Applications des lasers impulsionnels en biologie : résolution spatiale et génération de contraste

Transcription

1 Applications des lasers impulsionnels en biologie : résolution spatiale et génération de contraste Pierre-François LENNE Institut Fresnel - Marseille

2 The cell DNA contains the information for the production of proteins 10 µm 5 nm Mitochodrion 2 nm Energy Production Rough Endoplasmic Reticulum Protein production 5 nm

3 Plan I- Introduction - Rappels Résolution spatiale Microscopie de fluorescence excitée par l absorption de 1 et 2 photons II- Réduction du volume de détection et résolution sublongueur d onde : la microscopie STED III- Le contraste vibrationnel : la microscopie Raman stimulée CARS

4 Conventional contrast in optical microscopy Airy pattern The Airy pattern radius from the central peak to the first minimum is given by : λ R airy = NA R airy = 1.22 ( NA condensor λ + NA objective ) NA = nsinθ Condensor Object plane Tube lens Image plane θ

5 Critère de résolution : Résolution r xy =1.22 λ = λ 2NA NA

6 Fluorescence Microscopy

7 1P Fluorescence Microscopy

8 pinhole 1P Confocal Microscopy

9 I 2P ~(I exc ) 2 2P Fluorescence Microscopy 1-photon 2-photon molec s/molec = 10 GM

10 Ordre de grandeurs : Microscopie à 2 photons avec laser continu vs impulsionnel Laser continu I( t) = 1mW focalisé dans un échantillon de fluorescéine de 10 µm produit photons/s. Efficacité de détection 5 % 10 3 photons/s détectés. Laser impulsionnel Taux de répétition g p F /( Rτ) = 10 5 I( t) 2 = g p I( t) 2 Facteur de forme /( Rτ) Durée de l impulsion g p = 0.66 (profil gaussien), R = 80 MHz, τ= 100 fs 10 8 photons/s détectés.

11 Resolution in Fluorescence Microscopy Wide Field Confocal 1P 2P λ=0.5µm NA=1.2 r xy r z λ =0.61 NA = 2n λ 2 ( NA ) r z =0.4 λ NA = 1.4n λ r xy ( NA) 2 r xy r z =0.7 λ NA = 2.3n λ NA ( ) 2 n= µm / 0.16µm / 0.263µm 0.92µm / 0.65µm /1.07µm Wide Field 1P Confocal

12 Avantages de la M2P Excitation localisée photoblanchîment et photodestruction réduits Faible absorption Imagerie en profondeur (tissus)

13 II- Réduction du volume de détection et résolution sub-longueur d onde : la microscopie STED Non linear Optics: Stimulated emission depletion + = ~ 100 nm ~ 100 nm Excitation Stimulated emission depletion STED Confocal microscopy + STED T.A. Klar, PNAS USA 97, 2000

14 STimulated Emission Depletion - STED Utilisation d une non linéarité entre intensité d excitation et émission de fluorescence λ

15 STimulated Emission Depletion - STED Ti:Saph 76 MHz OPO + SHG strecher Fluo STED 40 ps 765 nm EX 0.2 ps 560 nm EX STED λ

16 STimulated Emission Depletion - STED fluorescence relaxation STED (40ps) excitation pulse (0.2ps) Cyclic process at a frequency of 40 MHz

17 STimulated Emission Depletion - STED E I STED N 1 dn dt dn 1 dt * 0 = N σi = N σi 1 1 STED STED / hω + / hω N N * 0 * 0 σi σi STED STED N N 1 k * 0 F1 k vib N 0 * k vib If σi STED <<k vib then N 0* =0 and re-excitation is negligible N1 Exp( τσi STED / hω) Strong non-linear dependence of N 1 with I STED Non linear Depletion

18 STimulated Emission Depletion - STED 97 nm FWHM, Z depth and XY!!! = 670 zeptoliters = 0.67 attoliter

19 STimulated Emission Depletion - STED Confoc STED Confoc STED Latex beads 100 nm S. cerevisiae yeast cell

20 Exogenous fluorophore Need to be introduced in the living sample. Quenching and Photobleaching Quenching Arises 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 p chemical damage and covalent modification. 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)

21 III- Contraste vibrationnel Coherent 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

22 Spontaneous Raman Scattering Anti-Stokes Stokes

23 Ω R Ω R ω S ω P ω AS ω P S =σ R I P σ R = cm 2 /molecule NB: σ F-1P =10-16 cm 2 /molecule σ F-2P =10-49 cm 4.s/molecule

24 Stimulated Raman Scattering FCARS ω p ω s ω p ω as Ω r If ω p -ω s =Ω r resonant effect: ω as is enhanced E-CARS

25 CARS versus Spontaneous Raman Laser ω p Stokes ω s Stokes ω s AntiStokes ω as AntiStokes ω as Spontaneous RAMAN Ω R Ω R wavelength

26 From the theory

27 From the theory NL I( ωas ) P ( ωas ) 2 P NL (3) 2 ( ω AS ) = χ EP ( ωp ) ES ( S ω ) * ω AS = 2ω P ω S F I CARS N 2 k = k AS ( 2k p k s ) k s k p k p k AS -k s

28 F-CARS and E-CARS FCARS E-CARS From Cheng et al, J. Opt. Soc. Am. B (2002)

29 F-CARS and E-CARS Melanine beads in agarose gel S: cm -1 (957nm) P: cm -1 (894nm) P:20mW S:10mW Agarose/glass interface Polystyrene beads in agarose gel S: cm -1 (908nm) P: cm -1 (711nm) 0.28µm 0.75µm From Cheng and Xie J. Phys. Chem. B, 108, 827 (2004)

30 Spectral shape Electronic state ω P ω S ω P ω P ω P ω S ω S ω AS ω AS ω P ω P ω AS Ω R Ω R χ (3) = Ω R AR ( ω ω ) + iγ p s R + χ (3) NR At + ω 2ω t p iγ t (3) χ R Far from two photons absorption

31 Spectral width Resonant and non resonant CARS As function of pulse width CARS line profile as a function of the pulse width Γ R =10cm -1 From Cheng and Xie J. Phys. Chem. B, 108, 827 (2004)

32 From the experiment

33 CARS Set Up BS : séparatrice 50/50 M: Miroir PZT : Piézoélectrique APD : Photodiode à avalanche APD F-CARS M BS Nd:Vd VERDI 10W Platine PZT XYZ ω AS Filtres Objective Microscope NA 0.5 PZT Echantillon MIRA Saphir Titane Esclave Synchro Lock MIRA Saphir Titane (Maître) M Filtre Objective Microscope NA1.2 ω S ω P ω P +ω S ω AS APD APD Filtres Monochromateur Pulse Select Pulse Select Delai M Télescope APD E-CARS BS Rétroréflecteur M (λ/2)+glan M

34 CARS Set Up

35 Y (µm) Imaging polystyrene beads C=C 1600cm -1 (ω p 750nm-ω s 850nm) F-CARS - 6µm beads 5 10 X (µm) 15 CARS Intensity (norm) X=7µm X = (7.458 ± 0.129) µm 10 X (µm) E-CARS 0.1µm beads X=0.57µm CARS Intensity (norm) Z=6.3µm 5 Z = ( ± ) µm 10 Z (µm) 15 Z=1.5µm 20 ω P ω AS z x ω S Y (µm) X (µm) 1.5 CARS Intensity (norm) X (µm) 1.5 FWHM=574 nm 2.0 CARS Intensity (norm) FWHM = 1.52 µm Z (µm ) ω AS 400 khz P: 160µW S: 80µW

36 Temporal detuning CARS signal at 660nm Delay between ω p and ω s pulses (in ps)

37 Lipid membranes Polar 2µm E-CARS spectrum of a multilamellar layer of DOPC on a clean glass coverslip. The pump beam was fixed at cm -1 (714nm). CARS signal from the lipids peaks at 2849 cm -1, (symmetric CH 2 vibrational mode). F-CARS image of erythrocyte ghost. Image was taken in the equatorial xy plane of the vesicle at a Raman shift of 2845 cm -1. From Potma et al, J. Raman. Spec. 34, 624 (2003)

38 Lipid membrane and H 2 0 E 5µm E E E F-CARS images of POPS multilamellar onions prepared at 27 C. ω p -ω s 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 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)

39 Cheng et al, Biophys. J. 83:502 Living cells C-H strech NIH 3T3 cells in interphase. Aliphatic C-H stretching 2970 cm -1 Pump cm -1 (711nm) and the Stokes cm -1 (894nm). P: 40mW; S: 20mW Interphase NIH3T3 cell mitochondria F-CARS: C-H Strech Fluo: mitotracker Red. Adapted from Cheng et al Biophys. J. 83, 502 (2002)

40 3D sectioning C-H strech Three dimensional distribution of lipids in epithelial cells. CH2 stretching vibration (2845 cm -1 ). Lipid granules and plasma membranes.

41 Living cells PO 2- strech F-CARS images of a NIH 3T3 cell in metaphase at different depths. PO 2- symmetric stretching vibrational frequency at 1090 cm -1. Pump cm -1 (735nm) and Stokes cm -1 (800nm). P: 40mW, S: 20mW, 400kHz

42 Intracellular hydrodynamics O-H strech 3300 cm -1 OH strech 3300 cm -1 OD strech 2800 cm -1 D 2 O H 2 O H 2 O living D. discoideum cells D 2 O t=0 H 2 O 3300 cm -1 OH strech ω P ω S Permeability of the plasma membrane P d =2.2 µm/s D w =5 µm 2 /s (10%-20% of the cell diameter) D w >500 µm 2 /s (central cell region) D w Exceptionally low Dw due to the presence of densely packed actin filaments in this region that provide an additional barrier in the process of water diffusion. From Potma PNAS 98, 1577 (2001)

43 - 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 bioobjects. - 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.

44 Institut Fresnel STIC Centre d Immunologie de Marseille Luminy (CIML) SDV H. Rigneault F. Beloni N. Djaker P.-F. Lenne S. Monneret D. Marguet A.Sergé M. Fallet A. Boned L. Wawrezinieck O. Wurtz