Two-photon multiplexing bio-imaging using a combination of Eu- and Tb-bioprobes

Article abstract of DOI:10.1039/c4dt03115f

During the course of our research studies aiming to combine the unique photophysical properties of lanthanide complexes and the intrinsic advantage of a nonlinear two-photon excitation, we described the proof-of-concept of a biphotonic multiplexing bio-imaging experiment. To that end, a new Tb-luminescent bioprobe [TbL²] was designed using the TACN-trispicolinate platform. The biphotonic imaging of T24 fixed cells stained either with [TbL²] or with the already described [EuL¹] complexes is reported. Finally the biphotonic multiplexing experiment was performed using a combination of both probes using the spectral mode of the two-photon microscope. This journal is

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Two-photon multiplexing bio-imaging using a combination of
Eu- and Tb-bioprobes.
Virginie Placide,a Anh Thy Bui,a Alexei Grichine,b,c Alain, Duperray,b,c Delphine Pitrat,a Chantal
Andraud,a* and Olivier Maurya*
During the course of our researches aiming to combine the unique photophysical properties of
lanthanide complexes and the intrinsic advantage of a nonlinear twoꢀphoton excitation, we
described the proofꢀofꢀconcept of biphotonic multiplexing bioꢀimaging experiment. To that
end, a new Tbꢀluminescent bioprobe [Tb
platform. The biphotonic imaging of T24 fixed cells stained either with [Tb
already described [EuL1
complexes is reported. Finally the biphotonic multiplexing
L
2] was designed using the TACNꢀtrispicolinate
2] or with the
L
]
experiment was achieved using the combination of both probes using the spectral mode of the
twoꢀphoton microscope.
Introduction
For about one decade, the tremendous development of biphotonic microscopy for bioꢀimaging applications triggered the
emergence of a new field of research aiming at combining the intrinsic advantages of twoꢀphoton excitation and the unique
photophysical properties of lanthanide ions.1 Twoꢀphoton (2P) excitation allows a 3D resolution, with an excitation in the nearꢀ
infrared spectral range and results in reduced photoꢀbleaching and photoꢀdamage and numerous organic dyes featuring optimized
twoꢀphoton brightness have been reported for this application.2 On the other hand, fꢀblock elements are well known for their sharp
emission bands characteristic of each element with long lifetime enabling timeꢀresolved and multiplexing imaging.3 The
combination of these properties in a new generation of biphotonic lanthanide luminescent bioꢀprobes (LLB) should strongly
enhance the potentialities of biphotonic microscopy. Following initial proofꢀofꢀconcept of sensitisation of lanthanides
luminescence by twoꢀphoton antenna effect,4 in 2008 were independently reported the first biphotonic bioꢀimaging experiments
using Eu(III) or Tb(III) probes.5 After these benchmark experiments many other Eu, Tb complexes were used as 2PꢀLLB,6 and
their scope was extended to Yb(III) luminescent compounds emitting in the near infrared spectral range7 enabling thick tissues 3D
bioꢀimaging8 towards the biphotonic detection of ultrabright single nanoꢀobjects.9 Very recently, we demonstrated the feasibility of
msꢀlifetime imaging including 2PꢀFRET experiment (TSLIM) and we extended the timeꢀresolved imaging (PSLIM) with a two
photon excitation using a conventional microscope.10 Herein we reported the proofꢀofꢀconcept of 2Pꢀmultiplexing experiment
using a combination of Tb(III) and Eu(III) 2PꢀLLB.
Multiplexing experiment consists in recording simultaneously the luminescence of several lanthanide bioꢀmarkers using the same
excitation and in reconstructing in silico the image related to each marker thanks to the spectral sharpness of the fꢀelement
transitions. This technique has been widely used for multicolour 1Pꢀbioimaging or for high throughput multiꢀlabels
fluoroimmunoassay11 but was never combined with a twoꢀphoton excitation. To that end, we designed an original 2Pꢀterbium(III)
bioprobe, [TbL2] based on the very stable trisꢀpicolinateꢀtriazacyclononane platform using the same approach successfully
employed for ytterbium(III)8 and more recently for europium(III) derivatives (including [EuL1], chart 1).12 The
πꢀconjugated
5
charge transfer (CT) antenna in [TbL2] was shorted compare to that of [EuL1] in order to sensitise the higher energy D4 terbium
emitting state. This article described the synthesis of [TbL2] and compared its photophysical properties with those of [EuL1]. The
biphotonic cells imaging was performed with either the terbium or europium markers alone or using the combination of both
probes.
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Chart 1. Structure of the 2Pꢀbioprobes, [EuL1] and [TbL2].
Results and discussion
A Synthesis
The preparation and characterisation of [Eu
achieved using the convergent pathway described in scheme 1.13 In a first step the iodinated trisꢀpicolinate TACN platform (
was prepared by alkylation of 1,3,7ꢀtriazacylononaneꢀtrishydrochloride ( ) with the mesylated compound in dry CH3CN. This
platform prepared on large scale is becoming a key synthon for the preparation of a large variety of functionalised ligands using
crossꢀcoupling reactions.14 In the present case,
was prepared with a 40% yield using the SuzukiꢀMiyaura coupling of anisole
boronic acid in DMF at 90°C with Pd(Ph3)4 as catalyst and Cs2CO3 as a base. The terbium complex was prepared using similar
procedure than that used for its europium analogous: (i) a saponification of the ester moieties of by sodium hydroxide
L 4 precursor of [TbL
1] has already been described.12 The synthesis of ligand 2] was
3
)
1
2
4
4
followed (ii) by the acidification of the reaction media with aqueous hydrochloric acid and (iii) a final reaction with a terbium
salt in the presence of carbonate. The complex was purified by several triturations and precipitations, isolated as a white
microcrystalline powder and characterized by HPLC and highꢀresolution mass spectrometry.
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Scheme 1. Synthesis of the [TbL2].
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B Spectroscopic properties
1,0
0,5
0,0
300
400
500
600
700
wavelength (nm)
Figure 1. Normalised absorption (dashed line) and emission (full line) of [EuL1] in red and [TbL2] in green at room temperature in
MeOH solution.
The photophysical properties of both compounds have been studied in diluted methanol solution, absorption and emission spectra
are reported in the figure 1. The [EuL1] complex is one of the brightest europium derivatives with a strong absorption at 338 nm (
ε
= 58000 L.Molꢀ1.cmꢀ1), a high quantum yield of 48% (
nm.12 The [TbL2] complex exhibited a less conjugated antenna composed of a simple biꢀaryl conjugated chain substituted by a
weak electroꢀdonating methoxy fragment. As a result the charge transfer absorption band (308 nm,
= 33000 L.Molꢀ1.cmꢀ1) is blue
shifted by 30 nm compared to the europium analogous. This antenna is suitable to sensitize efficiently the Tb(III) luminescence as
illustrated by the absence of any residual ligand centred emission. The quantum yield and lifetime remain rather modest ( = 12%,
= 0.24 ms) compared to related nonꢀsubstituted complex.15 Although back energy transfer cannot be completely excluded, these
τ = 0.95 ms) and an acceptable twoꢀphoton crossꢀsection of 49 GM at 700
ε
Φ
τ
low values can be explained by the free arylꢀaryl rotation, increasing nonꢀradiative relaxation pathways. This hypothesis was
corroborated by the significant exaltation of the lifetime to 0.63 ms in glycerol, a more viscous solvent where this rotation is more
difficult. The twoꢀphoton crossꢀsection of this derivative was not precisely measured since it lies mainly out of the Ti:sapphire
laser spectral range (700ꢀ1050 nm) and only the redꢀtail can be qualitatively observed. As illustrated by the figure 1, it is worth
noting that these two [EuL1] and [TbL2] compounds present very distinct transitions making them suitable for a multiplexing
5
5
experiment. In particular the D4→7F6 and D4→7F5 transitions at 490 and 543 nm respectively are perfectly isolated from the
signals of europium and are a clear signature of the presence of the terbium derivative. On the other hand, the hypersensitive
5D0→7F2 at 615 nm is representative for [EuL1] in spite of an overlap with the weak 5D4→7F3 transition of [TbL2].
C. Two-photon microscopy imaging.
These two complexes are not well soluble in water consequently the staining experiments were carried out by addition of a small
amount (10ꢀ100 µL) of a concentrated DMSO solution (10ꢀ3ꢀ10ꢀ4 M) to PBS (1 ml). In a first time, T24 cancer cells fixed with
paraformaldehyde, were stained either with [EuL1] or [TbL2] alone (figure 3). The cell images including in situ probes emission
spectra were registered under a biphotonic excitation (λex = 720 nm) as described in the experimental section. A single wide
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spectral detection channel was used in these cases and no spectral deconvolution was performed. It should thus be noted that the
small punctuate structures at the cell periphery may result from the contribution of autofluorescence. The two probes clearly
stained the endoplasmic reticulum and the nucleosol with a higher concentration of [TbL2] in the nucleoli. The emission spectra of
both LLB can be monitored in cellulo using the spectral detection mode of the microscope with a resolution of ca.10 nm. In spite
of this quite low resolution, the characteristic Eu(III) and Tb(III) signals appear very distinctly. For sake of comparison, cells
stained with [EuL1] were imaged with a single photon (1P) excitation at 405 nm and the emission spectrum was recorded (figure
2). Whereas cells images look very similar to that recorded with a twoꢀphoton excitation at, the emission spectrum exhibits a broad
signal centred at 470 nm in addition to the Eu(III) sharp bands. This wideꢀband signal, due to the contribution of cellular autoꢀ
fluorescence is reduced under 2P excitation and the 2Pꢀimages present consequently a higher signalꢀoverꢀnoise ratio.
Figure 2. Images of T24 cells after paraformaldehyde fixation and staining with [EuL1] (a) or [TbL2] (b). Images were taken in
transmitted light differential interference contrast (DIC, up) and twoꢀphoton laser scanning (λex = 720 nm, down). c) In cellulo
emission spectra of [EuL1] (red) and [TbL2] (green) under 2Pꢀ720 nm excitation and of [EuL1] under 1Pꢀ405nm excitation (blue).
The multiplexed imaging was then possible due to the distinct spectral signatures of the two lanthanide complexes. Their sharp
emission peaks and firm peakꢀtoꢀpeak intensity ratios allowed a quantitative discrimination of the relative contribution of each
complexe after spectral deconvolution, even if some minor peaks do overlap. It should be stressed here, that the spectral unmixing
is possible not only for the dyes of different “colours” but for any combination of lanthanide derivatives with the intermingled
emission peaks. In fact, the unmixing acts not only as a tuneable dichroic or bandꢀpass filter, but takes into account the whole
spectral shape and may be sensitive to small variations in any part of the spectrum. The limited spectral resolution of a confocal
microscope (ca. 10 nm) was however sufficient for the faithful discrimination of species and presented the advantage of increasing
the number of photons per channel. This feature is especially important in confocal microscopy where the measured volume in
each voxel is very small (0.2 fL) and the concentration of the species is variable and may be very low. In the present case, the cells
were stained with a mixture of [EuL1] and [TbL2] and the 2Pꢀimages were acquired using the spectral mode of the microscope.
The resulting emission spectra (figure 3) is composed of the Tb(III) and Eu(III) transitions and of a weak contribution of cellular
autoꢀfluorescence. The spectral deconvolution (or linear unmixing) procedure allows reconstruction of an image related to each
single component (figure 3) after elimination of parasitic autoꢀfluorescence. It is then possible to normalise and recombine these
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two images affected with a green (Tb) and a red (Eu) colours, respectively. In the reconstructed image, the green and red areas
indicate the major presence of [TbL2] and [EuL1], respectively whereas the yellow areas indicate a mixing of both dyes.
Figure 3. Twoꢀphoton multiplexing imaging experiments of fixed T24 cells stained with [EuL1] and [TbL2]. (top) multiplexed
emission spectra. (Middle) 2Pꢀimages obtained after linear unmixing procedure corresponding to Tb (green) and Eu (red) spectral
deconvolution. The contribution of the weak 2Pꢀautoꢀfluorescence has been previously subtracted. (bottom) reconstructed
multiplexed images by normalisation and addition of the two aforementioned images.
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Conclusion
In conclusion this article described the synthesis and characterisation of a new [TbL2] 2PꢀLLB based on the triazacyclononane
trispicolinate platform, belonging to the same family as the benchmark [EuL1] complex. These two derivatives were successfully
used as markers for the 2Pꢀscanning microscopy imaging of fixed cells. Finally the multiplexed imaging was possible using a coꢀ
staining with the two probes thanks to the distinct spectral signatures of the two lanthanide ions. This type of experiment is
classical in linear microscopy but was never achieved under twoꢀphoton excitation. Combined with the recently demonstrated long
luminescence lifetime imaging (TSLIM) and time gated (PSLIM) 2Pꢀmicroscopy,10 it is now possible to fully exploit the unique
luminescence properties of lanthanides in biphotonic microscopy and to achieve both spectral and temporal resolution. These
combined detections associated with specific functions for the targeting of different cellꢀcompartments would open the way for a
highꢀcontent multiꢀparameters multiplexed labelling.
Experimental
General. Starting materials were purchased from Sigma Aldrich®, Acros Organics® or Alfa Aesar® with the best available
quality grade and were used without further purification.
2
has been prepared using the procedure described in the literature.16
NMR spectra (1H, 13C) were recorded at room temperature on a BRUKER® Advance operating at 500.10 MHZ and 125.75 MHz
1
for H and 13C, respectively. Data are listed in parts per million (ppm) and are reported relative to residual solvent peaks being
used as internal standard (1H (CDCl3): 7.26 ppm, 13C (CDCl3): 77.2 ppm; H (CD3OD): 3.31 ppm, 13C (CD3OD): 49.0 ppm). UVꢀ
1
visible spectra were recorded on a Jasco® Vꢀ670 spectrophotometer in spectrophotometric grade methanol (ca. 10ꢀ5 or 10ꢀ6 molLꢀ
1). Molar extinction coefficients were determined at least two times. Emission spectra were measured using a HoribaꢀJobinꢀYvon
Fluorologꢀ3 fluorimeter. The steadyꢀstate luminescence was excited by unpolarized light from a 450 W xenon CW lamp and
detected at an angle of 90° for diluted solution measurements (10 mm quartz cuvette) by Hamamatsu R928 photomultiplier tube.
Spectra were corrected for both the excitation source light intensity variation and the emission spectral response. Phosphorescence
lifetimes were obtained by pulsed excitation using a FLꢀ1040 UP Xenon Lamp. Luminescence decay curves were fitted by leastꢀ
squares analysis using Origin®. Luminescence quantum yields
Q were measured in diluted water solution with an absorbance
lower than 0.1. Here, reference is quinine bisulfate in 1N aqueous sulfuric acid solution (Qr = 0.546). Excitation of reference and
sample compounds was performed at the same wavelength. Column chromatography was performed with Acros Organics®
(0.035ꢀ0.070 mm) silicagel. HPLC was performed on a Agilent Technologies 1260 Infinity apparatus equipped with a Waters
XBridge RPꢀC18 column, (3.5 µm, 4.6
× 100 mm). Experimental condition ammonium formate 0.25MꢀMeCN (v/v) as eluents:
isocratic MeCN (2 min), linear gradient from 15 to 100% MeCN (18 min), isocratic 100% MeCN (4 min) at a flow rate of 1
mL.minꢀ1, UV detection at 330 nm. High resolution mass spectrometry measurements were performed at Centre Commun de
Spectrometrie de Masse (Villeurbanne, France)
2. To a solution of methyl 6ꢀ(hydroxymethyl)ꢀ4ꢀiodopicolinate (1.56 g, 5.33x10ꢀ3 mol, 1.0 eq) in distilled THF (50 mL) was added
triethylamine (1.63 mg, 16x10ꢀ3 mol, 3.0 eq) and mesyl chloride (976 mg, 8.53x10ꢀ3 mol, 1.6eq). Reaction mixture was stirred
under nitrogen atmosphere at room temperature for 4h. Then the solvent was evaporated under reduce pressure, the crude residue
was dissolved in ethyl acetate and the solution was washed with a saturated aqueous NaHCO3 solution, then with water. The
organic layer was dried over Na2SO4 and the solvent removed at reduce pressure. The crude product was purified by flash
1
chromatography (CH2Cl2/ MeOH from 0 to 5%) to give the desired product as a white solid (1.40g, 71%). H NMR (500 MHz,
CDCl3) δ: 8.47 (d, 4J=1.5 Hz, 1H), 8.05 (d, 4J=1.5 Hz, 1H), 5.37 (s, 2H), 4.00 (s, 3H), 3.16 (s, 3H). 13C NMR (125 MHz, CDCl3)
+
δ: 164.1, 155.2, 148.0, 134.4, 134.3, 107.3, 70.0, 53.5, 38.3. HRMS (ESI ) : (Calc for C9H10NO5S : 370.93) ; [MH]+ : m/z=
393.9217
3. To a suspension of Na2CO3 (1.59 g, 15x10ꢀ3 mol, 10eq) in acetonitrile (60 mL) were added under argon atmosphere TACN.3HCl
(356 mg, 1.5x10ꢀ3 mol, 1.0 eq) and compound 2 (1.4 g, 4.79x10ꢀ3 mol, 3.2 eq). The reaction mixture was stirred at 60°C for 48
hours. The mixture was then cooled down and filtered. The filtrate was concentrated to dryness and the residue was purified by
precipitation from ethyl acetate to give the desired product as a white solid (814 mg, 57%). 1H NMR (200 MHz, CDCl3) δ: 8.35 (d,
J
=1.4 Hz, 3H), 8.21 (d,
147.6, 147.6, 135.6, 132.8, 106.6, 64.0, 56.1, 53.2. HRMS (ESI +) : (Calc for C30H33I3N6O6 : 954.9627); [MH]+ : m/z= 954.9668.
4. In a Schlenk tube were introduced compound
(150 mg, 1.57x10ꢀ4 mol, 1.0 eq) and 4ꢀmethoxyphenylboronic acid (79 mg,
J
=1.4 Hz, 3H), 3.97 (s, 9H), 3.93 (s, 6H), 2.90 (s, 12H). 13C NMR (75 MHz, CDCl3) δ: 164.6, 161.9,
3
5.18x10ꢀ4 mol, 3.3 eq) to a solution of anhydrous DMF (4 mL). The mixture was degassed under argon during 30 min. Then,
Cs2CO3 (215 mg, 6.59x10ꢀ4 mol, 4.2 eq) and Pd(PPh3)4 (10 mg, 9.42x10ꢀ6 mol, 0.06 eq) were added. The reaction mixture was
stirred under argon atmosphere during 48h at 100°C. The mixture was cooled down and Cs2CO3 filtered. The filtrate was
concentrated to dryness and the residue was dissolved in CH2Cl2. The solution was washed with a saturated aqueous NaCl
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solution, then with water. The organic layer was dried over Na2SO4 and the solvent removed under reduced pressure. The crude
product was purified by several washing in CH2Cl2/Et2O mixtures and precipitation with pentane and isolated as a pale yellow
solid (58 mg, 41%). 1H NMR (200 MHz, CDCl3) δ: 8.19 (s, 3H), 8.02 (s, 3H), 7.58 (d, 6H,
J= 8.8Hz), 6.94 (d, 6H, J= 8.9Hz), 4.02
(s, 6H), 3.99 (s, 9H), 3.85 (s, 9H), 3.02 (brs, 12H). 13C NMR (50.32 MHz, CDCl3) δ: 166.3, 161.8, 161.0, 149.4, 147.9, 129.6,
128.3, 123.3, 121.2, 114.8, 65.0, 56.5, 55.6, 29.9. HRMS (ESI +) : (Calc for C51H55N6O9: 895.4032 ) ; [MH]+ : m/z= 895.4025.
[TbL2]. To a solution of
4
(35mg, 3.91x10ꢀ5 mol, 1.0eq) in MeOH (10 mL) was added 10 mL of an aqueous solution of NaOH
(1M). The reaction mixture was stirred at room temperature for 1h. An aqueous solution of HCl (1M) was then added to the
reaction mixture until pH=2. K2CO3 (3.3 eq) and TbCl3.6H2O (44 mg, 1.17x10ꢀ4 mol 3.0 eq) were added to the solution and the
mixture was stirred at 50°C for 16h. The solution was cooled down and the solution evaporated to dryness. The crude product was
purified by several triturations and precipitations from water. The purity of the complex was established by HPLC analysis (RT =
9.17 min). HRMS (ESI +) : (Calc for C48H45N6Na2O9Tb: 527.1144); [M+2Na]2+ : m/z= 527.1143.
Cell culturing and treatment. We used T24 human epithelial bladder cancer cell line (ATCC No HBTꢀ4). In our experiments, T24 cells
were cultured in 25 cm2 tissueꢀculture flasks (T25) at 37°C, in a humidified atmosphere with 5% CO2. They were incubated in RPMI 1640
supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum (complete medium). Cells were grown to near
confluence in the culture flasks and then suspended with 0.05% trypsin–EDTA solution (Sigma).
Twentyꢀfour hours before experiments, cells were placed on LabTek I chambered cover glass (Nunc) at low cell density in complete culture
medium. After being washed with the phosphate buffer saline (PBS), cells were fixed using paraformaldehyde (PFA 3% in PBS) for 10 min,
permeabilized with PBS containing 0.5% Triton X100 for 10 min, and then washed again with PBS.
Microscopy. All confocal experiments were performed using a LSM710 NLO (Carl Zeiss) confocal laser scanning microscope based on the
inverted motorized stand (AxioObserver). The excitation was selected by the acoustoꢀoptical modulator and provided by either 405 nm DPSS
cw laser or Ti:Sa femtosecond tunable laser (Chameleon, Ultra II, Coherent) for 2P excitation in descanned detection mode. In the former
case the pinhole was closed to 1 Airy Unit and in the latter one it was fully open. Multiplexed imaging was realized using a spectral detection
mode of LSM710 in the range 422ꢀ655 nm with the resolution of 9.7 nm and a subsequent spectral unmixing with the reference spectra of
each compound.
Acknowledgement. Authors thank Lyon Science Transfer agency for financial support and for a grant to V.P.
Notes and references
a
Laboratoire de Chimie UMR UMR 5182 CNRS-University Lyon- ENS Lyon, 46 allée d'Italie 69364 Lyon, France.
olivier.maury@ens-lyon.fr
b. INSERM, IAB, F-38000, Grenoble, France
c Univ. Grenoble Alpes, IAB, F-38000, Grenoble, France.
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