Neutral push-pull chromophores for nonlinear optical imaging of cell membranest

Article abstract of DOI:10.1039/b915654b

A new class of push-pull molecules was recently identified, based on pyridine dicarboxamide as an electron acceptor group and bearing a fluorenethynyl π-conjugated bridge. The molecules present good second and third order nonlinear properties, with a stat

Full text of DOI:10.1039/b915654b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Neutral push-pull chromophores for nonlinear optical imaging of cell
membranes†
Cyril Barsu,a Rouba Cheaib,b,c Ste´phane Chambert,b,c Yves Queneau,b,c Olivier Maury,a Davy Cottet,d
Hartmut Wege,d Julien Douady,d Yann Bretonnie`re*a and Chantal Andraud*a
Received 31st July 2009, Accepted 29th September 2009
First published as an Advance Article on the web 30th October 2009
DOI: 10.1039/b915654b
A new class of push-pull molecules was recently identified, based on pyridine dicarboxamide as an
electron acceptor group and bearing a fluorenethynyl p-conjugated bridge. The molecules present good
second and third order nonlinear properties, with a static quadratic hyperpolarisability mb (at 1.907
mm) of 320 ¥ 10-48 esu (mb0 = 249 ¥ 10-48 esu) and a maximum two-photon absorption cross-section of
1146 GM. Starting from this generic structure, we designed a series of eight amphiphilic nonlinear
probes, varying the length of the lipophilic tail and the nature of the polar head, and tested their cell
membrane affinity by nonlinear optical imaging. A good membrane affinity was observed with probes
bearing short alkyl chains and carbohydrate moieties as the polar head, emphasizing the importance of
the lipophilic–hydrophilic balance, as well as the role of the polar head. This original approach based
on carbohydrate head groups is an important advancement in the design of membrane probes, since
these highly versatile functional groups confer adequate hydrophilicity and yet conserve overall
molecular neutrality.
Introduction
The description of the electrical activity of neuron networks in
the brain necessitates simultaneous measurement of the activity
in a great number of neurons in vivo,1 taking into account not
only the action potential with a large amplitude of 100 mV,
but also the small graduated potentials of lower amplitude
(5 mV).2 Only optical imaging methods, and especially fluo-
rescence, meet these requirements. A great deal of work has
been dedicated to the design of efficient fluorescent fast-response
voltage-sensitive optical probes showing good affinity for the
cell membrane.3 This engineering was based quasi-exclusively
on hemicyanine dyes featuring a charged pyridinium ring as an
electron acceptor group, such as the now commercially available
ANEPs (amino naphthyl ethynyl pyridinium), or the longer
dialkylaminophenylpolyenylpyridinium RH dyes (Chart 1). These
are amphiphilic dyes with hydrocarbon chains acting as membrane
anchors, and a hydrophilic group attached to the pyridinium
ring that aligns the probes perpendicular to the membrane–water
interface. Systematic variations of the alkyl chain lengths and of
the polar head enabled the identification of probes presenting
the requisite water solubility and membrane affinity in neuronal
Chart 1 Chemical structures of some voltage-sensitive fast response
probes.
aUniversite´ de Lyon, Laboratoire de Chimie de l’ENS Lyon, UMR
5182 CNRS-ENS Lyon, 46 alle´e d’Italie, 69364, Lyon, France. E-mail:
cells,4 and the most recent works have successfully extended the
Chantal.Andraud@ens-lyon.fr
bINSA Lyon, Laboratoire de Chimie Organique, Baˆtiment J. Verne, 20 av
class of available hemicyanine dyes to the near infrared.5 Styryl or
A. Einstein, 69621, Villeurbanne, France
naphthylstyryl dyes with short alkyl chains (such as di-4-ANEPPS)
have been shown to be quickly internalized by the cell, whereas
longer hydrocarbon tails tend to anchor the dyes into the outer
leaflet, preventing flip-flop (i.e. the inversion of the probes from one
leaflet to another resulting in the staining of both of the leaflets).
The hydrophilic groups introduced consisted principally of ionic
groups, such as sulfonium or polyammonium, giving overall
cCNRS, UMR 5246, ICBMS, Universite´ Lyon 1, INSA-Lyon, CPE-Lyon,
Baˆtiment CPE, 43 bd du 11 novembre 1918, 69622, Villeurbanne, France
dLaboratoire de Spectrome´trie Physique, UMR 5588 CNRS-Universite´
Joseph Fourier (Grenoble I), 140 avenue de la physique, 38402, Saint Martin
d’He`res, France
† Electronic supplementary information (ESI) available: All experimental
procedures and characterization. Spectroscopic data for all compounds
2–5, including Lippert–Mataga plot and fit. See DOI: 10.1039/b915654b
142 | Org. Biomol. Chem., 2010, 8, 142–150
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zwitterionic or polycationic molecules. The molecular mechanisms
resulting in voltage-dependent fluorescence properties have been
discussed and, in the case of these fast-response probes, certainly
arise from electrochromism—electric field-driven changes in the
intramolecular charge distribution.6
for direct time-resolved measurement of membrane potential in
living cells.14 These studies have shown the proof of concept, but
also pointed out the need for new chemical probes specifically
designed for that purpose.15 In a recent report, Anderson, Clays
and co-workers specifically designed a series of amphiphilic
porphyrin-based second order nonlinear chromophores for SHG
imaging of cell membranes, but still bearing a charged pyridinium
polar head.16 Although a very strong SHG signal was observed in
lipid vesicles for all compounds, the in vitro membrane localization
strongly depends on the nature of the polar head. Therefore,
despite the push in establishing pyridinium dyes as effective
SHG sensors of membrane potential, there is room for improved
molecules presenting largely different structures.
The same molecules have large second order nonlinear
coefficients,7 and have set the basis of the recent development of
new membrane potential imaging techniques based on nonlinear
optics: two-photon excited fluorescence microscopy (TPM) com-
bined with second-harmonic imaging microscopy (SHIM). Both
microscopy techniques originate from fundamentally different
nonlinear optical phenomena, two-photon excited fluorescence
(TPEF) and second harmonic generation (SHG), respectively.
TPEF involves the simultaneous absorption of two photons
(TPA) of lower frequency to trigger the fluorescence of the
molecule, whereas in SHG, two incident photons are scattered
into one coherent output photon with exactly twice the incident
frequency. As the amplitude of both nonlinear phenomena are
proportional to the square of the incident light intensity, they
only occur at the focal point of a tightly focused laser, providing
an intrinsic confocality, which results in a high z resolution.
Furthermore, since the incident laser wavelengths are in the
near infrared (NIR, typically in the tissue transparency region
between 750–850 nm), lower tissue autofluorescence or self-
absorption, increased penetration depth, reduced out-of-plane
photobleaching and phototoxicity can be achieved.8,9 The SHG
signal requires a non-centrosymmetric local environment, and
only molecules asymmetrically distributed in the cell membrane
will contribute to the observed signal,9–11 without any background
signal originating from isotropic distribution in the surrounding
medium. On the other hand, TPEF is an incoherent process and
imposes no restriction on symmetry. The observation of TPEF
will only depend on the presence of an emissive molecule, giving
information about molecular distribution.
Results and discussion
We have recently identified a new class of push-pull molecules,
based on pyridine dicarboxamide as an electron acceptor group
and bearing a fluorenethynyl p-conjugated bridge (1, Chart 2),
that displayed good second and third order nonlinear properties,
with a static quadratic hyperpolarisability mb (at 1.907 mm) of
320 ¥ 10-48 esu (mb0 = 249 ¥ 10-48 esu) and a maximum TPA
cross-section of 1146 GM, and whose intense fluorescence (with a
quantum yield of 89% as reported in Table 1) is characterized
by a strong positive solvatochromism, a key requirement for
efficient voltage-dependent nonlinear probes.17 The absorption of
this reference chromophore 1 is centered around 410 nm (Table 1),
thus enabling efficient TPEF and resonance-enhanced SHG by
working at 800 nm, and showed only little change with the
solvent polarity. The present work focuses on the modification
of this basic structure to tailor the dyes for cell membrane
imaging, concentrating especially on the nature of the polar
head.
Fluorescent probes with charged groups were found to occupy
only a shallow location within the polar headgroup region of
the bilayer, no matter how lipophilic the molecule to which they
are linked.18 Therefore, in order to obtain a deeper membrane
penetration, we incorporated two carbohydrate entities (glucosyl
or lactosyl residues) into the probe, with the aim of providing
an appropriate hydrophilicity while keeping overall neutrality.
Carbohydrates are major constituents of cell membrane surfaces
Some membrane-bound dyes of the ANEP family were found to
engender a SHG signal highly sensitive to membrane potential,9,12
and others to present sub-millisecond response times to voltage
change, which was shown to arise from electron redistribution
or molecular tilt mechanisms.13 These assets make combined
TPM/SHIM a unique and specific tool for the optical imaging of
cellular membranes providing complementary information, and
Table 1 Absorption and emission properties of chromophores 2a–5a and 2b–5b in dichloromethane and in water. Maxima of linear absorption lmax and
of steady-state fluorescence emission lem, molar absorption coefficients e at lmax ( 20%), Stokes shift Dv˜, quantum yields of fluorescence UF ( 10%) and
solvatochromic slopes s derived from the fit of the Stokes shift vs. Df using the Lippert–Mataga model
a
CH2Cl2
Water
e(lmax)/103 M-1 cm-1 em/nmb
c
c
n
l
max/nm
e(lmax)/103 M-1 cm-1
l
em/nmb
Dv˜/cm-1
UF
l
max/nm
l
Dv˜/cm-1
UF
s/cm-1e
1d
411
389
396
391
390
390
394
388
387
37.0
12.2
50.0
24.8
18.4
35.7
50.0
32.9
21.8
525
543
530
529
542
537
531
525
529
5438
7290
6348
6808
7190
7019
6477
5884
6936
0.89
0.69
0.75
0.60
0.49
0.79
0.78
0.69
0.50
—
—
—
—
—
19385
19700
15552
24819f
20718f
20879
31497
24779f
30588f
2a
3a
4a
5a
2b
3b
4b
5b
385
402
390
397
393
397
395
388
10.1
30.0
11.9
10.1
24.7
27.9
27.5
18.8
570
575
598
584
561
552
574
561
8368
7824
8720
8129
7620
7458
2299
7783
0.05
0.09
0.07
0.08
0.16
0.20
0.17
0.14
a Except for 1 in CHCl3. b lexc = 410 nm. c lexc = 410 nm, reference Coumarin 153 in MeOH (UF = 0.45).26 d Ref. 17. e Excluding water and methanol.
f Excluding cyclohexane.
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Chart 2 Structure of new pyridine dicarboxamide nonlinear probes.
as glycoproteins or glycolipids. Carbohydrates mediate cell–cell
contacts through sugar–protein specific interactions, and as such,
are involved in many significant biological processes at the cell
surface such as cell adhesion, cell recognition and differentiation,
development of the neuronal network, inflammation, metasta-
sis, and viral or bacterial infection.19 By grafting carbohydrate
residues into the precedent nonlinear chromophores, we sought
to mimic the overall structure of natural glycolipids and give
access to neutral probes with good affinities for the cell membrane.
To the best of our knowledge, no specially designed non-ionic
carbohydrate-containing probes have been developed to date for
nonlinear membrane imaging. The only example is a ribose-
containing ANEP dye (JPW1259, Chart 1) in which the sugar
cycle has been introduced as a chiral group to help assure a
non-centrosymmetric environment.12 Carbohydrates are highly
versatile scaffolds offering huge structural variation and can
easily be introduced to primary amine groups through the use
of carboxymethylglycoside lactones (CMGLs).20 In this article,
we describe the synthesis and appraisal in nonlinear optical
cell imaging of eight new amphiphilic compounds 2a/2b–5a/5b
(Chart 2) bearing either neutral carbohydrate entities (two glucosyl
or lactosyl residues) or simple amino and polyethyleneglycol
(PEG) motifs for comparison. These different moieties enable
considerable variation in hydrophilicity, whereas the length of the
alkyl chains on the pyridinecarboxamide groups and the presence
of two n-hexyl chains on the fluorenyl ring served to modulate the
lipophilic character.
Synthesis
As shown in Scheme 2, all the final compounds 3–5 were
obtained from the key diamines 2a–b, the synthesis of which
involved two successive Sonogashira cross-coupling reactions
(Scheme 1). Direct alkylation of 6 by the commercially available
N-(-2-bromoethyl)phthalimide yielded only sparse amounts of
dialkylated product. Thus, alkylation of 2-amino-7-iodo-9,9-di-
n-hexylfluorene (6)21 with 2-bromoethanol in DMF produced the
diol 7, which was further transformed to the diphthalimide prod-
uct 8 under Mitsonobu reaction conditions with phthalimide. For
the synthesis of the acceptor side of diamines 2, chelidamic acid
(9) was converted to 4-chloro-2,6-bis(dialkylcarbamoyl)pyridine
(10a–b) by treatment with thionyl chloride followed by reaction
of the acyl chloride in dichloromethane with excess diethylamine
(to give 10a), or two equivalents of di-n-octylamine and excess
triethylamine (to give 10b) . 10a–b were then converted to the
4-iodo analogues 11a–b as previously described,21 with careful
control of the reaction conditions to avoid cleavage of the amide
groups. The first Sonogashira coupling with trimethylsilylacety-
lene followed by classical trimethylsilyl deprotection (K2CO3
in methanol) afforded 4-ethynyl-2,6-bis(diakylcarbamoyl)pyridine
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Scheme 1 Synthesis of the diamino compounds (2a) and (2b). Reagents and conditions: (i) 1-bromoethanol, NaI, Na2CO3, DMF, 95 ◦C, 48 h, 66%;
(ii) N-phthalimide, PPh3, DEAD, THF, rt, 18 h, 95%; (iii) R = ethyl: SOCl2, DMF cat., reflux, 2 h, then Et2NH, CH2Cl2, reflux, 2 h, 93% or (iv) R =
n-C8H17: SOCl2, DMF cat., reflux, 2 h, then NH(n-C8H17)2, Et3N, CH2Cl2, reflux, 2 h, 90%; (v) 57% HI, H3PO3, 80 ◦C, 3.5 h, 72% (11a)/76% (11b);
(vi) trimethylsilylacetylene, CuI, PdCl2(PPh3)2, THF, Et3N, rt, 4 d; (vii) K2CO3, MeOH, rt, 2 h, for the two steps 86% (12a)/61% (12b); (viii) CuI,
PdCl2(PPh3)2, THF, Et3N, 50 ◦C, 48 h, 60% (13a)/65% (13b); (ix) NH2NH2·H2O, crotyl alcohol, THF, reflux, 18 h, 90% (2a)/95% (2b).
(12a–b) in moderate to good yields (86% and 61% for the two
steps for 12a and 12b, respectively). The second Sonogashira
coupling between 8 and 12a or 12b gave 13a and 13b in 65%
and 60% yield, respectively. Hydrazinolysis of phthalimides 13a–
b was performed in THF with the addition of 15 equivalents of
crotyl alcohol (2-buten-1-ol) to quench the diimide formed by the
oxidation of hydrazine.22 Under these conditions, compounds 2a–
b were obtained exclusively without reduction of the triple bond.23
Conversion to the PEG derivatives 3a–b was achieved by coupling
with the carboxylic acid 14 using DiC/HOBt. The sugar moieties
were introduced by the ring opening of lactones 1524 and 16,25
followed by removal of the acetyl groups using catalytic sodium
methoxide in methanol, to give probes 4a–b and 5a–b, respectively
(Scheme 2).
The molecules varied considerably in their water solubility. The
six PEG groups in molecules 3a–b for instance provided very high
solubility. Short chain 3a is soluble at 10-2 M and a clear solution
was obtained, whereas the n-octyl analogue at 10-3 M in water gave
a cloudy solution but no precipitate, suggesting the formation of
micelles. On the other hand, the solubility in water of the sugar
compounds 4a–b and 5a–b remains very low (a precipitate was
observed in water at 10-6 M) even for the short-chain lactose
compound 5a possessing four sugar residues.
Spectroscopic properties
Table 1 summarizes the data obtained for all compounds in
dichloromethane and water. Spectroscopic data in chloroform for
the reference molecule 1 are also included for comparison, but no
data were obtained in water due to the very low solubility in this
solvent.
The absorption spectra of all compounds displayed an intense
broad band in the range 350–450 nm with maxima at around
390 nm in dichloromethane. It is worth noting that the position
of the absorption band allows an enhancement of the SHG signal
by resonance at around 800 nm. The 20 nm blue shift of the linear
absorption of 2–5 with respect to that of 1 could be related to
the decreased donor strength due to the existence of hydrogen-
bonding between the aniline nitrogen and the primary amine
protons (compounds 2a–2b) or the amide hydrogen (3–5), as
suggested for push-pull chromophores where only two or three
carbon atoms separate the nitrogen donor atom from a hydrogen
donor functionality such as a hydroxyl or an amide group.27
The molar extinction coefficients, at the wavelength of maximum
absorption (emax) ranged from 12.2 ¥ 103 M-1 cm-1 for the diamine
2a to 50.0 ¥ 103 M-1 cm-1 for the PEG compounds 3a and 3b in
dichloromethane. A substantial decrease is observed in water for
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and the refractive index of the solvent, respectively. The Stokes
in cm-1) is correlated to Df according to eqn
Dn =n −n
shift (
A
E
(1), where K is a constant, c and h are the light speed and the
Planck constant, and a is the radius of the spherical cavity of the
solute.
2
Dm2
(1)
Dn =
Df
+ K
hc
a3
The solvatochromic slopes s derived from the Lippert–Mataga
model are reported in Table 1. Even if this model is limited to a
point dipole approximation, which is not the case here, a good
linear correlation with relationship (1) was found if hydrogen
bonding solvents (methanol and water) and, in the case of sugar-
based molecules, cyclohexane are excluded (Fig. 2, and S2–S10 in
the ESI†). This points out that the emission process occurs from
a dipolar excited st ate, and is indicative of a rather high variation
of the dipole moment between the excited and the ground state
(Dm). It is interesting to note that the emission wavelength is more
sensitive to solvent than the absorbance, which indicates that the
relaxed excited state may have a significantly different structure to
that of the ground state. The solvatochromism was not analyzed
further, as the shape and the dimension of the solute cavity a
can hardly be simply estimated for such chromophores. More
importantly, very close values have been found for all molecules 2–
5, which are similar to that of the reference compound 1. This led
us to think that the substitution does not significantly modify the
chromophore’s electrochromic properties, and by extension, the
nonlinear properties. The ETN scale29 emphasized that the points
for protic solvents are outside the linear regression zone (see
the ESI, Fig. S11–S12†). This was observed for all compounds
including the reference 1, showing the existence of a hydrogen bond
involving the electron acceptor group (pyridine dicarboxamide) of
the chromophores and the solvent.
Scheme 2 Synthesis of the target probes 3–5. Reagents and conditions:
(i) DiC, HOBt, CH2Cl2, rt, 2 d, 51% (3a)/58% (3b); (ii) CH2Cl2, rt, 12 h;
(iii) MeONa cat., MeOH, rt, 16 h, 45% (4a)/74% (4b), 30% (5a)/63% (5b)
overall yield for the 2 steps.
all systems with values decreasing from 50.0 ¥ 103 M-1 cm-1 in
CH2Cl2 to 30.0 and 27.9 ¥ 103 M-1 cm-1 in water for 3a and 3b,
respectively, for instance (Table 1).
All the studied compounds exhibited an intense emission with
a structureless band centered at 525–545 nm in dichloromethane
(Fig. 1). No residual emission was observed at 400 nm for each
compound, suggesting an unambiguous separation of TPEF and
SHG signals, which is a relevant condition for nonlinear imaging.
The quantum yields measured in dichloromethane were high
(UF = 0.50 for 5a and 5b to UF = 0.79 for 3b and 2b) and similar to
that measured for the reference 1 (UF = 0.89), independent of the
nature of the polar head. No differences were observed between
short chain and long chain amides. Only the presence of four sugar
groups (lactose compounds) resulted in a decrease of the quantum
yield. In water, however, long chain compounds displayed a much
higher quantum yield than the short chain analogs, suggesting a
different organization in solution for both cases.
Nonlinear optical imaging
Cellular staining was investigated using Chinese hamster ovarian
cancer cells (CHO) and F98 glial cells grown on borosilicate
microscope cover slips, which were clamped for microscopy imag-
ing into a custom-made circular Teflon/aluminium observation
chamber. A few microlitres of concentrated (1–10 mM) aqueous
(compounds 3a–b) or DMSO (compounds 2a–b, 4a–b and 5a–
b) dye solutions were injected into the culture medium-filled
chamber to yield final dye concentrations of ca. 10-7 M. At
higher concentrations, the low water solubility of most compounds
resulted in the formation of fluorescent aggregates after final
dilution in the aqueous culture media.
The influence of the functional changes on the chromophores’
optical properties was studied by recording the absorption and
fluorescence spectra in solvents of different polarity and proton
donating ability at room temperature. Fig. 1 shows the absorption
as well as the fluorescence spectra of the short chain compounds
2a–5a in the various solvents. Data for other probes are given
in detail in the ESI.† An increase in solvent polarity induced a
marked red shift of the emission band. The positive solvatochromic
properties were analyzed following the Lippert–Mataga model.28
The solvent polarity parameter is thereby defined by the factor
Cell staining was observed by TPEF imaging immediately
after dye injection, without washing. Experiments were run on
a modified upright microscope adapted for two-photon imaging
with an external scan head and pulsed laser illumination, with
direct immersion of the objective into the cell culture medium.
Irradiation was carried out at 810 nm in the TPA band17 with a
mode-locked Ti:sapphire laser, characterized by an output pulse
width of ~100 femtoseconds and a repetition rate of 78 MHz.
Surprisingly, the most lipophilic long chain compounds (2b–5b)
did not stain the cell membranes. No intracellular fluorescence
could be observed at any concentration, but a high background
signal arising from the surrounding liquids suggested that the dyes
e −1
n2 −1
Df =
−
, where e and n are the dielectric constant
2e +1 2n2 +1
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Fig. 1 Normalized absorption and emission spectra for compounds 2a (a), 3a (b), 4a (c) and 5a (d) in different solvents: (—) cyclohexane, ( ) THF,
(
) CH2Cl2, ( ) acetone, ( ) DMSO, ( ) methanol, ( ) water.
Fig. 3 TPEF images of CHO cells stained externally (0.1 mM) by 2a (left)
and 3a (right). The average excitation power was 10 mW at the sample and
the total acquisition time was 0.9 s. Scale bars are 20 mm.
Fig. 2 Variations of the Stokes shift (cm-1) Dv˜ with Df for compounds
4a–5a and reference compound 1.
Conversely, showing the role of the peripheral substituents and
of the balance between hydrophilicity and lipophilicity, a good
membrane staining was achieved with dyes 4a and 5a bearing
two and four pyranoside structures, respectively. It was observed
immediately after dye injection and remained as long as the
extracellular medium was not washed. The cytoplasm, where
no fluorescence was observed immediately after injection, was
progressively stained, as shown in Fig. 4. This indicates that the
dyes were able to cross the membrane bilayer and diffuse into
the cytoplasm. As for the PEG compound, the cell nucleus was
not stained. When stained cells were washed by fresh culture
medium after a few minutes of incubation, fluorescence from
the membrane vanished as the dyes progressively internalized.
Focusing the laser beam on a small area of the cell resulted in
stayed in the extracellular media, even after long incubation times
(overnight). For the short chain compounds 2a–5a, marked
differences were observed depending on the nature of the polar
head, as shown in Fig. 3 and 4. The diamino compound 2a
(protonated at physiological pH) and compound 3a bearing six
PEG groups quickly entered the cell and accumulated in the cell
cytoplasm where fluorescence was observed, as shown in Fig. 3.
The cell nucleus remained unstained. The exact localization of
the dyes, which did not evolve with time, has not been further
investigated.
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kinetics of internalization also resulted in cell death for the same
reasons.
Similar results were obtained for both compounds 4a and
5a, bearing a glucosyl and a lactosyl structure, respectively. On
the other hand, although quantification could not be obtained,
membrane staining with compounds 4a and 5a was long-lasting
and the signal was observed for a long time span by comparison
with di-4-ANEPPS. In the same imaging experiment, the latter
showed a stronger signal, almost immediately seen, but it vanished
quickly as the dye was internalized.
Fig. 4 TPEF images of CHO cells stained externally by 4a (10 mM)
showing the progressive internalization of the dye: immediately after dye
injection (left) and after 10 min (right). The average excitation power was
10 mW at the sample and the total acquisition time was 0.9 s. Scale bars
are 10 mm.
Conclusion
In order to develop efficient probes for cell membrane imaging
and to find adequate polar end groups, as well as the right
balance between hydrophilicity and lipophilicity, we synthesized
a series of novel push-pull probe molecules based on pyridine
dicarboxamide bearing lipophilic alkyl chains of varying length as
an electron acceptor group, a fluorenethynyl p-conjugated bridge
and a dialkylamino with different hydrophilic attachments as
a donor moiety. We studied their optical properties and their
efficiency for TPEF and SHG imaging using two different cell
lines, CHO and F98 glial cells, and showed that membrane
imaging depends strongly on the peripheral substitution pattern,
and that a small variation in the structure of the probe can induce
dramatic changes in the cell uptake or efficiency of the membrane
staining. The most hydrophilic probes diffuse with ease through
the medium, but show no affinity for the membranes, resulting in
fast cellular internalization. On the other hand, the molecules with
the longest lipophilic acyl tail groups are difficult to deliver to the
membranes, at least if delivery is based on molecular dissolution in
a biocompatible solvent. Different alternative methods, based on
liposomes, poloxamers and/or cyclodextrins as stabilizing agents
or delivery vehicles are currently under research.
Good membrane staining was observed on CHO and F98
glial cells for the dyes 4a and 5a bearing short alkyl chains and
containing two and four pyranoside structures, respectively, while
no significant difference was found between glucose- and lactose-
based groups. The approach based on carbohydrate head groups
is an important advancement in the design of membrane probes,
since these highly versatile functional groups confer adequate
hydrophilicity and yet conserve overall molecular neutrality.
Membrane specificity of the most promising dyes is currently being
tested in neuronal cells and brain slices in order to test the voltage
dependence in physiological conditions. With the huge structural
variety offered by carbohydrate scaffolds, more selective staining,
as well as nonlinear imaging of specific sugar receptors on the cell
membrane surface can be envisaged.
too strong absorption and rapid cell death. These encouraging
results prompted us to try to simultaneously record TPEF and
second harmonic generation signals. In SHG, being a coherent
process, the signal propagates roughly in the same direction as the
incident beam9,10 and was therefore collected in a transmitted light
configuration. With incident light at 810 nm in the TPA band,
the SHG was considerably enhanced by resonance, and the signal
was also spectrally separated from the fluorescence which allowed
for its selective detection by means of a 405 nm narrow band-
pass filter. The TPEF signal was collected in epi-configuration.
We also used commercially available di-4-ANEPPS as a reference
membrane-staining compound to assess instrument performance
and to adjust the recording configuration.
Fig. 5 shows the compared TPEF (left)–SHG (right) images
recorded with compound 5a on F98 glial cells. The TPEF signal
of the stained membrane was very strong and a single scan was
sufficient for recording a good quality image, while the SHG
signal observed at 405 nm, arising only from molecules in the
cell membrane, was obtained after averaging of 3 to 10 successive
scans, due to the weak intensity of the signal. It is worth noting
that similar results were obtained with CHO cells. Although the
SHG signal strength depends quadratically on incident intensity,
it could not be improved by increasing the laser power, due to
a simultaneous enhancement of the fluorophore TPA, resulting
in increased photo-induced damage in the cells, and limiting the
experiment to a laser power of 5–15 mW under the objective.
Focusing and scanning a reduced area of the cell to assess the
Experimental
Cell-cultures
Cells were cultured as adherent monolayers in 30 mL polystyrene
cultures dishes (Nunc) and on 28 mm circular borosilicate
glass microscope cover slips (Mentzel) in DMEM/high glucose
Dulbecco’s modified Eagle’s medium (HyQ), containing an added
10% v/v standard fetal bovine serum (Perbio), 1% v/v MEM/non-
essential amino acids (HyQ) (for CHO only) and 1% v/v of
Fig. 5 Simultaneous TPEF (left) and SHG (right) images of F98 cells
stained externally with 5a (2 mM) and excited at 810 nm. The SHG image
has been contrasted for better visualization. The average excitation power
was 15 mW at the sample, and the total acquisition time was 0.9 s for the
TPEF image and 3.6 s for the SHG image. Scale bars are 20 mm.
148 | Org. Biomol. Chem., 2010, 8, 142–150
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Penicillin and Streptomycin solution (DB Difco) in a humidified
incubator (Sanyo IncuSafe) at 37 ◦C and 5% CO2. Once the
monolayer reached 80–90% confluence (every 3–4 d), the cells were
resuspended as follows. The old growth medium was discarded
and the dishes were washed 3 times with PBS (PAA) to remove
from the cell monolayer all of the trypsin inhibitors contained
in the growth medium. After that, the dishes were incubated at
Images were acquired using the Biorad exploitation system and
displayed using ImageJ.30
Acknowledgements
This work was supported by funding by re´gion Rhoˆne-Alpes
through the CPER “Nouvelles approches physiques des sciences
du vivant”, from “Cluster de recherche Chimie de la re´gion Rhoˆne-
Alpes” for a PhD grant (R.C.), and from the RTRA “Fondation
nanosciences: aux limites de la nanoe´lectronique”.
◦
37 C for 3–5 min with a solution of trypsin/EDTA (Invitrogen
Gibco), which detaches the cells from the surface via proteolysis.
The resulting suspension was gently agitated with a pipette and
10% of it was transferred into duplicate wells of fresh, warmed
media.
Notes and references
1 R. Cossart, D. Aronov and R. Yuste, Nature, 2003, 423, 283–288.
2 (a) J. H. Goldberg, R. Yuste and G. Tamas, J. Physiol., 2003, 551,
67–78; (b) S. Charpak, J. Mertz, E. Beaurepaire, L. Moreaux and K.
Delaney, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 1230–1234.
3 (a) A. S. Waggoner, Annu. Rev. Biophys. Bioeng., 1979, 8, 47–68;
(b) L. M. Loew, Pure Appl. Chem., 1996, 68, 1405–1409; (c) M.
Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen, Y.-W. Lam,
S. Antic and D. Zecevic, Biol. Bull., 2000, 198, 1–21; (d) A. L. Obaid,
L. M. Loew, J. P. Wuskell and B. M. Salzberg, J. Neurosci. Methods,
2004, 134, 179–190.
4 Y. Tsau, P. Wenner, M. J. O’Donovan, L. B. Cohen, L. M. Loew and
J. P. Wuskell, J. Neurosci. Methods, 1996, 70, 121–129.
5 (a) P. Yan, A. Xie, M. Wei and L. M. Loew, J. Org. Chem., 2008,
73, 6587–6594; (b) J. P. Wuskell, D. Boudreau, M.-d. Wei, L. Jin, E.
Reimund, R. Chebolu, A. Bullen, K. D. Hoffacker, J. Kerimo, L. B.
Cohen, M. R. Zochowski and L. M. Loew, J. Neurosci. Methods, 2006,
151, 200–215.
Combined SHG-TPEF microscopy
Biphotonic laser scanning microscopy was performed with a
confocal set-up consisting of a Biorad MRC 1024 scanhead and
an Olympus BX50WI microscope. The fluorescence signal was
directly epicollected, as shown in Fig. 6, whereas collection of
SHG was performed on the other side of the sample because
the SHG signal is directional and more efficient in transmission.
An excitation beam at 810 nm (Tsunami femtosecond Ti:Sa laser
pumped by a 5 W Spectra-Physics Millenia V) was focused on the
sample using a 60¥ water immersion objective with a 0.9 numerical
aperture (LumPlanFl/IR Olympus). The back aperture of the
objective was slightly underfilled (~70%), giving rise to a voxel
size of 0.5 mm ¥ 0.5 mm in the horizontal plane and 3 mm in
the vertical dimension. The pulse length at the entrance of the
microscope is 100 fs but the light is dispersed, mainly by the
lenses of the objective. Therefore, we estimate a 500 fs length
at the focal plane. The beam was scanned in the x–y plane to
acquire 512 ¥ 512 pixels images in 0.9 s. To vary the observation
depth, z-scan was performed by vertical motion of a motorized
objective. The incident laser intensity was adjusted by a half-
wave plate and a polarizer placed before the microscope so that
the total average power delivered at the surface ranged from 1–
100 mW. Two channels could be simultaneously recorded using
two added external photomultiplier tubes and appropriate filters.
6 L. M. Loew, G. W. Bonneville and J. Surow, Biochemistry, 1978, 17,
4065–4071.
7 J. Y. Huang, A. Lewis and L. M. Loew, Biophys. J., 1988, 53, 665–670.
8 (a) V. E. Centonze and J. G White, Biophys. J., 1998, 75, 2015–2024;
(b) P. T. C. So, C. Y. Dong, B. R. Masters and K. M. Berland, Annu. Rev.
Biomed. Eng., 2000, 2, 399–429; (c) A. Hopt and E. Neher, Biophys. J.,
2001, 80, 2029–2036; (d) L. Sacconi, D. A. Dombeck and W. W. Webb,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 3124–3129; (e) G. McConnell,
J. Biomed. Opt., 2006, 11, 054020.
9 P. J. Campagnola, M. Wei, A. Lewis and L. M. Loew, Biophys. J., 1999,
77, 3341–3349.
10 L. Moreaux, O. Sandre and J. Mertz, J. Opt. Soc. Am. B, 2000, 17,
1685–1694.
11 L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce and J. Mertz,
Biophys. J., 2001, 80, 1568–1574.
12 O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell and L. M. Loew,
Biophys. J., 1993, 65, 672–679.
13 A. C. Millard, L. Jin, M.-d. Wei, J. P. Wuskell, A. Lewis and L. M.
Loew, Biophys. J., 2004, 86, 1169–1176.
14 L. Sacconi, J. Mapelli, D. Gandolfi, J. Lotti, R. P. O’Connor, E.
D’Angelo and F. S. Pavone, Opt. Express, 2008, 16, 14910–14921.
15 (a) D. A. Dombeck, L. Sacconi, M. Blanchard-Desce and W. W. Webb,
J. Neurophysiol., 2005, 94, 3628–3636; (b) T. Z. Teisseyre, A. C. Millard,
P. Yan, J. P. Wuskell and M.-d. Wei, J. Biomed. Opt., 2007, 12, 044001.
16 J. E. Reeve, H. A. Collins, K. De Mey, M. M. Kohl, K. J. Thorley, O.
Paulsen, K. Clays and H. L. Anderson, J. Am. Chem. Soc., 2009, 131,
2758–2759.
17 C. Barsu, R. Fortrie, K. Nowika, P. L. Baldeck, J.-C. Vial, A. Barsella,
A. Fort, M. Hissler, Y. Bretonnie`re, O. Maury and C. Andraud, Chem.
Commun., 2006, 4744–4746.
18 K. Kachel, E. Asuncion-Punzalan and E. London, Biochim. Biophys.
Acta, Biomembr., 1998, 1374, 63–76.
19 For reviews concerning the function and biological role of cell surface
sugars see: (a) A. Varki, Glycobiology, 1993, 3, 97–130; (b) R. A. Dwek,
Chem. Rev., 1996, 96, 683–720. For reviews dealing with cell surface
oligosaccharides and their implication in cell–cell interactions, and cell
adhesion in cancer metastasis and angiogenesis see: (c) K. J. Yarema
and C. R. Bertozzi, Curr. Opin. Chem. Biol., 1998, 2, 49–61; (d) E.
Gorelik, U. Galili and A. Raz, Cancer Metastasis Rev., 2001, 20, 245–
277; (e) N. Sharon and H. Lis, Glycobiology, 2004, 14, 53R–62R; (f) R.
Kannagi, M. Izawa, T. Koike, K. Miyazaki and N. Kimura, Cancer
Sci., 2004, 95, 377–384.
Fig. 6 Schematic representation of the microscopy set-up.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 142–150 | 149
©

View Online
20 S. Trombotto, M. Danel, J. Fitremann, A. Bouchu and Y. Queneau,
J. Org. Chem., 2003, 68, 6672–6678.
21 A. Picot, C. Feuvrie, C. Barsu, F. Malvolti, B. Le Guennic, H. Le Bozec,
C. Andraud, L. Toupet and O. Maury, Tetrahedron, 2008, 64, 399–411.
22 B. E. Maryanoff, M. N. Greco, H.-C. Zhang, P. Andrade-Gordon, J. A.
Kauffman, K. C. Nicolaou, A. Liu and P. H. Brungs, J. Am. Chem.
Soc., 1995, 117, 1225–1239.
23 Hydrazinolysis of phthalimides 13a–b using hydrazine in ethanol
without adding crotyl alcohol gave an approximately 3 : 1 mixture of
the desired alkynes 2a–b and of the products arising from the partial
reduction of the triple bond, which could not be separated by column
chromatography.
25 R. Cheaib, A. Listkowski, S. Chambert, A. Doutheau and Y. Queneau,
Tetrahedron: Asymmetry, 2008, 19, 1919–1933.
26 N. Boens, W. Qin, N. Basaric´, J. Hofkens, M. Ameloot, J. Pouget,
J.-P. Lefe`vre, B. Valeur, E. Gratton, M. vandeVen, N. D. J. Silva, Y.
Engelborghs, K. Willaert, A. Sillen, A. J. W. G. Visser, A. van Hoek,
J. R. Lakowicz, H. Malak, I. Gryczynski, A. G. Szabo, D. T. Krajcarski,
N. Tamai and A. Miura, Anal. Chem., 2007, 79, 2137–2149.
27 I. Liakatas, C. Cai, M. Bo¨sch, M. Ja¨ger, C. Bosshard, P. Gu¨nter, C.
Zhang and L. R. Dalton, Appl. Phys. Lett., 2000, 76, 1368–1370.
28 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edn,
Academic/Plenum, New York, 1999, pp. 205–226.
29 C. Reichardt, Chem. Rev., 1994, 94, 2319–2358.
30 ImageJ, v.1.36 Public Domain Software, available at http://rsb.info.
nih.gov/ij/.
24 S. Trombotto, A. Bouchu, G. Descotes and Y. Queneau, Tetrahedron
Lett., 2000, 41, 8273–8277.
150 | Org. Biomol. Chem., 2010, 8, 142–150
This journal is
The Royal Society of Chemistry 2010
©