Olivier Mongin et al- Brilliant organic nanodots: novel nano-objects for bionanophotonics

8/3/2019 Olivier Mongin et al- Brilliant organic nanodots: novel nano-objects for bionanophotonics

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Brilliant organic nanodots: novel nano-objects for bionanophotonics

Olivier Mongina, Cdric Rouxel

a, Anne-Claire Robin

a, Anna Pla-Quintana

b, Tathavarathy Rama

Krishnab, Galle Recherc, Franois Tiahoc, Anne-Marie Caminadeb, Jean-Pierre Majoralb,

Mireille Blanchard-Desce*a

a Molecular Chemistry and Molecular Photonics (CNRS UMR 6510), Universit de Rennes 1,Campus de Beaulieu, Bt. 10A, F-35042 Rennes Cedex, France.

bLaboratoire de Chimie de Coordination, CNRS, 205 route de Narbonne,

F-31077 Toulouse Cedex 4, France.cEquipe SCANING, CNRS UMR 6026, Universit de Rennes 1, Campus de Beaulieu,

F-35042 Rennes Cedex, France.

ABSTRACT

Semiconductor quantum dots are recognized to provide a particularly effective approach to bright nano-objects for

bioimaging. However, these inorganic systems suffer from several drawbacks such as toxicity, dispersity, blinking and raise a number of questions with respect to environmental issues. With this in mind, we have developed an

innovative route towards purely organic nanodots showing exceptional one and two-photon brightness by confining a

large number of optimized fluorophores within nano-objects of defined and controlled structure. These novel "soft"

nano-objects offer major promises for bio and nanophotonics.

Keywords: biophotonics, biological imaging, nanosciences, fluorescence, two-photon absorption, dendrimers

1. INTRODUCTION

Recently semiconductor quantum dots (QDs) have been shown to provide a particularly effective approach to fluorescent

nano-objects for biological imaging. Indeed, these inorganic nanoparticles exhibit large one- and two-photon absorption

cross-sections, reasonable fluorescence quantum yields, broad excitation but narrow emission bands, and high

photostability. These properties make them of particular interest for in vitro and in vivo imaging, and they have found

applications in specific labeling of cells1-5 and tissues6,7. In addition, because their emission spectra can be tuned by

playing on their size and composition, they can be used for multicolor imaging.8-10 They have also found applications in

two-photon excited fluorescence (TPEF) imaging,11 which has gained widespread popularity in the biology community

due to the many advantages it provides for biological microscopic imaging, including intrinsic three-dimensional

resolution and increased penetration depth in tissues.12-14

However, these inorganic nanocrystals suffer from several drawbacks such as biological toxicity 15 (due in particular to

the presence of heavy metals such as cadmium), polydispersity16,17 and blinking.18 These nano-objects also raise a

number of questions with respect to environmental issues. With this in mind, we have developed an alternative route

towards all-organic nanodots showing exceptional one and two-photon brightness. Our approach is based on the

confinement of a large and discrete number of optimized fluorophores within nano-objects of defined and controlled

structure.19-21 Such organic nanodots (OND) can be obtained by grafting the fluorophores on the surface of a dendrimericplatform (Figure 1). This strategy is highly modular and allows adjunction of supplementary layers, decoration of the

periphery with hydrosolubilizing groups, and insertion of functional groups that could be utilized for bioconjugation

purposes. Moreover, it offers several potential advantages: photoluminescence (PL) characteristics can, in principle, be

tuned by playing on the nature of the fluorophore, while the dendrimer scaffold can be chosen so as to minimize toxicity

effects and control clearance ability. In particular, phosphorus-based dendrimers have been shown to have low toxicity

and are biodegradable.22,23 However, implementing such modular approach also requires taking into account interactions

*[email protected]; phone (+33) 223236277; fax (+33) 223236955


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between the various molecular building blocks including possible interactions between decorating fluorophores. The

confinement of fluorophores within the OND imposes close proximity between chromophores, which favors

interchromophoric interactions: (i) in the ground state with potential effects on the absorption characteristics and (ii) in

the excited state with impending marked effects on PL. Such interactions can significantly affect the optical responses

and photophysical properties of the OND which will then differ from the mere addition of the contributions of isolated

single fluorophores. For instance formation of dimers or aggregates between adjacent fluorophores could lead to

fluorescence quenching.24

Also interactions in the excited state can lead to changes of PL characteristics due to variousphenomena (energy transfer, excimer formation or exciton annihilation which is an important decay process in

chromophore decorated dendrimers at high excitation intensity25). Also it has been shown that interactions between

chromophores within dimers26 or aggregates27,28 can lead to significant change in two-photon absorption (TPA)

responses. Such effects are expected to be strongly dependent on both the nature of the chromophores and their

proximity and relative orientations.

2. METHODOLOGY

2.1 Optical measurements

All photophysical measurements have been performed with freshly-prepared solutions in air-equilibrated solvents atroom temperature (298 K). UV/Vis absorption spectra were recorded on a Jasco V-570 spectrophotometer. Fluorescence

measurements were performed on dilute solutions (ca. 106 M chromophore concentration, optical density < 0.1)

contained in standard 1 cm quartz cuvettes using an Edinburgh Instruments (FLS920) spectrometer in photon-counting

mode. Emission spectra were obtained, for each compound, under excitation at the maximum absorption wavelength.

Fluorescence quantum yields were measured according to literature procedures using fluorescein in 0.1 N NaOH as a

standard (quantum yield = 0.90).29,30

2.2 TPEF measurements

Two-photon absorption cross sections (2) were obtained from the two-photon excited fluorescence (TPEF) cross

sections (2) and the fluorescence emission quantum yield (). TPEF cross sections in toluene (10-4 M chromophore

concentration) were determined using a Ti-sapphire laser (Coherent Mira 900 pumped by a 5 W Verdi) delivering 150 fsexcitation pulses and operating between 700 and 990 nm, according to the experimental protocol established by Xu and

Webb.31 This experimental protocol allows avoiding contributions from excited-state absorption that are known to result

in largely overestimated TPA cross-sections. Fluorescein in 0.01 M NaOH, whose TPEF cross-sections are well-

known,31 served as the reference, taking into account the necessary corrections for the refractive index of the solvents.32

The quadratic dependence of the fluorescence intensity on the excitation intensity was verified for each data point,

indicating that the measurements were carried out in intensity regimes in which saturation or photodegradation do not

occur. More details about the experimental setup have been previously published.32

2.3 Labeling and two-photon in vivo imaging

Stage 53 (Nieuwkoop and Faber 1956)Xenopus laevis tadpoles (national breeding facility of xenopus animals in Rennes,

France) were anesthetized in MS222 (tricaine, Sigma, 0.5 mg/ml). OND (2L of a 750M solution, i.e. 1.5 pM) were

injected in the heart with a syringe and animals were allowed to recover in Marks Modified Ringer (MMR) as long as

necessary for the complete diffusion of the OND into blood vessels. They were then re-anesthetized and mounted in the

recording chamber between two coverslips.

Two-photon imaging experiments were performed on the PIXEL platform, University of Rennes 1. A 860 nm excitation

beam from a femtosecond laser (MAITAI Spectra Physics) was focused on the samples via a Leica objective HCPLAPO

20X (NA=0.7). The vessels were scanned in 3D. After collection, data were analyzed with the open source software

ImageJ (http://rsb.info.nih.gov/ij/). The stacks were either projected along the z-axis or exported to be reconstructed with

the freeware UCSF Chimera (http://www.cgl.ucsf.edu/chimera).


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3. RESULTS AND DISCUSSION

3.1 Multichromophoric lipophilic organic nanodots

We have investigated and compared the photophysical properties of a series of multichromophoric single-layer (Figure1) and double-layer (Figure 2) organic nanodots, built from the gathering of an exponentially increasing number of one-

and two-photon active quadrupolar fluorophores F on phosphorus dendrimeric platforms.19,21

OHC[P3N3] N N P

Me S

OHC N N P

Me S

BuBuN

Hex

HexN

OO

2 26

G2

OHC[P3N3] N N P

Me S

OHC N N P

Me S

OHC N N P

Me S

BuBuN

Hex

HexN

OO

2 2 26G3

OHC[P3N3] N N P

Me S

OHC N N P

Me S

OHC N N P

Me S

OHC N N P

Me S

BuBu

N

Hex

HexN

OO

2 2 2 2 6G4

BuBuN

Hex

HexN

OHO

OHC[P3N3] N N P

Me S

BuBuN

Hex

HexN

OO

2

6G1

F

N PN

PNP

N PNPN

P

G1

G2

F

Figure 1. Chemical structures and schematic representations of fluorophore F and single-layer organic nanodots (SL-OND) G1-G4.

NNBuBu

OOO

N

H

NP

Me

SNN

BuBu Hex

HexOO

DL

2

6

[P3N3]

N PN

PN

P

DL

Figure 2. Chemical structure and schematic representation of double-layer organic nanodot (DL-OND) DL.


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One-photon absorption

All multichromophoric OND are excellent one-photon absorbers, with a strong absorption band in the near UV visible-

blue region. The single-layer organic nanodots (SL-OND) G1-G4 show a nearly linear increase of the extinction

coefficient with the number of chromophores, leading to giant absorption coefficients, exceeding 7 000 000 for G4

(Table 1). As observed from Table 1 and illustrated in Figure 3a, the ONDs definitely show much higher absorptivitiesthan QD480, a commercially available core-shell CdSe/ZnS quantum dot (QD) emitting in the same range, providing

evidence that the nanodot route may lead to competitive nano-objects with larger brightness than QDs.

The absorption spectrum of double-layer organic nanodot (DL-OND) DL is broader and slightly blue shifted with

respect to that of isolated fluorophore F (Figure 3c) and SL-OND dendrimers (Table1). Comparison of the absorption

spectrum ofDL with that corresponding to that expected for the additive contributions of 6 inner layer and 12 outer layer

chromophores F (Figure 3c) also shows that DL exhibits a slightly lower effective maximum absorption coefficient per

fluorophore along with a slight broadening observed on the blue side and red tail of the absorption band most probably in

relation with inhomogeneous broadening.

Table. 1. Photophysical properties of CdSe/ZnS quantum dot QD480, fluorophore F, single-layer organic nanodots G1-G4and double-layer organic nanodot DL in toluene.

Number of

fluorophoresabs

max (nm) (M-1 cm-1) em

max (nm) a2

max

(GM)b

QD480 460 5 20 000 480 5 0.3-0.5

F 1 386 84 900 420 0.83 765

G1 12 385 1 004 000 423 0.75 8 880

G2 24 386 2 035 000 426 0.71 17 700

G3 48 386 3 785 000 441 0.62 29 800

G4 96 386 7 101 000 445 0.48 55 900

DL 6 + 12 381 1 414 000 420 0.43 8 500

aFluorescence quantum yield determined relative to fluorescein in 0.1 N NaOH.

b1 GM = 10-50 cm4.s.photon-1.

Fluorescence

Quite interestingly, in spite of the confinement of a large number of chromophores in a very reduced volume, OND

retain fluorescence and maintain reasonably high quantum yields (Table 1), even for the highest generation dendrimer

G4, leading to exceptional one-photon brightness (). OND decorated with fluorophore F are blue emitting, and

overall, their emission spectrum is only slightly modified by the generation and concomitant size increase (Figures 3b

and 3d), in striking contrast with quantum dots whose emission color is directly correlated with the size. The emission

color of OND depends only on the constituting chromophores. Only a slight change of the emission band shape is

observed with increasing generations (Figure 3b) or layers (Figure 3d). A loss of fine vibronic structure and a broadening

of the band are observed with increasing generation, revealing an increase in reorganization energy in SL-OND G1-G4

as compared to the isolated fluorophore.21

In the case of double-layer dendrimer DL, the emission can stem from both inner and outer layer chromophores,

corresponding to different environments felt by the emitting fluorophores. Nanodot DL exhibits an emission spectrum(Figure 3d) similar to that of G1, with a fine vibronic structure clearly visible, but with a slight blue shift of the

maximum and a more pronounced red tail than G1 (and even than G2) Such behavior is consistent with a larger

inhomogeneous broadening in the case ofDL as already noted from of its absorption spectrum. A neat decrease of the

emission quantum yield is also observed (Table 1) This might originate from more pronounced interchromophoric

interactions in the excited state inside and between layers, i.e. with a closer packing in DL than in SL-OND.


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(c)

Figure 3. Absorption (a, c) and emission (b, d) spectra of single-layer organic nanodots G1-G4 and double-layer organic nanodot DLin toluene. Absorption and emission spectra of fluorophore F and CdSe/ZnS quantum dot QD480 are also given for comparison.

Comparison (c) of experimental and theoretical (evaluated from additive contributions of 18 isolated constituting chromophores F)

absorption spectra ofDL in toluene.

Two-photon absorption

The TPA of the OND was studied by investigating their two-photon excited fluorescence (TPEF) in toluene. TPEF

measurements allow direct measurement of the two-photon brightness (or TPEF action cross-section) , the relevant

figure of merit for imaging applications. The TPA spectra of single-layer and double-layer OND in the near infra-red

range (700-1000 nm) are shown in Figure 4. The TPA cross-sections in the SL-OND series increase almost linearly with

the number of decorating fluorophores leading to giant TPA cross-sections, up to 56 000 GM for G4 and concomitantly

to strong two-photon brightness (). This additive behavior indicates that the response of each individual

chromophore located on the periphery is more or less unaffected (as observed in one-photon absorption), in spite of theconfinement which could lead to possible TPA loss, as was shown for individual chromophores in solvents promoting

aggregation27.

As a result, TPA cross-sections of OND G1-G4 are overwhelming those of most of the quantum dots, in particular of the

same emission color. As a comparison, blue (2.4 nm diameter), green (2.9 nm), yellow (3.9 nm) and red (4.8 nm)

emitting CdSe quantum dots exhibit TPA cross-sections at 800 nm of 780 GM, 1950 GM, 4980 GM and 10300 GM,

respectively.33 As a result G4 shows unprecedented TPA cross-sections for a spherical nanoparticle of about 4 nm size

and definitely superior to that of related QDs.


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Figure 4. Two-photon absorption spectra of fluorophore F,

single-layer organic nanodots G1-G4 and double-layer organic

nanodots DL.

Figure 5. Comparison of the TPA efficiency (normalized by the

number of fluorophores) of single-layer and double-layer

organic nanodots.

Double-layer OND DL with 18 chromophores exhibits TPA cross-sections almost similar to that of single-layer G1 with

only 12 chromophores (Figure 4 and Table 1). We observe a noticeable difference in the TPA spectra normalized by the

number of fluorophores ofDL and G1 or G2 in the 700-750 nm region, whereas they nicely overlap in the 750-850 nm

region (Figure 5). It appears that the higher degree of confinement in DL (in particular for the inner layer) as compared

to G1 and G2 and the difference in orientation and packing between chromophores induced by the double-layer

arrangement significantly affect the TPA response. However, it should be noticed that this effect is only visible on the

higher-energy band (below 750 nm) which corresponds to the strongly two-photon allowed transition. On the other hand

the lower-energy band which corresponds to an only slightly two-photon allowed transition (due to slight symmetry

breaking)34 remains almost unaffected.

3.2 Water-soluble monochromophoric organic nanodots

The highly modular design of organic nanodots allows decoration of the periphery with hydrosolubilizing groups. In that

way, we have designed a series of water-soluble monochromophoric nanodots with shielding dendrimeric layers and

peripheral cationic groups.20 To ensure its water-solubility, two-photon active lipophilic fluorophores (C and F) were

incorporated in a dendrimer shells whose periphery is covered by ammonium groups (Figures 6 and 7). An alternative

and simpler route consisting in grafting ammonium groups directly onto the chromophore (compound WS-C) that has

been frequently used as the easier and direct way to provide solubility27,35 was also implemented for comparison with the

OND approach (Figure 6).


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Bu Bu

O O[P3N3]

[P3N3] NCH

O N

Me

P

S HNN C

HON

Me

P

S

NEt2

HHN

Et2N

H

Cl ClWS-G12 2 5

Bu Bu

HO OH

Me3N NMe3

HO OH

Br Br

+ +

WS-C

+++++++

++

+

++

++

+ + + ++

+

++ + + + + +

++

+

++

++

+++++

+

+++

+

+

+

+ + + +

++ +

+

+

+

++++WS-G1

WS-G2

5

Bu Bu

O O[P3N3]

[P3N3] NCH

O N

Me

P

S

NCH

O N

Me

P

SN C

HON

Me

P

S

N CH

ON

Me

P

SHN

NEt2

HHN

Et2N

H

252

2Cl Cl 25

WS-G2

C

Bu B u

O O

[P3N3] NCH

O N

Me

P

S

NCH

O N

Me

P

S

NCH

O N

Me

P

S HN

NEt2

H

[P3N3]N C

HON

Me

P

S

N CH

ON

Me

P

S

N CH

ON

Me

P

SHN

Et2NH

WS-G32 2 2

5

22

25

Figure 6. Chemical structures and schematic representations of model chromophores C, WS-C and water soluble organic nanodots

(WS-OND) WS-G1 WS-G3.

BuBuNN

OHO O

OH

F'

++

+++++

++

+

++

++

+ + + ++

+

++

+ + + ++

++

+

++

++

+++++

+WS-G'2F'

BuBuNN

OO O

O[P3N3]O [P3N3] O

HC N N P

Me S

OHC N N P

Me SHN

NEt2

H

Cl

HCNNP

MeS

OHCNNP

MeSHN

Et2NH

ClWS-G'2

22

5

2 2 5

Figure 7. Chemical structures and schematic representations of model fluorophore F and water soluble organic nanodot WS-G2.

Photophysical properties

As clearly observed from Table 2 the adjunction of ammonium groups directly on the aliphatic chains of lipophilic

chromophore C (to give water-soluble chromophore WS-C) does not lead to retention of the PL efficiency in water

(Table 2). This fluorescence quantum yield decrease can be related to non-radiative processes mediated by watermolecules and/or to molecular aggregation phenomena. Indeed, the WS-C PL in water is restored upon addition of a

surfactant (sodium dodecyl sulfate). The water-soluble quadrupole WS-C also endures a marked decrease of its TPA

efficiency in water as compared to C in ethanol. As a result, although being water-soluble and based on a quadrupolar

chromophore that shows reasonable TPA efficiency in non-aqueous protic media, WS-C only shows very low two-

photon brightness (2) in water. This clearly evidences that the "simple" water solubilization strategy is detrimental to

both PL and TPA efficiency indicating that protection of the TPA chromophore from close proximity of water molecules

is required Indeed, the PL efficiencies of water-soluble nanodots WS-G1, WS-G2 and WS-G3 in water are clearly

much higherthan that of water-soluble chromophore WS-C, and in the case ofWS-G2 and WS-G3, comparable to that

ofC in ethanol. Interestingly, the differences in relative vibronic intensities in the emission spectra of dendrimers in

ethanol and water tend to disappear with increasing the generation number, and the emission spectrum of the highest

generation dendrimer WS-G3 shows similar vibronic intensities in ethanol and water indicating that the dendritic


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branches provide shielding layers that isolate the core chromophore from interactions with the external environment

(Figure 8). Moreover, the TPA cross-sections of the dendrimers are also restored at a significant level. These results

indicate that the dendritic branches efficiently isolate the chromophore from deleterious effects of water, preventing both

TPA reduction and PL quenching.

Table. 2. Photophysical properties of model compounds C, WS-C and water-soluble organic nanodots WS-G1 WS-G3.

Solvent absmax (nm)

max

(M-1.cm-1)em

max

(nm)

a 2b

(GM)c

C EtOH 379 78 000 435 0.79 155

WS-C water 349 354 0.22 8

WS-G1 water 377 72 700 442 0.53 104

WS-G2 water 381 62 400 444 0.71 119

WS-G3 water 383 63 100 443 0.66 127

a Fluorescence quantum yield determined relative to fluorescein in 0.1 N NaOH. b at 705 nm. c 1 GM = 10-50 cm4.s.photon-1.

Figure 8. Comparison of absorption and emission spectra of

water-soluble nanodot WS-G3 in water and ethanol.

Another water-soluble nanodot (WS-G2) was obtained by wrapping lipophilic fluorophore F with dendrimeric sheaths.

In the case ofF, the dendritic branches are not directly connected to the chromophore as for the organic nanodot derived

from C, but via spacers. The absorption spectrum ofWS-G2 in water is clearly broader than that of isolated fluorophore

F in acetonitrile, and concomitantly its absorption coefficient is lower than that of F (Figure 9 and Table 3). Such a

behavior, which was not observed for OND derived from C (Figure 8), reveals a larger inhomogeneous broadening, in

relation with the higher flexibility of the dendritic branches. The emission ofF being very sensitive to polarity,36 it can

be used as a probe to provide useful information on the local polarity at the core of the nanodot. 37 Indeed the emission

spectrum ofWS-G2 reveals that the local polarity at the WS-G2 core is close to that of acetonitrile (i.e. high polarity)most probably indicating that water molecules are not far (Figure 9). This is confirmed by the low PL of WS-G2 in

water (Table 3) which more probably originates from the proximity of water molecules responsible for non-radiative

decay routes. Hence, in that particular case, the core chromophore is not adequately isolated from the external

environment so that better (tighter) shielding is needed It should be stressed however that in contrast to WS-C, which

shows much lower PL and TPA in water than its lipophilic analogue C in ethanol (Table 2), the TPA response ofWS-G2 is overall maintained in water (Figure 11 and Table 3) and only slightly smaller than that of its lipophilic analogue

F' in acetonitrile , indicating that the main problem lies in the occurrence of competitive non-radiative decay channels.

Furthermore a broadening of the TPA band is observed in relation with inhomogeneous broadening, which eventuallyleads to an increase of the TPA cross-sections above 720 nm.


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Table. 3. Photophysical properties of model fluorophore F and water-soluble organic nanodot WS-G2.

Solvent abs (nm)max

(M-1.cm-1)em

(nm)

a 2 (max)(GM)b

F CH3CN 383 85 000 498 0.69 800

WS-G2 water 385 60 500 480 0.074 680

a

Fluorescence quantum yield determined relative to fluorescein in 0.1 N NaOH.b

1 GM = 10-50

cm4

.s.photon-1

.

Figure 9. Absorption and emission spectra of dendrimer

WS-G2 in water and fluorophore F in CH3CN.

Figure 10. Two-photon absorption spectra of dendrimer WS-G2

in water and comparison with model fluorophore F in CH3CN.

Two-photon in vivo imaging

Water-soluble organic nanodots can be used as contrast agents for in vivo 3D TPEF imaging on living animal.20 In Figure

11 are shown as examples 2D and 3D images of bloods vessels of the tail of the Xenopus tadpole. The vascular networkwas labeled after intracardiac injection of an aqueous solution ofWS-G2.

Figure 11. In vivo two-photon imaging of blood vessels of stage 53 Xenopus laevis tadpole, obtained after intracardiac injection of

1.5 pmol ofWS-G2 (excitation at 860 nm). Left: Projection of 93 m thick stack of a 94 x 115 m2 image. Right: 3D representation

of the same stack.


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4. CONCLUSION

We have developed an innovative route towards purely organic nanodots showing exceptional one and two-photon

brightness by confining a large number of optimized fluorophores within nano-objects of defined and controlled

structure. Organic nanodots thus represent a promising biocompatible and eco-friendly alternative to semiconductor

quantum dots as fluorescent labels for one- and two-photon fluorescence imaging applications including bioimaging as

evidenced from small animal in vivo imaging thanks to the use of these organic nanodots. In addition, the highly modular

design of organic nanodots allows adjunction of supplementary layers and decoration of the periphery with

hydrosolubilizing groups. This modularity is of high interest for the customization of nanodots designed for specific

applications, by changing the chromophore nature and thus tuning the fluorescence color, or by grafting biomolecules for

targeting or diagnostic applications. These novel "soft" nano-objects offer major promises for bio and nanophotonics.

ACKNOWLEDGMENTS

We acknowledge financial support from ANR (Projects Biodendridot and Moduloo). MBD thanks Rgion Bretagne

(ARED Project BIONAD) for the fellowship to CR and the Ministre de lEnseignement Suprieur et de la Recherche

(France) for the fellowship to ACR. JPM thanks the Ministre de la Recherche (France) for the post-doctoral grant to

TRK. Thanks are due to the Fundacin Ramn Areces for a grant to APQ. Access to multiphotonic microscopy facilities

(PIXEL platform, University of Rennes 1) is acknowledged. We also aknowledge financial support (equipment grants)

from Rennes Mtropole and CNRS.

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Olivier Mongin et al- Brilliant organic nanodots: novel nano-objects for bionanophotonics

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