Nonlinear microscopy 1

• Introduction• Wide-field and confocal (3D) microscopy• 2PEF microscopy• Applications: neurosciences, embryology• Imaging depth in scattering media• SHG microscopy• Endogenous bio SHG/2PEF contrast

Emmanuel BeaurepaireLab. for optics and biosciences - Ecole Polytechnique - Palaiseau

www.lob.polytechnique.fr

Impulsions femtosecondes: des concepts fondamentaux aux applications - Les Houches Janv 2009

Contributions: D Débarre, N Olivier, W Supatto, MC Schanne-Klein, JL Martin, M Joffre, M Strupler, T Boulesteix, AM Pena, G Labroille, R S.Pillai, C Boudoux, J Ogilvie, E Farge, N Desprat, PA Pouille, B Moulia, N Peyriéras, L Duloquin, PL Tharaux, B

Crestani , R Legouis, T Tordjmann, L Combettes, S Charpak, L Moreaux, J Mertz

Nonlinear microscopy 2

• THG microscopy• CARS microscopy• Epidetection of coherent signals• Microscopy with shaped broadband pulses • Coherent microscopy with shaped beams• 3D microdissection with fs pulses

THG microscopy – mechanisms of contrast

THG microscopy = relatively recent approach

First demonstrations : Barad, Eisenberg, Horowitz, Silberberg. Appl. Phys. Lett. 70, 922 (1997)

Müller, Squier, Wilson, Brakenhoff. J. Microsc. 191, 266 (1998)

• characterized by χ(3)(-3ω; ω,ω,ω)• may be obtained from any medium

Non-linear scattering (coherent)

THG

400 600 800 1000 1200 (nm)

THG

Exci

tatio

n(~

1200

nm

)

Excitation

TH signal buildup along z-axis

Intensity near focus

−π (Gouy shift)

0Phase near focus

No signal from a homogeneous medium (normally dispersive) (destructive interference between TH from the beginning and the end of the focal volume)

Signal ∝ (Δχ(3) )2 at interface between 2 “different” bulk media.

THG microscopy: contrast mechanism

“coherence length” for theTHG

Excitation volume

THG

χ(3)(medium 2)

χ(3)(medium 1)

Signal depends on sample structure AND on focal field

distribution

Ward & New (1969); Boyd (2003) Barad et al (1997) App Phys Lett

Using paraxial Gaussian beam model:

( )( )2

0

32

/3kwZb

nnckR ==

−=Δ ωωω z →∞

Constructive interference is possible only if sample is heterogeneous within the focal volume

No signal from a homogeneous medium(Effect of Gouy phase + dispersion)

0.5 1.0 1.5 2.0 2.5 3.00.0

0.2

0.4

0.6

0.8

1.0

THG

pow

er (n

orm

aliz

ed)

Bead diameter (µm)

δz= 2.0 µm

Bead diameter (µm)0.5 1.0 1.5 2.0 2.5 3.0

0.0

0.2

0.4

0.6

0.8

1.0

THG

pow

er (n

orm

aliz

ed)Experiment Theory

∝Ø6

Déb

arre

et a

lOpt

. Let

t. 30

, 213

4 (2

005)

Pha

se m

atch

ing

0.6 µm bead 3.0 µm bead

Detects: interfaces, inclusions

Acts as a band-pass filter on sample spatial frequencies

THG microscopy: contrast mechanism

Signal at the center of a spherical object

CfD

ébar

re e

t al

Opt

. Let

t. 25

, 288

1 (2

004)

Tota

l dur

atio

n~

80 m

in

2s/image, 30s between imgsλ=1180nm

THG: Structural imaging of a developing embryo+ quantification of morphogenetic movements

2PEF THG

2PEF+THG

15µm

Polytechnique CNRS INSERM

Glass Glass

Liquid

Measuring nonlinear susceptibilities χ(3) of liquids

Confocal parameter b~300µm

Liquid sample

thickness (µm)

150

80

10

z (µm)050

-50

0

1

Lateral cell displacement

z

Period ∝ (λ/3)(nω-n3ω)liquid

Here: Maker fringes measurementCf Kajzar & Messier, Rev. Sci. Instr. (1987)

[+] Accurate[-] Less control over sample state

THG ratio method (Cf Barille et al, PRE 2002)[+] Fast[+] Control over sample state (oxygenation…)[-] Prone to artifacts (spherical aberration, surface defects)

Amplitude ∝ (α(glass)- α(liquid))α ≈ χ(3) / n3ω(n3ω -nω)

Déb

arre

& Be

aure

paire

Bio

phys

J. 9

2, 6

03 (2

007)

Cells and tissues are mostly made of water→ THG contrast comes from structures with optical properties (χ(3), dispersion, n) very different

from water.

Weak signal at the interface between 2 aqueous structures

A lipid inclusion inside an aqueous environment (cytoplasm) is an efficient contrast source

1.0 ×10-41.69 ± 0.12polypeptidetriglycine 1M

lipids

lipids

protein

Amino acid

sugar

ions

-

2.2 ×10-21.83 ± 0.08glucose 1M

4.9 ×10-31.75 ± 0.13BSA 1mM

1.0 ×10-41.69 ± 0.13glycine 1M

2.71 ± 0.5

2.58 ± 0.5

1.79 ± 0.09

1.68 ± 0.08

χ(3)

(×10-22m2 V-2)

1.06

0.81

1.2 ×10-2

0

| χ(3) − χ(3)water |2

NaCl 1M

oil

triglycerides

water

λ=1180nm

Optical properties of biological liquids

Biop

hys

J. 9

2, 6

03 (2

007)

glycerolwater

Epithelial cell

But: THG is less specific than labeling or spectroscopic techniquesOther contrast sources: - dense mineralized structures; - absorbing structures; - interface between cell and extracell. space

Contrast can depend on mounting medium

Lipid droplets as THG sources

Lipid droplets are efficient THG source in many cells and tissues

10 µm

hepatocytes

(P=9

0mW

, NA

=0.8

, 250

fs, 7

6MH

z)

0 1 0 2 0 3 00

1 0

2 0

THG

sign

al (a

.u.)

P o s itio n (µ m )

Déb

arre

et a

l, N

at. M

etho

ds 3

, 47

(200

6)

Biophys J. 92, 603 (2007)

Poly

tech

niqu

e C

NR

S IN

SER

M

Poly

tech

niqu

e C

NR

S IN

SER

M

50 µm

50 µm

2PEF (700/860 nm): elastin, NADH, flavins, vit-A…

THG (1180 nm): lipid bodies air bubbles

SHG (1180 nm):collagen

fibers

Multimodal imaging of intact tissueFresh mouse lung tissue

Excitation

400 600 800 1000 1200

2PEFSHG

(nm)

ExcitationSHGTHG

400 600 800 1000 1200(nm)

Polytechnique CNRS INSERM

Nat. Methods 3, 47 (2006)

CARS microscopy:chemically-specific multiphoton imaging without labeling ?

CARS = coherent anti-Stokes Raman scattering

1965: first CARS

1982: CARS µscopy (non colinear)

1999: colinear CARS microscopy→ “CARS explosion”

Reviews:Müller & Zumbusch (2007) ChemPhysChem 8, 2156-2170Cheng & Xie (2004) J Phys Chem D 108, 827-840Volkmer (2005) 38, R59-R84

Excited state

Virtual state

Ground state

Wavelength

Spontaneous Raman scattering: energy transfer between photons and molecular vibrations

Raman spectra of cells contain rich information

k=1/λ (cm-1) wavenumber unitsRaman shift = (1/λincident) – (1/λscattered)

Advantages

• Minimally invasive technique

• Non-photobleaching signal for live cell studies

• Chemical imaging without exogenous tags

Difficulties

• Weak signal – long integration times

• Fluorescence interference

10-30cm2 cross-sections1 in 108 incident photons

are Raman scattered

Photons interacting with sample canlose/accept quanta of vibrational energy

CARS involves using two laser frequencies to interact with a specific molecular vibration

CARS signals are generated at wavelengths shorter than the excitation

wavelength (anti-Stokes)

Principles of CARS (coherent anti-Stokes Raman scattering)

CARS is a third-order nonlinear process → needs pulsed excitation

(typ. picosecond to match Raman lines width)

Signal usually mixed with

nonresonantcontributions (electronic

origin)Polarization:

CARS intensity:

Resonant (signal) Non resonant

ωAS = 2 ωpump – ωStokes

Che

ng &

Xie

, J P

hys

Che

mB

(200

4) 1

08, 8

27

Advantages of CARS imaging• Chemical specificity without staining (like spontaneous Raman imaging)

• Nonlinear: intrinsic 3D resolution, appropriate for tissue imaging

• Coherent signal buildup → strong signal if the density of oscillators is large enough

• Signal shifted to lower wavelengths → not polluted by fluorescence

(but presence of non-specific CARS background)

Coherence-enhanced signal (∝ N2)

A single DPPC bilayer contains 6×106 CH2 groups in a 0.25µm2 area

Current limitations:• Sensibility ~106 oscillators• Residual unspecific background

(∝ N2)

(∝ N)

Ex: long-term imaging of lipid droplets

Nan

, Che

ng a

nd X

ie, J

. Lip

. Res

. 200

3

3T3-L1 cell culture with induction medium.

CH2 stretching freq. 2845 cm-1

Evans et al, PNAS 2005

Visualization of chromosomes

Che

ng, J

ia, Z

heng

& X

ie, B

ioph

ys. J

. 200

2

PO2- stretching frequency. (1090 cm-1)

CARS – Examples of applicationsin vivo imaging of

lipids

Stratum corneum

Adipocytes of the dermis

Adipocytes of subcutaneous

layer

Other possible applications: hydratation measurements, changes in packing or

nature of molecules in organelles, spectroscopic imaging…

(Sunney Xie group @ Harvard)

Directionality THG, SHG and CARS emission is mainly forward-directed

d

d = 1nm d = 100nm d = 10nm d = 150nm d = 50nm d = 200nm

P

d

laser

Bac

kwar

d Forward

Normalized emission diagrams(slab / sphere)

For small dielectric objects (<80nm for THG), emission

becomes isotropic

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

0.0

0.2

0.4

0.6

d

Slab thickness (µm)

B-T

HG

F-TH

G

…but vanishingly small(e.g. d6 dependence for spheres)

Distance of constructive interference:

λ / 4n>λCARS

λ / 8n>λSHG

λ / 12n0.7 λTHG

BackwardForward

Distance of constructive interference in the backward direction is small due to the large wave-vector mismatch.

and: signal ~ (emitters)2

Déb

arre

et a

l Opt

Exp

15, 8

913

(200

7)

→ convenient epi-detected imaging of collagen SHG in tissues

One remarkable exception:SHG from collagen is very efficient → detectable backward-emitted signals

SHG EpiSHG Trans

Human corneaN. Morishige, J. Jester et al, Invest. Ophtal. Vis. Sci (2007)

Zipfel et al PNAS (2003)

XY

XZ

Directionality

In contrast with 2PEF in tissues, backscattered coherent forward emission is exclusively diffusive → absorption becomes a limiting factor

→ field-of-view becomes a critical parameter for efficient detection

Epidetection is possible only when ls’ < la for harmonic light

THG 2PEF

(photons need to travel a long distance without being absorbed to be backscattered)

10-4 10-3 10-2 10-1 1000.00

0.05

0.10

0.15

0.20

Epid

etec

ted

frac

tion

THG 2PEF

Monte-Carlo simulation

ls/(la+ls)

la= 2ls’

g=0.92, ls=25µm, NA=0.95, field of view=1mm, working distance=2mm, sample thickness=2.5mm

Coherent NL imaging of thick tissue: backscattering

Déb

arre

et a

l Opt

Exp

15, 8

913

(200

7)

Experimental confirmation

Lung tissue:epi-THG enhanced

in thick areas

epi-THG

~800µm

trans-THG

50 µm

lung

Absorbing tissue (liver): no backward-detected THG

20 µm

trans-THG

Blood !

z

epi

trans

Epidetection of backscatterd THG in thick tissues

Déb

arre

et a

l Nat

. Met

hods

3, 4

7 (2

006)

Nonlinear microscopy with broadband pulses ?

Goal: another way to obtain spectral resolution+ rapidly address several transitions

Exci

tatio

n(7

50 n

m)

2PEF

(CFP

)

(900

nm

)

2PEF

(GFP

)

Excitation

400 600 800 1000 1200 (nm)

Possible applications:•2PEF microcsopy•CARS microscopy•Two-photon absorption…

Processus à deux photons en l’absence de niveau intermédiaire

e

g

Absorption proportionnelle au carré de l’intensité, i.e. à E4.

∫∝πωωω

2)()( Signal

2)2()2( dEg

πωωω

222)()2( Ω

⎟⎠⎞

⎜⎝⎛ Ω−⎟

⎠⎞

⎜⎝⎛ Ω+= ∫

dEEE

(champ doublé en fréquence)∫ −=≡πωωω

2)exp()()()( )2(2)2( dtiEtEtEoù

2)2( )(ωE spectre du champ doublé:détermine l’efficacité de l’absorption à 2 photons

( ) ( ) ( )( )∫Ω

Ω−+Ω+Ω−Ω+=π

ωϕωϕωωω2

)2/()2/(exp2/2/)()2( diEEE

Pour une phase antisymétrique autour de ω0/2, on a 0)2/()2/( 00 =Ω−+Ω+ ωϕωϕ

)()( 0)2(00

)2( ωω ϕ ==⇒ EE

En explicitant la phase spectrale:

spectre d’excitation à deux photons (réponse) de l’échantillon)()2( ωg

Maximal ∀ω pour φ=0 (phase nulle, impulsion limitée par TF)

t t

2)2( )(ωSHGE

2nd-harmonic spectrum

of a transform-limited pulse

2)2( )(ωSHGEShaping of

→ modulation of two-photon absorption

ω

½ω1

ω1

ω2

½ω2

+ Poster by Guillaume Labroille

Coherent control of two-photon absorption

"blue"SH spectrum

"green"SH spectrum

TL pulse

Corresponding to phase antisym. around ½ ω1

See e.g.:Silberberg, Nature 1998Dantus, J Phys Chem 2002Ogilvie, Joffre, et al, Opt. Express 2006

ω1

ω2

ω0↔822nm

½ω0↔411nm

ωFL↔460nm

D. Meshulach et Y. Silberberg, Nature 396, 239 (1998)

Contrôle cohérent de l’absorption à deux photons

Phase symétrique par rapport à ω0/2

Phase antisymétrique par rapport à ω0/2

(Césium)

λ [nm]

365 375 385 395 405 415 425 435 4450

5

10

15

20

25

30

λ [nm]

Spec

tres

doub

lés

730 750 770 790 810 830 850 870 890

-5-4-3-2-1012345

Phas

e [ra

d]

365 375 385 395 405 415 425 435 4450

5

10

15

20

25

30

λ [nm]

Spec

tres

doub

lés

730 750 770 790 810 830 850 870 890

-5-4-3-2-1012345

Phas

e [ra

d]

λ [nm]

Coherent control of two-photon absorption in vivowith a rapidly switching pulse shaper

→ Poster by G. Labroille et al

Perspective: multiplexed imagingof several dyes withdifferent absorption

spectra

Phase [rad/2π]

Pixe

l

GFPendoTL TL

NA=0.83.5 s/image

Polytechnique CNRS INSERM

∫∝πωωω

2)()( Signal

2)2()2(2

dEg SHGPEF

e

g

∫∝πωωω

2)()( Signal

2)2()2( dEg ORCARS

Second-harmonic field : 2)2( )()( tEtESHG ≡

Two-photon excitation Raman excitation

Optically-rectified field : )()()( *)2( tEtEtEOR ≡

Coherent control ≡ Shaping of 2)2( )(ωORECoherent control ≡ Shaping of

2)2( )(ωSHGE

The population of a vibrational level at ΩR is proportional towhere

→ constructive interference when Φ(ω)=Φ(ω-ΩR) for all frequency components of the excitation pulse

CARS microscopy with shaped femtosecond pulses

e

gΩR

The population of a vibrational level at ΩR is proportional towhere

Constructive interference when Φ(ω)=Φ(ω-ΩR) for all frequency components of the excitation pulse→ always true with flat phase (TL pulse): no selectivity→ with periodic spectral phase (period Ω) : true for energy levels where ΩR = N Ω

τ=2π/Ω

Pulse with periodic phase TL pulse+

++

→ pulse split in time:can excite vibrations

with period T= τ/N

CARS microscopy with shaped femtosecond pulses

Dudovitch, Oron, SilberbergJ Chem Phys 118, 9208 (2003) N. Dudovich, D. Oron, Y. Silberberg, Nature 418, 512 (2002)

MéthanolCH2Br2

Dibromomethane CH2Cl2

Pulse shapemaximizing

CH2BR2 577cm-1

contribution

a - b TL pulse

Pulse shapeminimizing

CH2BR2 577cm-1

contribution

CARS microscopy with shaped femtosecond pulses

Vectorial formulation of the field near focus

( ) ( ) ( )

( ) ( ) ( )

( ) ( ) ( )⎪⎪⎪

⎩

⎪⎪⎪

⎨

⎧

=

=

=

∫∫∫

α

α

α

θθθθθθ

θθθθθ

θθθθθθ

02

22

011

00

20

cosexpsinsin2

sincos,

cosexpsinsincos,

cosexpsinsin2

coscos,

dikzkrJzrI

dikzkrJzrI

dikzkrJzrI

The field distribution near focus is actually vectorial.Result for an incident linear polarization along Ox:

( ) [ ]( ) [ ]( ) [ ]⎪

⎩

⎪⎨

⎧

−=−=

+−=

φφφφ

φφ

cos,,2sin,,

2cos,,

1

2

20

IEzrEIiEzrE

IIiEzrE

z

y

x

where

Resulting intensity distribution:

( ) ( ) ( ) ( ) 222 ,,,,,,,, zrEzrEzrEzrI zyx φφφφ ++=

E0

y

x

z

r φ

θ

Richards & Wolf (1959) Proc Roy Soc A 253 358Born & Wolf (1980) Principles of optics×15 ×3×1

Field distribution in focal plane for a focused Gaussian beam

nk πω2=αsinnNA =

[ ]µmµmyxNA 1,1,,4.1 +−∈= Calcul: N Olivier, LOB

y z

xy z

Zoology of focal fields

-2 -1 0 1 2

Pha

se (x

π ra

d)

HG00 NA=1.4 HG20 NA=1.4 HG00 NA=1.2

-1.6

1.6On-axis phase distribution dependson beam mode (and polarizationcomponent)

Olivier & Beaurepaire (2008) Opt. Express ×1.4×1.4 ×1

×15 ×3×1

Polarizationproperties

Intensity, phase and polarization distribution near focus can be modulated by engineering the fieldbefore focusing. Examples with focused Hermite-Gaussian and Laguerre-Gaussian beams.

( ) ( )zyxxx

wzyx m

m

n

nmnH

nm ,,,, 0 EE∂∂

∂∂

= +

×1.4×1.4 ×1

Can be usedto visualize

axial structures (ex: collagen SHG)

→

Yoshiki et al (2007) Opt. Lett. 32Yew & Sheppard (2007) Opt. Commun. 275

SHG with focused radially polarized beamsFocusing converts radial polarization into

axial component at focus

HG01

HG10

0.0

0.5

1.0

1.5

2.0

F-TH

G (n

orm

)

Interface angle (rad)

HG00 HG01 (x3) HG10 (x2) HG01/HG10 ratio

0 π/6 π/3 π/2 π2π/3 5π/6

HG00HG01 (×3)HG10 (×2)HG01 / HG10 ratio

χ(3) = 0

χ(3) = 1

φ

Sensitivity to XZ or YZ interface orientation

Oliv

ier &

Bea

urep

aire

, Opt

. Exp

ress

(200

8)

(Sim)

Far-field emission patterns (XY)

Excitation with asymetric HG modes: increased sensitivity to interface orientation

zNote: Gouy shift deflects emission off-axis (Cf Mertz JOSA 2001; Cheng & Xie JOSA 2002)

Krishnamachari & Potma(2007) Chem PhysKrishnamachari & Potma(2007) JOSA A 24

CARS: bulk background reduction using engineered beams“Optical bottle beam” (OBB) focal field

Focal fieldGauss OBB Gauss OBB

CARS polarization (x component)

Bulk

z-ed

ge

Radiation patterns Nonlinear microscopy + ablation → biomechanics

* *

2PE

F λ=

920n

m

Disruption of morphogenetic movements in vivo

→ disrupted compression of anterior pole cells

control photoablated

compressionexpansion

Sup

atto

et a

l.,

PNAS

(200

5)

An ultraslowpump-probe

experiment…

Collab. X-LOB / E. Farge group @ Inst. Curie Paris

ablated ablated+

rescued

ablations + magnetic micromanipulation + imaging were used to demonstrate the existence of endogenous

mechanical modulation of Twist gene expression by tissue deformations during morphogenesis

Des

prat

et a

l, D

evC

ell(

2008

)

Twist expr. level

Gen

e Ex

pres

sion

Tiss

ue

defo

rmat

ions

(Her

e: 2

PE

F im

agin

g)

Twist Twist

Restoringtissue

compressionwith

ferro-fluids

60 +/-20 nN force mimics endogenous

deformation rate

• Microscopies non linéaire → apport pour l’étude de tissus intacts

• Décryptage des signaux endogènes → nouvelles applications

• Combinaison naturelle des techniques femto (2PEF,SHG,THG,ablation…) →imagerie multimodale + manipulation

• Développements: • plus profond (optique adaptative, amplis…); • in vivo (fibres, endoscopes…)• plus spécifique (marqueurs génétiques fonctionnels, CARS, manipulation du

front d’onde, contrôle cohérent…)

Quelques messages

LOB Polytechnique PalaiseauDelphine DébarreNicolas Olivier Mathias StruplerGuillaume LabroilleAna-Maria Pena Thierry BoulesteixRajesh S.PillaiCaroline BoudouxJennifer OgilvieMarie-Claire Schanne-KleinManuel JoffreJean-Louis MartinXavier SolinasJean-Marc Sintes

Institut Curie CNRS ParisWilly SupattoEmmanuel FargeNicolas DespratPh-Alex. PouilleUniv Paris 5Serge CharpakLaurent MoreauxBoston UnivJerome Mertz

IBAIC Inserm OrsayLaurent CombettesThierry TordjmannINAF CNRS GifNadine PeyriérasLouise DuloquinCGM CNRS GifRenaud LegouisCRC Lariboisière ParisP.-L. TharauxHôp Bichat Inserm ParisBruno CrestaniINRA Clermont-FBruno Moulia

Contributions

A few referencesNonlinear microscopy

“Nonlinear magic: multiphoton microscopy in the biosciences”Zipfel et al, Nature Biotechnology 21(11), 1369-1377 (2003).

Deep-tissue imaging“Deep tissue two-photon microscopy”Helmchen & Denk, Nature Methods 2, 932-940 (2005).

Endogenous SHG/2PEF contrast“Live tissue intrinsic emission microscopy using multiphoton excited intrinsic fluo. and SHG”Zipfel et al, PNAS 100(12), 7075-80 (2003).

SHG & THG microscopy“Microscopies multi-harmoniques pour l’imagerie structurale de tissus intacts”Débarre et al, Médecine/Sciences 22 (2006).

“SH imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms”Campagnola & Loew, Nature Biotechnology 21(11) 1356-60 (2003).

“Depth-resolved structural imaging by THG microscopy”Oron et al, J. Struct. Biol. 147: 3-11 (2004).

CARS microscopy“Coherent anti-Stokes Raman scattering microscopy”Müller & Zumbusch, ChemPhysChem 8, 2156, (2007).

Photoablation“Mechanisms of femtosecond laser surgery of cells and tissues”Vogel et al, Appl. Phys. B (2005).

“La nanochirurgie laser en biologie cellulaire”Colombelli et al, Médecine/Sciences 22(6-7) 651-8 (2006).