Molecular Molecular NanoprobesNanoprobes for for MultiphotonicsMultiphotonics as as New New toolstools for for BioimagingBioimaging

2nd Annual Symposium on IntegratingNanotechnology with Cell Biology and Neuroscience

The University of New Mexico, August 22nd 2008 UNM ICNCBN-IGERT

@ 488 nm @ 1032 nm

• intrinsic 3-D resolution

Fluorescence excited by :

1 photon 2 photons

Two-Photon Excited Fluorescence(TPEF or 2PEF)

⇒ Advantages in biological imaging :∝ I2∝ I

• increased penetration in tissues

• reduced photodamage• reduced background fluorescence

MultiphotonicsMultiphotonics: Two: Two-- photon absorption (TPA) photon absorption (TPA) ⇒⇒ TwoTwo--photon excitedphoton excited--fluorescence (TPEF)fluorescence (TPEF)

2PEF2PEFTwo-photon excited fluorescence

∝ σ2.Φ I2

pulsed lasers + focusing

Φ : fluorescence quantum yield

σ2 : two-photon absorption cross section(in GM = 10-50 cm4 .s. photon-1)σ2Φ TPEF cross -section (in GM)

⇒ molecular engineering of fluorophoreswith high σ2 in the biological spectral window (700-1200nm)

I

I

Fluorescent Markers and Probes for 2PEF imagingFluorescent Markers and Probes for 2PEF imaging

- Endogenous biological chromophores:NADH, riboflavins, retinol : 10-5 < σ2.Φ < 1 GM

- Classical one-photon fluorophores :DAPI : σ2.Φ < 1 GMCoumarin 307, Bodipy : σ2.Φ < 20 GMFluorescein : σ2.Φ < 40 GM

MOLECULAR ENGINEERING FOR MOLECULAR ENGINEERING FOR MULTIPHOTONIC BIOIMAGINGMULTIPHOTONIC BIOIMAGING :

examples and applicationsWhat is needed ?

• high fluorescence quantum yield• very large TPA cross-sections in the target spectral range• without residual one-photon absorption

⇒ 3D imaging

⇒ enhanced sensitivity

⇒ reduced photodamage

⇒ selective photo-addressing

IncreaselengthD/A strength type of spacer

Molecular engineering of quadrupoles

π co n jug a te dsys te m

D π co n jug a te dsys te m

Dθ

Adjust θ

Chain

SolubilityAffinity

• TPA enhancement• spectral tuning• (photo)stability

J. Chem. Phys. 2000, 113, 3951

R2N NR2

R2N NR2

X = NR2,: X = OMe

2.4 nm

2.3 nm

NonNon XX

NonNon SO2OctOctO2S

NonNon SO2CF3F3CO2S

NonNon NH2H2N

2.4 nm

2.4 nm

2.3 nm

2.3 nm

Non Non

Non Non NR2NonNonR2N

Non Non

SSR2N NR2

Non NonX X

R2N NR2

R2N NR2

X = NR2, : X = SO2R

3.8 nm

3.6 nm

4.5 nm

3.7 nm

3.7 nm

Non Non

NR2

3.6 nm

R2N

RodRod--likelike and bananeand banane--shapedshaped quadrupolarquadrupolar fluorophoresfluorophores

Chem. Eur. J. 2007, 13, 1481

ND filter wheel

photodiode

molecules(solution)

dichroic

NIR blockingfilter

spectro CCD

695695--980 nm, 150 980 nm, 150 fsfs, 76MHz, 76MHz λexcitation=750 nm

Photochem. Photobiol. Sci. 2005, 4, 531..

Measurement of two-photon absorption cross sections by TPEF

major TPA amplification in the NIR fluorescence is maintained

Molecular optimization of quadrupolar fluorophores

Fluorophore λabsmax

(nm)

Φ τ

(ns)

σ2*

(GM)

2

RHex2N

431 0.850.83 2115

2

R

Bu2N

R R

2

R

SOct2N

429

470

0.780.73

0.470.79

3470

5480

*at 705 nm (toluene)

Chem. Eur J, 2007, 13, 1481-1498.

APPLICATION: 3-D BIOIMAGING APPLICATION: 3-D BIOIMAGING

TPEF cross-sectional image of a GUV labeled with BAQ1.The giant unilamellar vesicles (GUV) were prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)

Angew. Chem., Int. Ed. 2001, 40, 2098

@ 750 nm

polarheadgroup

hydrophobicchain

lipids

bolaamphiphilic fluorophore

hydrophobic part hydrophilicpart

N NOHHO

N NOHHO

N NOHHO

HO OH

l = 30 Å

l = 35 Å

N+ N+

SO3--O3S

N+ N+

From model lipidic membranes…..

APPLICATION: 3-D BIOIMAGING APPLICATION: 3-D BIOIMAGING

C.R. Physique, 2002, 3, 439.

TPEF image of LLC-PK1 cells* labeled with with BAQ1, (excitation at 740 nm with less than 1mW excitation power)

To non-damaging cell imaging

FROM IMAGING TO SENSINGTowards Medium Responsive Two-Photon

Nanoprobes

⇒⇒sensitive twosensitive two--photon pH probes in the NIRphoton pH probes in the NIR

⇒⇒⇒ sensitive twosensitive twosensitive two---photon photon photon micropolaritymicropolaritymicropolarity probesprobesprobes

⇒⇒⇒ fast voltage probesfast voltage probesfast voltage probes

Recent examples of TP probes

NNC

N

OO

OO

B. R. Cho and coworkers, JOC, 2004, 5749-5751

Metal-ion sensor(Ca2+, Ba2+, Mg2+, Na+, K+)

J. W. Perry and coworkers, JACS, 2004, 12, 9291-9306

N

N

OO

OO

CN

NC

Metal-ion sensor(Mg2+)

H+ sensors ⇒ 2-photon pH probes ?

CL4

Diapositive 13

CL4 TP probes for metal-ions have been recently synthesized. This fluorophore with crown ether is a sensor for Mg2+.This another one is based on the same design and can sense different ions :…..At last, this quadrupole is sensitive to the pH of solution.céline; 27/06/2005

SMART TWOSMART TWO--PHOTON FLUOROPHORES AS PHOTON FLUOROPHORES AS SENSITIVESENSITIVE pH PROBES IN THE NIRpH PROBES IN THE NIR

J. Am. Chem. Soc. 2004, 126, 16294.

AA

high TPA

low TPAR R

NH3H3N

+ H+ - H+

R R

NH2H2N

D' D'

+ +

high TPA

low TPAR R

OHHO

D D

+ H+ - H+

R R

OO- -

D" D"

D D

D" D"

AA

D' D'

⇒ Two-photon pH sensing at membrane interface:two-photon microspectroscopy

OHHO

2 photon excitation @780 nm

2PEF emission when the membrane is stainedunder neutral and basic conditions

Towards Medium Responsive Two-Photon Nanoprobes

Towards sensitive twoTowards sensitive twoTowards sensitive two---photon pH probes in the NIRphoton pH probes in the NIRphoton pH probes in the NIR

Towards sensitive twoTowards sensitive twoTowards sensitive two---photon photon photon micropolaritymicropolaritymicropolarity probesprobesprobes

Towards fast voltage sensitive probeTowards fast voltage sensitive probe

Incoherent processes

TPEF

Coherent processes

Second HarmonicGeneration : SHG

Visualizationlocalization

Local orderAssymetry

TPEF SHG

σ2 σSHG

Molecular engineering of NLO molecular probes

MultiphotonicsMultiphotonics : Second : Second harmonicharmonicgenerationgeneration (SHG)(SHG)

SHG requires asymmetric sourceD Aπ

Amphiphilic Push-pull Chromophores

lipophilic tails

π−conjugated system AD

hydrophilic headgroups

objectives : from static imaging of cells to dynamic imaging of membrane processes

n↑ ⇒ σ SHG↑ (Chem. Comm., 2000, 353 )

σ2 ↑ in the NIR (Optics Lett., 2000, 25, 3220)

molecularmolecular engineering of engineering of chromophores for combined chromophores for combined TPEF and SHG imaging of membrane dynamicsTPEF and SHG imaging of membrane dynamics

AmphiphilicAmphiphilic PolyenicPolyenic PushPush--pull Chromophores (APPC)pull Chromophores (APPC)

lipophilic tails

N+

R2NR'

π−conjugated system AD

n

hydrophilic headgroups

LIPIDIC BILAYER

APPC(n, R, R')

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

.u.)

0.0

0.5

1.0

430 435 440 445 450S

HG

Pow

er (a

.u.)

0.0

0.5

1.0

fluorescence

SHG

SHG 2PEF

APPLICATIONS: NON-LINEAR IMAGING OF BIOLOGICAL CELLS

APPLICATIONS: NON-LINEAR IMAGING OF BIOLOGICAL CELLS

Simultaneous TPEF and SHG cross-sectional images of isolated Ncad1 cells labeled with an amphiphilicpush-pull polyenic chromophore. Internalized dye molecules become randomly oriented in the cytoplasm

and generate no SHG, whereas they continue to generate fluorescence

Biophys. J. 2001, 80, 1568.N+

Bu2N

SO3-

3

@ 880 nm

time (min)

0 50 100 150 200 250

norm

aliz

ed

0.1

1

2PEF 2HG (2HG)1/2 / 2PEF

time

Biophys.J. 2001, 80, 1568.

APPLICATIONS: NONAPPLICATIONS: NON--LINEAR IMAGINGLINEAR IMAGINGOF OF ““FLIPFLIP--FLOPFLOP”” DYNAMICSDYNAMICS

SHG

0.6 w0 d

destructive constructive none

SHG produced by two labeled GUV membranes.When they are separated by 0.6 times the excitation beam waist

(w0 ), SHG constructively interferes resulting in “hot spots”.

Measurement of Measurement of intermembraneintermembrane distancedistanceat at subwavelengthsubwavelength resolutionresolution

E

(Lewis, Loew, 1993)

SHG for membrane voltage sensing ?SHG for membrane voltage sensing ?

Membrane ~ 50-100 ÅPotential 100 mV

Membrane potential105 V/cm

Proc. SPIE-Int. Soc. Opt. Eng. 2002, 4812, 24-32.C. R. Acad. Sci. Paris, Série IV, 2001, 2, 1153–1160.

E

Voltage sensors?Voltage sensors?

Potentiel trans-membranaire [mV]-200 -150 -100 -50 0 50 100 150 200

SHG Intensité [a.u.]

0

10

20

30

40

50

60

70

80

+100mV

0mV

-100mV

NHex2N

SO3

SHG probes for imaging of neuronal activity

J. Neuroscience, 2004, 24, 999

Imaging ~400 µm deep into intact neurons ⇒ No photodamage

λ = 940 nm

NHex2N SO3-

Optically recording of action potentials⇒ linear dependence of SHG on E⇒ 0.833 ms temporal resolution⇒ 0.6 µm spatial resolution

Collab. W. Webb

quantum dots (QDs) ⇒ bright nanoobjects

⇒ tuneability, photostability

⇒ very large one (ε Φ) and two-photon

(σ2Φ) brilliance*

* W. W. Webb et al, Science, 2003, 300, 1434.

but toxicity, clearance, degradation ?…

Soft substitutes for semiconductor QD's ?

Soft All-Organic Alternative to QD's ?• biocompatibility / degradability• environmental friendly

quantum dots (QDs) ⇒ bright nanoobjects

⇒ tuneability, photostability

⇒ very large one (ε Φ) and two-photon

(σ2Φ) brilliance*

but toxicity, clearance, degradation ?…

Soft substitutes for semiconductor QD's ?

An "organic" alternative ? : modular route towards organic nanodots:

⇒ optimized fluorophore

NR2N

OHO

=

⇒ phosphorous-based dendrimersJP Majoral (Toulouse)

NanodotsNanodots : : ModularModular approachapproach

NPN

P NP

NPN

P NP

NPN

P NP

G1 (n = 12) G2 (n = 24) G3 (n = 48)

Controlled Grafting of Optimized Fluorophores on a branched (dendritic) platform

⇒ nano-object of controlled size, geometry, number of flurophores.

Chem. Commun. 2006, 915-917; New J. Chem., 2007, 31, 1354-1367.

Organic Organic nanodotsnanodots ::a valid strategy towards bright a valid strategy towards bright nanonano--objectsobjects

7 100 0003 800 0002 000 0001 000 000

85 000

ε (M-1 cm-1)

964824121

nF

0.5G4

0.6G3

0.7G2

0.75G1

0.81

Φ

⇒ huge extinction coefficients

⇒ fluorescence is maintained

Organic Organic nanodotsnanodots ::a valid strategy towards bright a valid strategy towards bright nanonano--objectsobjects

"super" bright nano-objects : record brilliance : ε . Φ

− ΦF > 50 % (QD 30-50 %)− ε → 7 000 000 M-1 cm-1

PCT Int. Appl. 2007, WO 2007080176

⇒ Fluorescent nanodots with giant TPA cross-sections…

New. J. Chem, 2007, 31, 1354

"super" bright nano-objects : record two-photon brilliance : σ2 Φσ2 → 60 000 GM (QD 700-10 000 GM)*

* Lin, Chou Small, 2006, 2, 1308.

PCT Int. Appl. 2007,WO 2007080176

Lin, Chou Small, 2006, 2, 1308-1313

Nanodots Two-Photon Brilliance as compared to QD's

⇒biocompatible biphotonic contrast agents for in vivo imaging

Two-photon imaging of the vascular network in the dorsal part of the rat olfactory bulb. Vessels were labeled after injecting intravenously a small bolus of 500 µM G2 nanodots in water. The image was taken at ~200 µm depth. No obvious toxic effects were observed during the experiment

Excitation @ 710 nmDetection @ 440 nm

Angew. Chem. Int. Ed., 2006, 45, 4645.

Collab. S. Charpak,L. Moreaux(INSERM, Paris Descartes)

+

+

+++++

++

+

+

+

+

++ + + +

++ +

++ + + +

+

++

+

++

+++++

+

+

+

+

+

σ2 = 130 GMλem = 440 nm

APQ 145 (λex : 860 nm)

0

1

2

3

4

5

400 450 500 550 600 650

λem (nm)

ΔF/

F (A

. U.)

2-photon in vivo small animal imaging

Xenopus laevis (stage 53)Gaëlle Recher, François Tiaho (Rennes)

σ2 = 1000 GMλem = 530 nm

SPIE Proc. 2008, 7040, 704006-704017.

Nanodot 2-photon tracer, 0.1 pmoleExcitation : 860 nmEmission 530 nm

NandotsNandots: versatile : versatile nanonano--objectsobjects

- Fluorescence Tuning :

- Shape Modulation :

- Modular approach ⇒ Water solubility :

quantum dots : size

nanodots : fluorophore

+

+

+++++

++

+

+

+

+

++ + + +

++ +

++ + + +

+

++

+

++

+++++

+

+

+

+

+

NPN

P NP

N PN

PNP

++++

++

+

+

+

+

+

++

++

+

+

+

+

++

+ + +- Surface functionalization⇒ targeting

Molecular and Supramolecular Photonics(Rennes)

Laurent PorrèsMarina CharloCéline Le DroumaguetCédric RouxelAnne-Claire RobinJean-Marie Vabre

Dr Olivier MonginDr Claudine KatanDr. Martinus H. V. Werts

Plate-forme d’imagerie PIXEL (Rennes)

LCC (Toulouse)Jean-Pierre MajoralAnne-Marie CaminadeThatavarathy Rama KrishnaAnna Pla-Quintana

Neurophysiologie et NouvellesMicroscopies (Paris)Laurent MoreauxJerome MertzSerge Charpak

Cornell UniversityD. DombeckW.W. Webb

SCANING (Rennes)François Tiaho, Gaëlle Recher

ACKNOWLEDGMENTSACKNOWLEDGMENTS