styryl derivatives; (ii) boron substitution using the
appropriate Grignard reagents to afford the novel dyes in good
yields.9 The use of short methoxyethyleneglycol was inspired
by the previous use of PEG (PEG-PE or PEG-DSPE) for drug
vectors10 or Bodipy4 cell internalization and phototherapy. Single
photon excitation around 647 nm resulted in strong emission in
the red (662 nm). The brightness, expressed as ffluoe was found to
lie at 90 750 for 1 and 67 900 for 2 (Table 1). The redox behaviour in
solution was consistent with reversible radical anion and radical
cation formation and successive irreversible styryl oxidation
(Table 2).11 Little redox quenching is expected in standard
biological conditions keeping in mind the modest oxidation
Fig. 1 Absorption (blue) and emission (green) spectra for compound 1
in CH2Cl2 at rt. TPA action cross section (GM) for 1 (red). The excitation
power was 1 mW at the sample level and was kept constant for all
wavelengths.
∑
potentials of dyes 1 and 2 in the excited state (E0 B*/B- ª 0.82 V
The absolute two-photon absorption (TPA) action cross section
(fd) was determined using interferometric autocorrelations and
eqn (1):15
vs SCE, E00 = 1.95 eV) and the weak reducing environment of a
aLaboratoire de Chimie Organique et Spectroscopie Avance´es (UMR7515-
T
⎡
⎢
⎢
⎣
⎤
⎥
⎥
⎦
´
CNRS), Ecole de Chimie, Polyme`res, Mate´riaux (ECPM), 25 rue Bec-
f
F(t )−2TF∞
querel, 67087 Strasbourg Cedex, France
∫
−T
bLaboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS,
Universite´ de Strasbourg, Faculte´ de Pharmacie, 74 route du Rhin, 67401
ILLKIRCH Cedex, France
(1)
fd =
8n
2
2ch
P0(t)
pl
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Table 1 Spectroscopic data in dichloromethane solution at 298 K
∑
E◦ B*/B- eVd
a
c
Compds
l abs (nm)
e (M-1cm-1)
l F (nm)
UF
e¥UF
tF (ns)
krb (108 s-1)
knrb (108 s-1)
DGES eV
1
2
643
646
121 000
97 000
663
662
0.75
0.70
90 750
67 900
5.6
6.7
1.34
1.04
0.45
0.45
1.95
1.95
+0.82
+0.87
a At a concentration of 5 ¥ 10-7 M using cresyl violet as a reference U = 0.51 in ethanol, l exc = 578 nm.14 All UF are corrected for changes in refractive
index. b Calculated using the following equations: kr = UF/tF, knr = (1 - UF)/tF, assuming that the emitting state is produced with unit quantum efficiency.
c Excited state energies were estimated ( 5%) by drawing a tangent on the high energy side of the emission. d Calculated excited state oxidation potential
∑
∑
vs SCE, E◦ (B*/B- ) = E◦ (B/B- ) + DGES. B accounts for Bodipy.
Table 2 Electrochemical data for the BODIPY dyesa
Cmpds
E◦¢(ox, soln) (V), DE (mV)
E◦¢(red, soln) (V), DE (mV)
HOMO–LUMO gap (eV)
1
2
+0.67 (60), + 1.09 (irrv.),* + 1.25 (irrv.)**
+0.69 (70), + 1.10 (irrv.),* + 1.25 (irrv.)**
-1.13 (60)
-1.97 (irrv.)
-1.08 (70)
-1.88 (irrv.)
1.80
1.77
a Potentials determined by cyclic voltammetry in deoxygenated dichloromethane solution, containing 0.1 M TBAPF6, [electrochemical window from
+1.6 to -2.2 V], at a solute concentration of ca. 1.5 mM and at rt. Potentials were standardized versus ferrocene (Fc) as internal reference and converted
to the SCE scale assuming that E1/2 (Fc/Fc+) = +0.38 V (DEp = 60 mV) vs SCE. Error in half-wave potentials is 10 mV. For irreversible processes the
peak potentials (Eap or Ecp) are quoted. All reversible redox steps result from one-electron processes. * Irreversible oxidation of one styryl unit and **
Irreversible oxidation of the second styryl unit.
where f is the quantum yield of the chromophore, d is the TPA
action cross section, f the repetition rate of the laser, T the total
acquisition time window, F• is the fluorescence signal for infinite
delay, h the detection efficiency of the experimental set-up, c the
concentration of the chromophore, n the refractive index of the
solvent, l the laser wavelength and P0(t) the average incident power
on the sample. The TPA action spectra are given in Fig. 2 for
dyes 1 and 2 in DMSO, as a function of the laser wavelength.
Tetramethylrhodamine (TMR) was used as a reference compound
(Fig. 2). The two novel dyes have high TPA action cross sections (72
and 60 GM respectively for 1 and 2) around 780 nm, approximately
twice the wavelength of the linear absorption of the S0–S2
transition band of the chromophores (Fig. 1). This two-photon
transition takes place from the ground state S0 to the lowest excited
state with similar geometry, being S2 in this centrosymmetric
system.16 Note that both dyes exhibit a two fold enhancement of
the TPA action cross section compared to TMR in water (Fig. 2).
fluorescent species within a femtoliter volume (defined by the
laser excitation), several physical parameters such as the diffusion
time, local concentration and the molecular brightness, which are
related to the hydrodynamic and photophysical properties, can be
monitored. The molecular brightness (i.e. the number of photons
emitted by a particle per second for a given excitation intensity) of
1 and 2 in DMSO (lexc = 780 nm) was compared to the molecular
brightness of TMR in water (lexc = 860 nm), as a function of the
laser power at the sample level (Fig. 3).
Fig. 3 Molecular brightness measured by FCS as a function of laser
power at the sample level for 1 (blue) and 2 (green) in DMSO (lexc = 780 nm)
and TMR (red) in water (lexc = 860 nm). The solid lines correspond to a
second order polynomial fit (y = aP2).
As expected from the TPA measurements, the molecular
brightness displayed a quadratic dependence with respect to the
applied laser power. The molecular brightness of 1 and 2 showed a
strong increase compared to that of TMR, which is auspicious for
use in two-photon excitation microscopy. Indeed, in non-linear
microscopy the molecular brightness of the fluorescent dye is
generally a limiting factor for cellular imaging.
Fig. 2 Two-photon absorption action cross section spectra of TMR
(water), compounds 1 and 2 (DMSO) determined by interferometric
autocorrelations.
To further characterize the non-linear optical properties of
the compounds, fluorescence correlation spectroscopy (FCS)
measurements were carried out to characterize the translational
dynamics of the dyes.17 By using the intensity fluctuations of the
To demonstrate the possible utility of the newly synthesized
chromophores for two-photon excitation microscopy, fluorescence
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lifetime imaging microscopy (FLIM) experiments were performed
on Hela cells incubated for 6 h in an aqueous solution with
0.2% of DMSO containing the dyes. The cells were then washed
several times with a buffer (PBS) solution and imaged under two-
photon excitation. The FLIM images obtained for 1 and 2 are
reported in Fig. 4a and b respectively.
The fluorescence quantum yield (fexp) was calculated from eqn
(2). Here, F denotes the integral of the corrected fluorescence
spectrum, A is the absorbance at the excitation wavelength, and
n is the refractive index of the medium. The reference systems
used were rhodamine 6G in methanol (fref = 0.78, lexc = 488 nm)
and cresyl violet in ethanol (fref = 0.50, lexc = 546 nm)14 in air-
equilibrated water and de-aerated solutions.
F 1−exp(−A ln10) n2
{
}
ref
Fexp = Fref
(2)
Fref 1−exp(−A ln10) n2
{
}
ref
Luminescence lifetimes were measured on a PTI QuantaMas-
ter spectrofluorimeter, using TimeMaster software with Time-
Correlated Single Photon Mode coupled to a stroboscopic system.
The excitation source was a thyratron-gated flash-lamp filled with
nitrogen gas. No filter was used for the excitation. An interference
filter centred at 550 nm selected the emission wavelengths. The
instrument response function was determined by using a light-
scattering solution (LUDOX).
Fig. 4 HeLa cells were incubated for 6 h in a aqueous solution with
0.2% of DMSO containing the dyes. FLIM images for 1 (a) and 2
(b) were obtained with a 2 mW excitation power at the sample level. The
corresponding lifetime colour scale is given at the bottom.
The two-photon absorption cross section (TPACS) measure-
ments were performed on a home-made system. Briefly, the set-
up is based on a modified widefield epi-fluorescence microscope
(IX70, Olympus, Japan) with an Olympus 40¥ 0.9NA air objective.
Two-photon excitation (TPE) was provided by a titanium:sapphire
laser (Tsunami Spectra-Physics). To perform absolute TPACS
measurements between 740 and 920 nm, a Michelson interferom-
eter was incorporated in the optical path between the laser and the
microscope. By applying a slowly varying voltage with a frequency
generator on one arm of the interferometer, an interference pattern
is generated in the focal plane of the microscope. Photon detection
was performed with an Avalanche Photodiode (SPCM-AQR-14-
FC, Perkin Elmer) connected to a time-correlated single photon
counting (TCSPC) module (SPC830, Becker & Hickl, Germany)
running in the FIFO mode. In the TPE regime, the solution of
fluorescent molecules acts as a non-linear medium that allows the
measurement of an interferometric autocorrelation from which it
is possible to deduce the absolute TPACS of the dyes.
FCS measurements were performed on a home-built two-
photon system set-up.20,21 Photons were detected with an
Avalanche Photodiode (APD SPCM-AQR-14-FC, Perkin Elmer)
and the normalized autocorrelation function was calculated on-
line with a hardware correlator (ALV5000, ALV GmbH). FCS
measurements were sequentially repeated, typically 20 ¥ 30 s with
an excitation power around 3 mW at the sample level. Each
FCS curve was then fitted independently with the standard 3D
diffusion model G(t)=(1/N)*(1 + t/tD)-1*(1 + t/(S2tD)-1/2, where
N is the average number of fluorescent species in the focal volume,
t the lag time, tD the average residence time in the focal volume
and S is the ratio between the equatorial and axial radii of the
focal volume. The experimental curves were analysed with an
Igor Pro (Wavemetrics) function written to automatically process
the data. The molecular brightness of the fluorescent species
diffusing through the excitation volume was obtained by dividing
the average fluorescence intensity <F> by N.
From these experiments, many interesting features were estab-
lished: (i) these novel dyes can be efficiently internalized within
the living cells; (ii) no change in the cell morphology is induced by
incubating the dyes for at least 3 h, suggesting the absence of signif-
icant cytotoxicity; (iii) the lifetime distribution over the cells is not
homogeneous and likely due to the aggregation of the dye, as con-
firmed by brightness analysis in FCS experiments within the cells.
The lifetime decrease in the nucleus could be due to a higher aggre-
gation rate in proximity to DNA and a probable electron transfer
to nucleic bases such as guanine.18 We have previously shown that
aggregation of Bodipy in DNA results in complete extinction of
the fluorescence in the major groove of the polynucleotide.19
Due to the relative ease of synthesis of these E-Bodipy dyes, their
relatively high emission quantum yield and brightness in the red
spectral region (70–75%), their improved TPA action cross section
compared to TMR and their lack of cytotoxicity, they represent
promising candidates for imaging application and diagnostic tools.
The good results obtained with these model compounds prompted
us to investigate the preparation of efficient water-soluble TPA
active Bodipy dyes, able to be bioconjugated. This research is
currently in progress.
Experimental
Materials and methods
Chromatographic purification was conducted using 40–63 mm
silica gel. Thin layer chromatography (TLC) was performed on
silica gel plates coated with fluorescent indicator.
Spectroscopic measurements
300 and 400 (1H), 75.47 (13C) MHz NMR spectra were recorded
at room temperature using perdeuterated solvents with residual
protiated solvent signals providing internal references. UV–is
spectra were recorded using a Shimadzu UV-3600 dual-beam
grating spectrophotometer with a 1 cm quartz cell. Fluorescence
spectra were recorded on a HORIBA Jobin-Yvon Fluoromax
4P spectrofluorimeter. All fluorescence spectra were corrected.
Time-correlated single-photon counting FLIM was performed
using a home-made multi-photon laser scanning microscope
sharing the same core as the system described for TPACS
measurements.22 For FLIM, a 60X 1.2NA water immersion
objective was used and the laser power was adjusted to give count
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rates with peaks up to as few as 106 photons s-1, so that the
pile-up effect can be neglected. Imaging was carried out with a
laser scanning system using two fast galvo mirrors (Model 6210,
Cambridge Technology), operating in the descanned fluorescence
collection mode. The fluorescence was directed to a fiber coupled
APD (SPCM-AQR-14-FC, Perkin Elmer), which was connected
to a time-correlated single photon counting (TCSPC) module
(SPC830, Becker & Hickl), operated in the reversed start-stop
mode. Typically, the samples were scanned continuously for
about 30 s to achieve appropriate photon statistics to analyse
the fluorescence decays. Data were analysed using a commercial
software package (SPCImage V2.9, Becker & Hickl, Germany),
which uses an iterative reconvolution method to recover the
lifetimes from the fluorescence decays.
1
7.9 Hz, n0d = 73.8 Hz); 13C { H} NMR (CDCl3, 75 MHz): 14.9,
55.3, 58.7, 59.4, 68.2, 71.5, 91.6, 114.5, 118.2, 118.9, 125.9 (q, J =
3.8 Hz), 128.8, 129.5, 129.9, 130.8, 131.1, 131.5, 134.3, 136.1,
139.6, 139.8, 152.3, 160.4. EI-MS (nature of the peak, relative
intensity): 816.2 ([M], 100); C48H48BF3N2O6 (Mr = 816.71) C,
70.59; H, 5.92; N, 3.43; Found C, 70.35; H, 5.78; N, 3.31.
Acknowledgements
We thank CNRS and UdS for partial financial support and Drs
C. Andraud and O. Maury (ENS Lyon) for fruitful discussions.
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1
Hz); 13C { H} NMR (CDCl3, 75 MHz): 15.1 (CH3), 55.4 (CH3),
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3642 | Org. Biomol. Chem., 2009, 7, 3639–3642
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©