applications des lasers impulsionnels en biologie...

Applications des lasers impulsionnels en biologie :

résolution spatiale etgénération de contraste

Pierre-François LENNEInstitut Fresnel - Marseille

The cell

5 nm

5 nm

2 nm

10 µm

Mitochodrion

Energy Production

RoughEndoplasmicReticulum

Protein production

DNA contains the informationfor the production of proteins

PlanI- Introduction - Rappels

• Résolution spatiale

• Microscopie de fluorescence excitée par l’absorption de 1 et 2 photons

II- Réduction du volume de détection et résolution sub-longueur d’onde : la microscopie STED

III- Le contraste vibrationnel : la microscopie Raman stimulée CARS

The Airy pattern radius fromthe central peak to the firstminimum is given by : NA

Rairy 222.1 λ

=

θsinnNA =

Object plane Image plane

θ

Tube lens

)(22.1

objectivecondensorairy NANA

R+

=λ

Conventional contrast in optical microscopy

Airy pattern

Condensor

RésolutionCritère de résolution :

NANArxyλλ 61.0222.1 ==

Fluorescence Microscopy

1P Fluorescence Microscopy

1P Confocal Microscopypinhole

2P Fluorescence Microscopy

1-photon 2-photon

I2P~(Iexc)2

molec s/molec = 10 GM

Ordre de grandeurs :Microscopie à 2 photons avec laser continu vs impulsionnel

•Laser continu focalisé dans un échantillon de fluorescéine de 10 µM produit 20000 photons/s. Efficacité de détection 5 % 103 photons/s détectés.

•Laser impulsionnel

)/()()( 22 τRtIgtIF p=∝

mWtI 1)( =

Facteur de formeDurée de l’impulsion

Taux de répétition

510)/( =τRgp

108 photons/s détectés.

fs 100 MHz, 80 R gaussien), (profil 66.0 === τpg

Resolution in Fluorescence Microscopy

Wide Field Confocal 1P 2P λ=0.5µmNA=1.2n=1.33

0.25µm / 0.16µm / 0.263µm

0.92µm / 0.65µm /1.07µm2)(

2NA

nrzλ= ( )24.1

NAnrz

λ=

NArxyλ4.0=NArxy

λ61.0=NArxyλ7.0=

( )23.2NA

nrzλ=

Wide Field 1P Confocal

Avantages de la M2P•Excitation localisée

photoblanchîment et photodestruction réduits

• Faible absorptionImagerie en profondeur (tissus)

II- Réduction du volume de détection et résolution sub-longueur d’onde : la microscopie STED

Excitation Stimulated emission depletion

STED

+ =

~ 100 nm

~ 100 nm

Confocal microscopy + STED

T.A. Klar, PNAS USA 97, 2000

Non linear Optics: Stimulated emission depletion

STimulated Emission Depletion - STED

Utilisation d’une non linéarité entre intensité d’excitation et émission de fluorescence

λ


Ti:Saph76 MHz

OPO + SHGEX0.2 ps560 nm

STED40 ps765 nmstrecher

EX STED

Fluo

λ


fluorescence relaxation

STED(40ps)

excitation pulse (0.2ps)

Cyclic process at a frequency of 40 MHz


E

vibSTEDSTED

FSTEDSTED

kNININdt

dN

kNININdt

dN

*0

*01

*0

11*

011

/

/

−−=

−+−=

σωσ

σωσ

h

hN1

ISTED

If σISTED<<kvib then N0*=0 and re-excitation is negligible

N0*

kvib)/(1 ωτσ hSTEDIExpN −∝

Strong non-linear dependence of N1 with ISTED Non linear Depletion


97 nm FWHM , Z depth and XY !!! = 670 zeptoliters = 0.67 attoliter


STEDConfoc

S. cerevisiae yeast cell

STEDConfoc

Latex beads 100 nm

Exogenous fluorophoreNeed to be introduced in the living sample.QuenchingArises from a variety of competing processes that induce non-radiative relaxation of excited state electrons to the ground state.Photobleaching Occurs when a fluorophore permanently loses the ability to fluoresce due to pchemical damage and covalent modification.

Quenching and Photobleaching

Typical example of photobleaching (fading) observed in a series of digital images captured at different time points for a multiply-stained culture of bovine pulmonary artery epithelial cells. - Nuclei stained with 4,6-diamidino-2-phenylindole (DAPI; blue fluorescence)- Mitochondria stained with MitoTracker Red (red fluorescence)- Actin cytoskeleton stained with phalloidin derivative (green fluorescence)

III- Contraste vibrationnelCoherent Anti-Stokes Raman Microscopy

CARS

A. Zumbusch, G. R. Holtom, and X. S. Xie, Three-Dimensional Imaging by Coherent Anti-Stokes Raman Scattering, Phys. Rev. Lett. 82, 4142, 1999

Spontaneous Raman Scattering

Anti-Stokes

Stokes

ωωP ωASωS

ΩR ΩR

PS=σR IPσR=10-31 – 10-29cm2/molecule

NB: σF-1P=10-16cm2/moleculeσF-2P=10-49cm4.s/molecule

Stimulated Raman Scattering

ωpωpωs

ωas

Ωr

If ωp-ωs=Ωr resonant effect:ωas is enhanced

FCARS

E-CARS

CARS versus Spontaneous Raman

wavelength

Laser ωp

Spontaneous RAMAN

Stokes ωs

AntiStokes ωas

Stokes ωs

AntiStokes ωas

ΩR ΩR

From the theory…

From the theory…2

)()( ASNL

AS PI ωω ∝

*2)3( )()()( SSPPASNL EEP ωωχω =

SPAS ωωω −= 2

ICARS∝ N2F

ks

-ks

kp kp

kAS

)2( spAS kkkk −−=∆

F-CARS and E-CARS…

From Cheng et al, J. Opt. Soc. Am. B 19 1363 (2002)

FCARS

E-CARS

F-CARS and E-CARS

Melanine beadsin agarose gelS: 10450 cm-1(957nm)P: 11177 cm-1(894nm)P:20mWS:10mW Agarose/glass interface

0.75µm

0.28µmPolystyrene beadsin agarose gelS: 11009 cm-1(908nm)P: 14054 cm-1(711nm)

From Cheng and Xie J. Phys. Chem. B, 108, 827 (2004)

Spectral shape…Electronic state

tpt

tNR

RspR

R

iA

iA

Γ−−++

Γ+−−Ω=

ωωχ

ωωχ

2)()3()3(

ΩR ΩR

ωS

ωS

ωPωP

ωP

ωP

ωS

ωAS ωASωAS ωPωP

R)3(χ Far from two photons absorption

Spectral width

CARS line profile as a function of the pulse widthΓR=10cm-1

Resonant and non resonant CARSAs function of pulse width

From Cheng and Xie J. Phys. Chem. B, 108, 827 (2004)

From the experiment…

CARS Set Up

Nd:Vd VERDI 10W

MIRASaphirTitane

(Maître)

MIRASaphirTitane

Esclave

PulseSelect

PulseSelect

SynchroLock

APD

Platine PZT XYZ

Echantillon

Objective Microscope NA1.2

FiltreM

BS

M

M

M

Delai

(λ/2)+Glan

ωP+ωSωAS

PZT

Filtres

Monochromateur

Télescope

APD

Filtres

Objective Microscope NA 0.5

E-CARS

F-CARS

M BS

APDAPD

ωPωS

ωAS

Rétroréflecteur

BS : séparatrice 50/50M : MiroirPZT : PiézoélectriqueAPD : Photodiode à avalanche

CARS Set Up

Imaging polystyrene beadsC=C 1600cm-1 (ωp750nm-ωs850nm) ωAS

x

z

ωP

ωAS

ωS

15

10

5

0

Y (µ

m)

151050X (µm )

1.0

0.8

0.6

0.4

0.2

0.0

CA

RS

In

ten

sity (

no

rm)

151050X (µm)

∆X = (7.458 ± 0.129) µm

1.0

0.8

0.6

0.4

0.2

0.0

CA

RS

Inte

nsity (

norm

)

20151050Z (µm)

∆Z = (6.2028 ± 0.0911) µm

∆X=7µm ∆Z=6.3µmF-CARS - 6µm beads

E-CARS – 0.1µm beads ∆X=0.57µm ∆Z=1.5µm

1.0

0.8

0.6

0.4

0.2

0.0

CAR

S In

tens

ity (n

orm

)

2 0151 050Z (µm )

FWHM = 1.52 µm

1.0

0.8

0.6

0.4

0.2

CAR

S In

tens

ity (n

orm

)

2.01.51.00.50.0X (µm)

FWHM=574 nm

1.5

1.0

0.5

0.0

Y (µ

m)

1.51.00.50.0 X (µm)

400 kHzP: 160µWS: 80µW

Temporal detuning

Delay between ωp and ωs pulses(in ps)

CARS signal at 660nm

Lipid membranes…

Polar

2µm

E-CARS spectrum of a multilamellar layer of DOPC on a clean glass coverslip. Thepump beam was fixed at 13990 cm-1

(714nm).CARS signal from the lipids peaks at 2849 cm-1, (symmetric CH2 vibrational mode).

F-CARS image of erythrocyte ghost. Image was taken in the equatorial xy plane of the vesicle at a Raman shiftof 2845 cm-1.

From Potma et al, J. Raman. Spec. 34, 624 (2003)

Lipid membrane and H20

E

E E

E5µm

F-CARS images of POPS multilamellar onions prepared at 27°C. ωp-ωs was tuned to 2845 cm-1 (C-H strech) and 3445 cm-1 (O-H strech). The number of bilayers was estimated to be 500.The pump frequency was fixed at 14212 cm-1 (704nm). P: 100 mW and S: 50 mW - repetition rate 80 MHz

POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) Adapted from Cheng et al PNAS 100, 9826 (2003)

Cheng et al, Biophys. J. 83:502Living cells…C-H strech

NIH 3T3 cells in interphase. Aliphatic C-H stretching 2970 cm-1

Pump 14054 cm-1(711nm) and the Stokes 11184 cm-1(894nm). P: 40mW; S: 20mW

Interphase NIH3T3 cell mitochondriaF-CARS: C-H Strech

Fluo: mitotracker Red.

Adapted from Cheng et al Biophys. J. 83, 502 (2002)

3D sectioning C-H strech

Three dimensional distribution of lipids in epithelial cells.CH2 stretching vibration (2845 cm-1).Lipid granules and plasma membranes.

http://bernstein.harvard.edu/research/cars.html

Living cells…PO2- strech

F-CARS images of a NIH 3T3 cell in metaphase at different depths. PO2

-symmetric stretching vibrational frequency at 1090 cm-1.Pump 13593 cm-1(735nm) and Stokes 12503 cm-1(800nm). P: 40mW, S: 20mW, 400kHz

Intracellular hydrodynamics O-H strech 3300 cm-1

living D. discoideum cellsOH strech 3300 cm-1

OD strech 2800 cm-1

D2O H2OH2O

From Potma PNAS 98, 1577 (2001)

D2O

t=0H2O

ωP ωS3300 cm-1 OH strech

Permeability of the plasma membrane Pd=2.2 µm/sDw=5 µm2/s (10%-20% of the cell diameter)Dw>500 µm2/s (central cell region) Dw

Exceptionally low Dw due to the presence of densely packed actinfilaments in this region that provide an additional barrier in theprocess of water diffusion.

- CARS addresses molecular intrinsic vibrational transition and does not requires staining with fluorophore or radioactivity.

-CARS is a coherent process which builds an anti-stokes wave on a large number of molecular bonds. This coherent process permits to obtain a signal orders of magnitude larger than spontaneous Raman scattering. Small laser powers (1mw) can be used which are compatible with bio-objects.

- CARS is selective of a certain molecular bond (by adjusting the detuning between laser and Stokes beam)

- CARS is a non linear process which takes place only at the focal point of the microscope lens (diffraction limited) . Therefore the confocal effect is automatic and permits 3D imaging of bio-objects.

- Working in IR limits the absorption and diffusion of bio- tissue. Image as far as 0.3mm in depth can be obtained in living tissues.

- CARS is an elastic process which does not store energy into the system. It is therefore insensible to bleaching as fluorescence is.

- Finally, CARS is not affected by endogenous fluorescence because the Anti-Stokes signal is at lower wavelength than the pump lasers.

Institut Fresnel

STICCentre d’Immunologie de Marseille Luminy (CIML)

SDV

D. MarguetA.SergéM. FalletA. BonedL. WawrezinieckO. Wurtz

H. RigneaultF. BeloniN. DjakerP.-F. Lenne S. Monneret

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