Applications à la microscopie non-linéaire

Transcription

1 Applications à la microscopie non-linéaire Sophie Brasselet Institut Fresnel, Marseille

2 Nonlinear Microscopy and Pulse Shaping 100µm In vivo mouse spinal cord: Myelin (CARS) Axons (2P) LysM cells (2P) Cd11c cells (2P) I. Fresnel - INT, Marseille 1. Nonlinear contrast mechanisms for imaging 2. Optimizing spectral conditions in two photon processes 3. CARS single pulse imaging 4. Short pulses and tissue imaging

3 1. Nonlinear contrast mechanisms for imaging

4 Contrasts : fluorescence and nonlinear optics p 2ω µ abs µ em Ω Incoherent processes: 1PF, 2PF Coherent nonlinear processes : SHG, FWM, CARS - Single molecule detection - Biological systems are labelled - In-depth detection in tissues - No labeling

5 Linear contrast (1 photon) Staining Confocal type imaging Multi-λ imaging possible 1PF

6 Nonlinear contrasts (2 photon) 2PF necessity of staining in non-fluorescence media, autofluorescence (Flavin, NADH,..) SHG 7 mm x7 mm I. Fresnel CIML: mouse skin tissue slice imaging non-centrosymmetric media (labels in membranes, collagen,..)

7 Near-IR excitation reduces scattering in tissues µm : enhanced penetration in tissues 1 ω 0 ω ω 1 0 n E. Beaurepaire, LOB Palaiseau

8 Two photon excited fluorescence (2PF) imaging ( 1 photon) Pabs I 1-photon 2-photon Pabs ( photon) I and 2 photon fluorescence in a solution 1-photon 0.81µmx0.37µm z r Excitation volumes at 750nm, NA 1.2: 2-photon 0.52µmx0.26µm Reduced volume by about 2

9 Two photon excited fluorescence (2PF) efficiency 1-photon excitation 1molecule in bright day light : 1 event /s 2-photon excitation : virtual ultra-short lived (<1fs) intermediate state 1molecule in bright day light : 1 event / 10 million years Pabs ( 1 photon) µ 01. E Absorption probabilities : 2 P µ. E µ. E = ( 2 photon) ( )( ) 2 abs 0n n1 P abs ( 1 photon ) ( 1) 2 = σ. E P abs ( 2 photon ) ( 2 ) 4 = σ. E σ ( 1) 16 2 ( ) Absorption cross sections : cm σ ( 2) 50 4 ( ) 10 cm s / ph Usual unit : Göppert-Mayer 1 GM = cm 4 s/ph

10 1 photon fluorescence signal from a single molecule Volume d excitation (surface: A) I(t) P(t) = C Φf hν σ A ph/s ~ 300nm P(t) excitation Collection factor: C 2 10% Fluorescence quantum yield: Φf Molecule absorption cross section: σ 3 10 cm 16 2 σraman 10 cm 28 2 P ( t) = 10µ W I ( t) = 6300 ph / s 2 A 1m µ I 10ph / s Raman signal noise

11 2 photon fluorescence signal from a single molecule Volume d excitation (surface: A) I 2 ph P( t) 1 ( )... σ ν t = C Φ f h 2 ph A 2 (2) ph/s ~ 300nm P(t) excitation Collection factor: C 2 10% Fluorescence quantum yield: Φf σ Molecule 2 photon absorption cross section: = ( 2) 50 4 cm s / ph P( t) = 10mW 2 A 1m µ In the continuous (CW) excitation regime : 2 ph I ( t) = 0.6 ph / s Signal << noise!

12 Short laser pulses are required for two-photon imaging: Optimal excitation with minimum average power ( ) = σ ( ) TPEF 2 2 I E t dt 2 ( t ) = P f ( t ) P peak. Over-simplified form : 1 f τ Rectangle shape Width τ, frequency f : Ppeak = 1 τ. f P ( ) P I TPEF 2 0 P = τ. f 2 Typically : f = 76MHz, τ = 220fs, for same average power: ( pulsed ) 4 ( CW ) I = 6 10 I Hell et al. 1994

13 Two photon excited fluorescence (2PF) microscopy Advantages / 1 photon: - Less background noise - Deeper penetration length - Better λ ex λ em separation

14 Two photon excited fluorescence (2PF) microscopy Deep two-photon imaging in mouse neocortex, obtained from a transgenic mouse expressing a genetically-encoded chloride indicator Helmchen and Denk, Nat. Meth. 2, (2005)

15 E Absorption 3ω Fluorescence SHG and THG Microscopy imaging 2ω Intensity (counts) ω THG SHG 2ω wavelength (nm) TPF ω F E (t) E fluo() t p 2 ω E () t 3 ω E () t P 2ω ( 2 ) ω ω = χ : E E P 3ω ( 3) ω ω ω = χ : E E E

16 SHG from biological molecules Collagen I is non-centrosymmetric SHG image of collagen in a muscle tissue 60 µm Univ. Exeter σ ( SHG) cm s / ph!

17 Pabs ( P) ( ) = σ. E molecule : ph/s 1 molecule : ph/s 10 nm molecular nanocrystal ~1000 dipoles

18 Nonlinear contrasts (3 photons) 3PF Autofluorescence UV range detection Inst. Fresnel THG Interface between media of different refractive indexes LOB FWM label-free contrast mechanism, non-resonant contribution of CARS Inst. Fresnel CARS label-free, chemical specific Inst. Fresnel

19 Coherent Anti Stokes Raman Scattering (CARS) Probing Vibrational modes : A. Volkmer, J. Phys. D: Appl. Phys. (2005) C-H streching imaging Pump wave ω P Anti-Stokes ω AS = ω P +Ω R Stokes ω S = ω P -Ω R Ω R

20 Coherent Anti-Stokes Raman Scattering : CARS Spectral behaviour of the χ (3) tensor: χ (3) R and χ (3) NR CARS as a third-order nonlinear process: P E E E ( ω ) = χ ( ω ) : ( ω ) : ( ω ) : ( ω ) (3) (3) * as as p p p p s s (3) 2 (3) 2 as I( ω ) P ( ω ) χ as Phase shift of π/2 of the χ (3) at resonance χ (3) decomposition into two parts : For an isolated Raman line : χ (3) R χ (3) Electronic response spectrally independent = p χ : (3) R χ s (3) NR + χ a = ( ω ω Ω (3) NR R Oscillator strength ) + iγ is real & constant Raman line half-width Vibrational frequency χ (3) spectral behaviour

21 CARS

22 CARS as a resonant process 1003cm -1 Source: Polystyrene spontaneous Raman spectrum CARS intensity (kcps) Raman shift (cm-1) Polystyrene CARS spectrum 1080 CARS resonance Off-resonance

23 Multimodal nonlinear imaging Forward detector APD Multimodal imaging in a molecular crystal 2PF FWM/CARS SHG filters sample 50x x z x BS Pump Stokes S. Brustlein et al. JBO 16 (2011) Epi detector APD Pump : nm / 1.5m W Stokes : nm / 750µW

24 100µm 2PF / CARS imaging in the mouse spinal cord Axon imaging (CFP, exc 800nm) / myelin imaging EAE mice, INT I. Fresnel Marseille Fixed spinal cord, 30µm depth

25 Short pulses and nonlinear microscopy At 800nm : Fused silica : GVD = fs 2 /mm BK7 : GVD = fs 2 /mm t A microscope objective : typically 3000 fs 2

26 A typical nonlinear microscope working with ultra-short pulses Spectral resolution of the shaper : 0.53nm/px Max time shift : 1.9ps Max GVD : 5000fs 2 x100, NA 1.4 (sub-10fs pulses, M Pawlowska et al. OE 2014)

27 Short pulses and nonlinear microscopy Obtaining a TL pulse maximizes a NLO process - Interferometric autocorrelation / interferometric frequency resolved optical gating (FROG) traces NLO nanocrystal From R. Trebino Extermann et al. OE 16 (2008)

28 Short pulses and nonlinear microscopy Obtaining a TL pulse maximizes a NLO process - Interferometric autocorrelation / interferometric frequency resolved optical gating (FROG) traces - Multiphoton intrapulse interference phase scan (MIIPS) (phase only modulation) From M. Dantus

29 Short pulses and nonlinear microscopy Obtaining a TL pulse maximizes a NLO process - Interferometric autocorrelation / interferometric frequency resolved optical gating (FROG) traces - Multiphoton intrapulse interference phase scan (MIIPS) (phase only modulation) - Optimization (evolutionary) algorithms based on intensity measurements Random phase mask Binary phase mask P. Schön, I. Fresnel From M. Dantus

30 Short pulses and nonlinear microscopy Obtaining a TL pulse maximizes a NLO process - Interferometric autocorrelation / interferometric frequency resolved optical gating (FROG) traces - Multiphoton intrapulse interference phase scan (MIIPS) (phase only modulation) - Optimization (evolutionary) algorithms based on intensity measurements - Phase resolved interferometric spectral modulation Phase modulation is multiplexed in time frequency M Pawlowska et al. OE 22 (2014)

31 Short pulses and nonlinear microscopy Distorsion occurs both in space and time (space-time coupling) Nanoscatterers: gold nanorods (34 nmx25 nm) M Pawlowska et al. OE 22 (2014)

32 Short pulses and nonlinear microscopy Distorsion occurs both in space and time (space-time coupling) Nanoscatterers: gold nanorods (34 nmx25 nm) After pulse shaping optimization M Pawlowska et al. OE 22 (2014)

33 2. Optimizing spectral conditions in two photon processes (coherent control for selective nonlinear microscopy)

34 Nonlinear processes involve intra-pulse interferences SHG :

35 Two-photon processes : nonresonant TPA 2 nd Order Time-Dependent Perturbation Analysis ( ) a E t i t dt 2 f ( ) exp( ω fg ) f ( ω ) E( ω ω ) dω a f ( ) E fg g Many combinations of the frequency pairs determine the total excitation Y. Silberberg, Annu. Rev. Phys. Chem. 60 (2009)

36 Nonresonant TPA a f ( ) = dδω E dδω E ( ω + δω) E( ω δω) ( ω + δω) E( ω δω) = i e [ Φ( ω + δω ) +Φ( ω δω )] 0 0 Transition probability is controlled by the spectral phase of the incident field f Antisymmetric phase has no effect on transition probability at ω 0 Transform limited pulses are most efficient: g ω 0 = ω fg /2 Y. Silberberg, Annu. Rev. Phys. Chem. 60 (2009)

37 Nonresonant TPA: experiment 2π Φ ω 2π Meshulach & Silberberg, Nature, 396, 239 (1998)

38 Control of TPA in molecular systems f Atomic two-photon transitions can be controlled with excellent contrast g Can this concept be used for controlling organic chromophores with broad absorption bands? f g Y. Silberberg

39 TPA in bio-molecules (fluorescent proteins) 2PA (TPA) 1PA M. Drobizhev et al. Nat Meth 2011

40 Phase coherent control of nonlinear processes in molecular systems TPF σ ω 2 ω + 2 ( 2 ) ( ) E E d dω ω 2 TPA large spectrum 2-photon excitation spectrum - Flat phase : Optimum over the whole spectrum - Phase antisymmetric point : no destructive interference - Elsewhere : phase is not optimal : weak or zero-signal V.V. Lozovoy et al. J. Chem. Phys. 118 (2003)

41 Controlling the spectral phase can lead to controlled 2-photon spectra P. Nuernberger, Phys Chem Chem Phys (2007)

42 Pulse shaping in molecular systems Spectral regions in destructive interferences ϕ ( ω) = a cos( bω + Φ) tuned Read-out by SHG E phase profile φ(ω) ε(ω) E(2) excitation spectrum SHG in a BBO crystal E phase profile E(2) excitation spectrum I. Fresnel V.V. Lozovoy et al. J. Chem. Phys. 118(7) 2003

43 Molecular absorption profile Matching the molecular absorption profile ( ) ( ) ω + ω ω ω σ d d 2 E 2 E TPF 2 2 V.V. Lozovoy et al. J. Chem. Phys. 118(7) 2003

44 Coherent control for selective two-photon fluorescence microscopy of live organisms egfp Yolk 2PF ratio egfp/yolk measured with narrow band shaping Drosophila embryo TL Resulting 2TPA spectra J.P. Ogilvie,. E. Beaurepaire, M. Joffre, OE (2006)

45 Coherent control for selective two-photon fluorescence microscopy of live organisms Microscope Shaper J.P. Ogilvie,. E. Beaurepaire, M. Joffre, OE (2006) Also Marcos Dantus group, Michigan State

46 Linear combinations yield two selective images of Drosophila embryo Blue pulse Yolk emission 25 µm Red pulse GFP emission J.P. Ogilvie,. E. Beaurepaire, M. Joffre, OE (2006)

47 I. Pastirk et al. OE 11 (2003) Phase coherent control for specific imaging : ph selectivity Excitation Fluorescence

48 Optimizing the spectral selectivity: application to FRET Förster Resonant Energy Transfer DC Flynn et al. OL

49 Optimizing the spectral selectivity: application to FRET Förster Resonant Energy Transfer quick switching between excitation conditions (~ 35 ms) cells expressing linked construct mametrine-tdtomato plus excess mametrine. stochiometry DC Flynn et al. OL

50 3. CARS single pulse imaging

51 CARS Microscopy CARS Image tuned to DNA backbone vibration at 1090 cm -1 in mitosis CARS image of fibroblast cells that are stimulated to synthesize lipids. The lipid droplets are visualized with CARS tuned to the C-H vibration at 2845 cm -1 A. Zumbusch, Univ. Konstanz Xie s group, Harvard

52 CARS generation processes Scanned stokes/pum Multiplex CARS Single pulse CARS

53 Single pulse CARS A(Ω) is broadband Oron et al., Phys. Rev. A 65 (2002)

54 Broad-band excitation of a Raman transition ( Ω) = dωe A v * dω E ( ω) E ( ω Ω) ( ω) E( ω Ω) = i e [ ( ) ( )] Φ ω Φ ω Ω Ω Transform-limited pulses maximize transition rates Periodic phase functions maintain efficiency : nonresonant effects are much weaker Oron et al., Phys. Rev. A 65 (2002) Ω g v

55 Single-pulse CARS with periodic phase spectrum time τ Dudovich et al., Nature 418 (2002)

56 Single-pulse CARS microscopy Pulse bandwidth 1500cm -1 SLM 15 fs input pulse output pulse From Y. Silberberg blocker filtered signal Dudovich et al., Nature 418 (2002) λ

57 Spectroscopy by selective excitation Modulated spectral phase function Φ=1.25cos( cω ) λ λ Fourier transform t Ba(NO 3 ) 2 (1048 cm -1 ) Diamond (1333 cm -1 ) Toluene (788, 1001 cm -1 ) lexan From Y. Silberberg Dudovich et al., Nature 418 (2002) Nonresonant background is removed

58 Single-pulse CARS microscopy Pulses are shaped to maximize CARS signals from specific molecules Maximal resonant contribution Minimal resonant contribution Nonresonant Resonant + nonresonant Resonant contribution extracted exclusively Glass capillary plate with 10 µm holes filled with CH 2 Br 2 Maximal-minimal difference From Y. Silberberg Dudovich et al., Nature 418 (2002) Transform limited

59 4. Short pulses and tissue imaging

60 Adaptive Optics in Microscopy Third Harmonic Generation (THG) Microscopy Plant tissue Beaurepaire, Débarre & Olivier, LOB

61 Scattering media spatial distortions Speckle From Y. Silberberg

62 CW Wavefront Shaping Vellekoop and Mosk, 2007 Or, alternatively, measure the complex transmission matrix and compute the required SLM settings (Gigan, Fink, ESPCI)

63 Ultrashort pulses in a scattering medium spatial + temporal distortions Spatiotemporal speckle (Multi-path problem) space time Y. Silberberg τ time

64 CW vs.fs focusing CW fs White light speckles From Y. Silberberg

65 Spatiotemporal focusing of an ultrafast pulse through a multiply scattering medium Speckle is imaged on a 2D spectrometer After local phase compensation Space and time are TL D.J. McCabe,. S. Gigan, B. Chatel, Nat Comm (2011)

66 Spatiotemporal focusing by a spatial control τ 2PF screen EMCCD femtosecond pulse SLM 10X scattering medium 20X F Spatially resolved autocorrelation widths Initial temporal width Optimized y y x Katz et al. Nature Photonics 5 (2011) x Temporal control is possible by spatial modulation

67 From Y. Silberberg Katz et al. Nature Photonics 5 (2011) Mechanism for temporal control Space-time coupling by the random medium short pulse time

68 Spatiotemporal focusing through biological samples 1mm thick brain tissue: Initial excitation Optimized 500µm thick bone: Initial excitation Optimized Sample surface 100µm Katz et al. Nature Photonics 5 (2011) From Y. Silberberg

69 Conclusion Efficient nonlinar microscopy requires pulse shaping New contrasts and spectral specificity can originate from coherent control and Complex media couple spatial and time control