Focal field distribution - RESEAU FEMTOreseau-femto.cnrs.fr/IMG/pdf/Houches2009_cours15.pdf ·...

Nonlinear microscopy 1

bull Introductionbull Wide-field and confocal (3D) microscopybull 2PEF microscopybull Applications neurosciences embryologybull Imaging depth in scattering mediabull SHG microscopybull Endogenous bio SHG2PEF contrast

Emmanuel BeaurepaireLab for optics and biosciences - Ecole Polytechnique - Palaiseau

wwwlobpolytechniquefr

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

Contributions D Deacutebarre N Olivier W Supatto MC Schanne-Klein JL Martin M Joffre M Strupler T Boulesteix AM Pena G Labroille R SPillai C Boudoux J Ogilvie E Farge N Desprat PA Pouille B Moulia N Peyrieacuteras L Duloquin PL Tharaux B

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

Nonlinear microscopy 2

bull THG microscopybull CARS microscopybull Microscopy with shaped broadband pulses bull Coherent microscopy with shaped beamsbull Epidetection of coherent signalsbull 3D microdissection with fs pulses

bull 1590 Zaccharias amp Hans Janssen (2 lentilles)bull 1665 Robert Hooke cellules (liegravege)bull 1632-1723 Anton van Leeuwenhoekrarr premiegravere description des micro-organismes cellules sanguines etc

laquo Renaissance de la microscopie raquoMicroscope confocal (1957 1980s) imagerie cellulaire 3D agrave lrsquoeacutechelle du micromegravetre

+ deacuteveloppement de marqueurs fluorescents morphologiques et fonctionnels (potentiel membranaire calcium hellip)

hellipmicroscopie subcellulaire in vivo rarr microscopie multiphoton (1990s)

bull XXe siegravecle Profondes avanceacutees en biologie (biologie moleacuteculaire geacuteneacutetiquehellip)rarr besoin de comprendre lrsquoorganisation spatiale des eacutevegravenements intracellulaires

Microscopie optique en biologie(bref raccourcihellip)

Field distribution in the focal region of an objective lens

E0

objective

Paraxial approximation not always validTransverse beam profile not always Gaussian

Field near focus of an aplanetic lens from an arbitrary pupil profile

Focal field distribution

Richards amp Wolf Proc Roy Soc A 253 358 (1959)Born amp Wolf Principles of optics (1980)

Debye-Wolf (Richards-Wolf) diffraction integral

Note relation between incident and refracted fields in an aplanetic system

Intensity law(energy conservation)

nk πω2=

θ zdA1 dA2

dA1 = dA1 cosθ

( ) ( ) ( )( )244sin0 uuuI prop

Point spread function (PSF) of single lens

( ) ( ) ( ) ( ) PSFdiuvJPvuI =primeprimeprimeprimeprime= int21

0

20 2exp2 ρρρρρ

v equiv r middot NA middot 2π λu equiv z middot NA2 middot 2π nλJ0 = 0th order Bessel functionρrsquo = θ middot n NA (radial coordinate in pupil)P(ρrsquo) = pupil function

Intensity distribution in the focal region of an objective with numerical aperture NA=n sin(α)

( )int=cstu

dvvuI is constant along zNote

NAnr

2sin2λ

αλδ =asymp

αn

αsinnNA =

Numerical aperture (NA)

( ) ( ) 2120 vvJvI prop

Born amp Wolf (1980) Principles of optics Muumlller (2006) Introd to confocal fluorescence microscopy

Wilson amp Sheppard (1984) Theory and Practice of Scanning Optical Microscopy

Simplified expression (within Kirchhoff Debye paraxial and scalar approximations)

lateral width

~λ2NA

axialwidth

~2nλNA2

Assuming P(ρrsquo) = 1

bull Lateral resolution ~λ2NA (related to width of PSF amp OTF)bull No true axial resolutionMissing cone of spatial frequencies the axial position of a thinfluorescent plane (laterally uniform) can not be determined

Standard fluorescence microscope (wide field)

( ) ( ) ( )

( ) ( )xyxPSFzyxO

zdydxdzzyyxxPSFzyxOZYXI

otimes=

primeprimeprimeprimeminusprimeminusprimeminus= intintintinfin

Image formation process

Can also be described in spatial frequency spaceby means of the optical transfer function (OTF)

( ) )( vuPSFFOTF vu =ΩΩ

Muumll

ler

Intro

d to

con

foca

lflu

or m

icro

sc (

2006

)

Lateral cut-off frequency (nλ)

Axi

al c

ut-o

ff fre

quen

cy(n

λ)

laquo missing cone raquo

OTF

Lamp

camera

F

Laser

spatial filter

detector

(Note scanning microscope)

3D imaging by (linear) confocal microscopy

488

nm

~500

nm

z

2det excexcconf PSFPSFPSFPSF asympsdot=

( ) ( ) ( )( )444sin0 uuuI prop

( )int=cstu

dvvuI peaks for upropz=0

PSFexc PSFconf

OTF wide field OTF confocal

λ=500nmNA=13

n=15log scale

Dia

spro

et a

l Bi

omed

Eng

Onl

ine

(200

6)M

uumllle

r In

trod

to c

onfo

calf

luor

esce

nce

mic

rosc

opy

(200

6)

rarrTrue axial resolution

Laser

spatial filter

detector


3D imaging by (linear) confocal microscopy[+] Optical sectioning

⎩⎨⎧

propΔpropΔ

2NAnzNAr

λλ

[+] Many available fluorophores for biology

αsinnNA =

Wide field Confocal

Resolution

488

nm

~500

nm

z Excitation is not confined rarr photobleaching phototoxicityVery sensitive to scattering of visible light in tissues rarr limited penetration (~100microm)

4det

minuspropsdot= zPSFPSFPSF excconf

αn

3D microscopy in a biological tissue Diffraction-limited rArr relies on unscattered light

Howeverhellip visible light is strongly scattered in tissues

scattered photons

ballistic photons

DefScattering mean free path (Ls)= average distance between 2 scattering events (50-100 microm in biological tissues for visible light)

z

N0

N0 exp(-zLs) tissue

The number of ldquoballistic photonsrdquodecays exponentially with z

Ls

Confocal microscopy in scattering medium

bull Scattering of non-focal fluorescencebull Scattering of excitation light and of

focal fluorescence

rarr backgroundrarr signal attenuation

(focal point must be imaged on detector)

ExcitationFluoresc

Bbackground

(surface)

S

z

log (S+B)

transparentscattering

z

rArr Scattering limits imaging depthZmax ~ 50-150 microm

nonlinear excitation

F prop I 2PEF prop I2

1PEF1 photon excited

fluorescence


fluorescence

Nonlinear (=multiphoton) microscopy

exci

tatio

n(v

isib

le)

exci

tatio

n(IR

)

[+] preserves sub-cellular resolutioninside scattering medium

ExcitationFluorescence

near IR λ excitation rarr better penetration

scattered IR produce ldquonordquo fluorescencerarr reduced background

2PEF microscopy in scattering medium

[+] Excitation is localized in 3Drarr reduced photoxicity

Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear

S prop I2 (or I3)rArr excitationis confined

S prop I prop 1z2

rArr excitationis not confined ( )222 vuI

PSF =

Near-IR excitation λ asymp 07-12 microm[+] Reduced perturbabtion[+] Enhanced penetration in tissues

02 04 06 08 10 12 14 16 18 2001

1

10

100

Abs

orpt

ion

coef

ficie

nt (c

m-1)

Wavelength (microm)

Water (pure)Fat (pure)Hb02 (1mM)Hb (1mM)Melanin (1mM)Tryptophan (1mM)

Near-IR excitation reduces absorption and scattering laquo transparence window raquo of tissues

http

om

lco

gie

du

04 05 06 07 08 09 10 11 120

200

400

600

800

1000

1200

1400

Scat

terin

g co

effic

ient

(cm

-1)

Wavelength (microm)

confocal

2PEF SHGTHG

Multiphoton excitation tissue optics

Scattering mean free path (Ls) = average distance between 2 scattering events

Ls

g asymp 1 forward scatteringg asymp 0 isotropic scattering

θθcos=g

~100-200microm for λ asymp 07-12 microm

Scattering anisotropy

Biological tissues g ~ 08 ndash 095

F prop I


fluorescence

exci

tatio

n(v

isib

le)

Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2

rArr excitationis not confined

( )222 vuIPSF =


2PEF SHG prop I2


fluorescence

SHGsecond-harmonic

generation

THG prop I3

THGthird-harmonic

generation

CARScoherent anti-Stokes Raman

scattering

ωP ωSex

cita

tion

(IR)

CARS prop IP2 IS

[+] Several possible contrastmechanisms rarr different information

2PEF fluorescence SHG non-centrosymetryTHG χ(3) heterogeneitiesCARS vibration resonancehellip

[+] More robust in the presence of incoherent scattering (inside tissues)

2PEF penetration

100-600 microm


Nonlinear (=multiphoton) microscopies

g

e

g

e

Eeg

σabs asymp 10-16cm2 σ2p-abs asymp 10-49 cm4 sphoton

Usual unit Goumlppert-Mayer(1 GM = 10-50 cm4 sphoton)

1 event s 1 event 10 million years

Standard molecule in bright daylight

1-photon 2-photon

Absorption cross-section

Note Focusing a 1mm2 beam to a 1microm2 area increases the intensity squared by a factor (106)^2 rarr still not enough to enable rapid imaging (2-10 micros pixel dwell time) with such small cross-sections

22PEF2

12

2PEF21

p IτTσI

τTσ

TτF

TτF ⎟

⎠⎞

⎜⎝⎛=⎟

⎠⎞

⎜⎝⎛==

pI

Fluorescence during pulse

Averagefluorescence

laquo gain raquo Tτ

TiSaph laser

gain asymp 105T asymp 10 ns(100 MHz)τ asymp 100 fs

Gain with pulsed excitation

τ asymp 10-13 s T asymp 10-8 s

Pulsed lasers are used for optimal multiphoton excitation with minimal average power

time

pulses are typically τ ~100 femtoseconds FWHM

2P fluorescence depends on the average squared intensity (rather than on )2I ( )2I

Example withsquare pulses

Fp = frac12 σ2PEF Ip2 σ2PEF equiv η σ2p-abs (η radiative quantum efficiency)

More generally for pulsed excitation

( )22 IIτTgP= with gP depending on temporal shape

(066 for a Gaussian pulse shape)

( )22 II = second-order temporal coherence

wr

wz

2PEF cross-section orders of magnitude

σ2PEF = η σ2p-abs ~ 10-48 cm4 sphoton

202PEF2

10 IσF τ= T

20

0π

P2Iw

=

θω

ω

sinnλ032

=rw

)cos-(1nλ530

z θ=

ω

ωw

wr wz radius at 1e

NA2λ

asymp

2NAλn13

asymp

zr ww22

3

2πV ⎟

⎠⎞

⎜⎝⎛=

PSF (point spread

function)

Focal volume Gaussian-shape fit to

diffraction theory

Typically 2 microm times 04 microm


Example one molecule in focus

bull P = 1 mW asymp 1015 photonss224 cm sphotons10asymp

sphotons105asymp

bull σ2PEF = 100 GM

λ=1 μm n=13 NA=1 σ2PEF=100 GM

C=1 μM rarr N=1000 molecules

rarr F ~ 2 times 107 photonss

Example 2 several molecules

CVγIσF 202PEF2

1τ= T

average fluorescence produced by one molecule

in excitation volume

pulsed excitation

2PEF microscopy implementation(source scanning objective dispersion compensation)

700-1200 nm~80 MHz~100 fs

source

No need to produce a descanned image of the the focal spot on a spatial filter simpler than a confocal microscope (except for the laser source)

LASER

Beam scanning

Laser in

Laser

out

Point Scanning

to microscope

Δt

Typical pixel time ~2-10 micros (100-400 kHz)Typical line duration ~1 ms (1 kHz)Typical image acq time ~05-2 s

Some methods for faster scanningbull X-axis resonant scanning

http

pa

rker

lab

bio

ucie

du

128 micros

sinusoidalx-scan

pixel clockline sync

y-scan

frame sync66 ms

laser

y-galvo

32-sidedpolygon

obj

telescope1

tele

scop

e2

PMTbull X-axis polygon mirror

These 2 approaches canbe used to record images

at video rates

Limitation signal level

Multipoint excitation canalso be used in certain

cases

http

ce

llser

vm

edy

ale

edu

imag

ing

Dureacutee de vie ~ 1ns

photonss 10F 9max lt

Autres facteursbull Photoblanchimentbull Photo-ionisation bull laquo inter-system crossing raquo

10 ns

100 fs ltlt 1 ns

(limiteacute par la moleacutecule)


(taux de reacutepeacutetition de lrsquooscillateur)

1 photon de fluorescence max par molecule par

impulsion

Facteurs limitant le niveau de signal

M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)

Engineered fluorophores with enhanced 2PEF

2PEF action cross-sections

10-100 GMldquoQuantum dotsrdquo

104 GM

Endogenous fluorophores (NADH etc)

Standard fluorophores (Rhodaminehellip) 01-10 GM

10-3 - 10-1 GM

Fluorescent proteins (GFP etc)

100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)

Example 150 fs rarr (5000 fs2) rarr 177 fs80 fs rarr (5000 fs2) rarr 190 fs10 fs rarr (5000 fs2) rarr ~1 ps 80 fs rarr (20000 fs2) rarr 697 fs150 fs rarr (20000 fs2) rarr 403 fs

Dispersion of optics in a multiphoton microscope 2000-20000 fs2

Implementation prism pairs gratings chirped mirrors SLM-based pulse shaper

( )2200 2ln41 τϕττ primeprime+=Pulse broadening

τ0 = initial duration (transform-limited pulse)φrsquorsquo = group delay dispersion (fs2)

Dispersion compensation

In practice compensation is necessary mostly for pulses ltlt 100 fs

Note1 2PEF imaging is typically done with 100fs pulsesShort pulses are interesting for spectroscopy (next lesson) but may induce additional toxicityNote2 managing 10fs pulses at the focus of high NA objective is not trivial Radially varying group delay can be a few 10s fs in some objectives

2PEF prop 1τ

PSF degradation example for aqueous sample

60times NA 12water immersion

40times NA 13oil immersion

rarr Use water-immersion objectives (index matching)

PSFz PSFz

Index mismatch cause PSF spreading and signal loss

Diaspro Ed Confocal and 2P Microscopy Foundations Applications and Advances (2001)Jacobsen Hell et al Refractive-index-induced aberrations in 2P confocal fluorescence microscopy J Microsc 176 226 (1995)Booth amp Wilson ldquoRefractive-index-mismatch induced aberrations in single-photon and 2P microscopyrdquo J Biomed Opt 6 266 (2001)

For small aberrations

( ) ( )2221 ΔΦminusasymp λπrefaber II

mean-square deformationof the wave front

( )2ΔΦ

rarr rapid impact on NL signal

Incidence on signal level

NeuroscienceYuste Denk (1995) Dendritic spines as basic functional units of neuronal integration Nature 375 682-684Svoboda Tank Denk (1996) Direct measurement of coupling between dendritic spines and shafts Science 272 716-719Svoboda Denk Kleinfeld Tank (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons Nature 385 161-5

2PEF microscopy in biology some fields of application

Immunology

Developmental biology

ReviewsSvoboda amp Yasuda (2006) Principles of 2P excitation microscopy and its applications to neuroscience Neuron 50 823-839Mertz (2004) Nonlinear microscopy new techniques and applications Curr Opin Neurobiol 14 610-616

ReviewCalahan MD amp Gutman GA (2006) The sense of place in the immune system Nat Immunol 7 329-332

McMahon A Supatto W Fraser SE and Stathopoulos A (2008) Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration Science 3221546-50

laserTiSaphir

rarr 400 μm

50 microm

Application example in neurosciences in vivo 2PEF imaging of an olfactory neuron

50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)

Les PA spontaneacutes enregistreacutes dans le soma (trace du bas) induisent des entreacutees de calcium dans le bouquet dendritique

Pb marquage cellules multiples rarr marqueurs geacuteneacutetiques agrave base de proteacuteines fluorescentesex laquo cameleons raquo (constructions 2XFPs+cmd sensibles au calcium) etc

[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40

Two-photon-excited fluorescence (2PEF)

[+] Genetically encoded probes fluorescent proteins (GFPhellip)

Nonlinear microscopy of tissues 2PEF

GFP

~ns

700-

1000

nm

350-

600+

nm

Some important fields of application of 2PEF microscopy

Neurosciences in vivo neuronal activityImmunology lymphocyte trafficking

GFP labeling of nuclei

2PEF λ=920 nm 2 simage 10 mW 1 image every 30 s

100 microm

yolk

vitelline membrane

nucleilipid droplets

Live embryo imaging 2PEF microscopy

Supatto et al (2005) PNASSquirrell et al (1999) Nat Biotechnol

LOB Polytechnique Curie


2PEF λ=920 nm


Caltech bioimaging centerMcMahon Supatto et al (2008) Science

Example quantitative study of individualcollective cell motions in a developing embryo

1 XYZ image every 10s

z


[+] Endogenous fluorescents species

elastin(rat artery wall fresh)

Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)

Excitation 720nm 800nm 900nm Detection 350-505 505-560 560-650 + spectral unmixing

Note NADH fluorescence is an

indicator of metabolic state

Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164

NADH fluorescent NAD+ non-fluorescent

zmax

P0

( ))ex(s0 LzexpP minus

z

( )[ ] ( )zLzPTS exs Φsdotminussdotprop

2)(0 expτ

Signal

excitation detection

typically ~500 μm(layer 23 neocortex)

α - fluorophore efficiencyand detector noise

tissue scattering length average laser power

Φ - collected fraction of the generated fluorescence

inverse laser duty cycle

( )τα )(ln max)(

max TzPLz exs Φ=

Rat brain Ls~200microm

If limited by detector noise (no background)

zmax~50-200 μm with endogenous signals

What limits the imaging depthConfined excitation even in scattering media hellip but the number of ballistic excitation photons decreases exponentially with depth

PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=

A How to increase collection efficiency (Φ)

How to increase imaging depth

zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA

(1) first idea increase NA

This workshellip

Field-of-view (FOV)

(2) hellipbut what if sample is scattering

Scattered fluorescence seemsto originate from an

extended source

Field of view (related to the angular acceptance of the detection path) defines the depth where collection efficiency starts to drop

At large depths (diffusive light)

Oheim et al (2001) J Neurosci Meth 112 205Beaurepaire amp Mertz (2002) Appl Opt 41 5376

An objective with low magnification and high NA is advantageous for collecting scattered fluorescence

( ) 22 minussdotpropΦ zrFOV

Typical multiphoton microscope

laquo non-descanned raquo detection(close to objective)

700-1200 nm~80 MHz~100 fs

source

Diaspro et al (2006) Biomed Eng online

B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA

Δz asymp ln(rTSrRA)2 asymp 2-3 scattering lengths Δz

rTS = 80 MHz

rRA = 200 kHz

log F

log FTemporal redistribution of the

same excitation power

At large depths contrast (amp resolution)

loss

hellip in vivoexperiment

( )2balscat II +

Theer amp Denk (2006) JOSA A 23 3139

2PEF imaging depth fundamental limit

Note Zmax increases with staining heterogeneity Zmax is increased by ~Ls when stained fraction is reduced 6times

Zmax reached when

( ) intint ge+focusat

balfocusofout

balscat III 22

Contribution of the different planes Model accounting for the temporal distribution of scattered light and assuming that scattering is mostly forward-directed

Influence of pulse duration rarr Using pulse duration of 20fs instead of 200fs should increase the SB ratio by 25times resulting in an increase of 05 scattering MFP to the depth limit

( )τα )(ln max)(

max TzPLz exs Φ=


zmax

bull Regenerative amplification multiplies Tτ by 400rarr Equivalent to multiplying P by 20

bull Implement wavefront correction to correct for specimen-induced aberrations (adaptive optics)

bull Design background rejection schemes to remove light generated out-of-focus when doing large depth imaging

B Improving excitation

bull Low mag objective multiplies Φ by 10(Only equivalent to multiplying P by 3)

bull And always non-descanned detection

A Improving collection Φ

Theer amp Denk OL2003

2PEF from a thin fluorescent slab as a function of slab defocus Ratio betweensignal detected with an unaberrated and an aberrated phase

2PEF images of a labeled glomerulus (from mouse olfactory bulb)

Wavefront correction in microscopy

Φ-Φ

Φ

Possible strategiesMeasure aberrated wavefront with wavefront sensor rarr implementation eg Denk PNAS 2006

Iterative sensorless approach with merit functioneg Wright et al OE 2007 (applied to CARS)

Model-based sensorless approach eg Deacutebarre Botcherby Booth Wilson OE 2008 (applied to structured illumination microscopy)

reference

astronomy ophtalmology microscopy




( )2ΔΦ


LOB

-X

low-coherence interferometry+ wave-front sensing

4 images (I1hellipI4) recorded with reference path length shifts of 0 λ4 λ4 and 3λ4 For each pixel a complex amplitude A is calculated by

Reference

Sample

Low-coherence sourceTiS 915nm 100fs

CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=

22ln2expcos τνπδϕsrsrD iiiiI

z-resolution ~ 20 micromνΔ FWHM of source power spectral density

νΔprop 1

δϕ applied phase shift

τΔ delay due to path difference

+ ldquocompatiblerdquo with 2PEF microscopy

obj

(conjug with objective back focal plane)

Feieraband et al (2004) Opt Lett 29 2255Ruumlckel et al (2006) PNAS 103 17137Ruumlckel et al (2007) JOSA A 25 3517

δϕ

τΔ

6times 2PEF signal improvement

(2006) PNAS 103 17137

1P excitation 2P excitation

2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I

Fluoexcitation(visible) ωP ωS

3P excitation

3PEF

CARS prop IP2 IS

THG CARS

Different contrast mechanisms rarr different information

In particular SHG second harmonic generation ndash sensitive to symmetry at the sub-microm scaleTHG third harmonic generation ndash detects interfaces and microm-size inclusionsCARS laquononlinear Ramanraquo ndash chemical specificity

SHG THG specifically obtained from certain structures(little spectroscopic information)

Alternative contrast modes SHG THG CARS

E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)

Harmonic signal depends on the nature of the emitting medium

P(2ω)=frac12χ(2)(minus2ωωω) E(ω)E(ω)P(3ω)=frac14χ(3)(minus3ωωωω) E(ω)E(ω)E(ω)

Nonlinear microscopy harmonic generation

P = P(ω) + P(2)(2ω) + middot middot middot + P(n)(nω)= PL + PNL

avec P(n)(nω) =

Polarisable medium excited by an intense field components of order ngt1 in the induced polarization

rarr Emission at harmonic frequencies (nonlinear scattering)

non zero χ(2) =gt non centrosymmetric mediumχ(3) non zero everywhere (but weak)

Multiphoton microscope rarr combined contrast modes

2PEFsignal

SHGsignal

Osc

illato

r

THGsignal

Example of push-pull laquo harmonophore raquo molecule for SHG

(SHG)

Example stylbene derivative

hellip and amphiphilic version (for lipid membrane labeling)

M Blanchard-Desce (Univ Rennes)

SHG = coherent process (ne 2PEF)rarr possibility of constructive and destructive interferences

Example (labeled vesicle) parallel molecules rarr SHG

antiparallel molecules rarr no SHG (centrosymmetric medium)

Mertz amp Moreaux OL 2001

Constructive interference rarr enhanced signal Destructive interfeacuterence rarr null signal

Molecules emitting in phase Molecules emitting with opposite phases

ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF

Note in contrast 2PEF emission does not

depend on symmetry

SHG microscopy adapted for

membrane imaging

hellipand some endogenousstructures (see later)

Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG

Spectrum radiated from a GUV labeledwith the styryl dye Di-6-ASPBS

Phase matching in (coherent) nonlinear optics

zLc

If Δkne0 (dispersion) I2ω(z) prop sin2Δkz2coherent signal buildup is limited to Lc

If Δk=0 (phase-matching case) I2ω(z) prop z2

Δk = k2ω - 2kω = wave vector mismatch

laquoClassicalraquo example SHG by plane wave propagating in a nonlinear medium

Note if Δkne0 phase-matching

can be forced in a birefringent crystal eg

where ne(2ω)ltno(ω)

keθ(2ω)=2ko(ω)neθ(2ω)=2no(ω)

The same applies for other NL processes

such as THG

Lc = coherence length

But what happens in a tightly focused geometry

bull Presence of transverse componentsbull Many possible k

kω = 2π nω λ

I Field near focus from arbitrary pupil profile (Cf Richards amp Wolf 1959 Born amp Wolf 1980)

III Far-field signal (Cf Novotny amp Hecht 2006)

II Induced polarization density near focus

For a homogeneous isotropic medium

expressed using Greenrsquos function

Signal generation in coherent NL microscopy

Example for THG

Solve wave equation taking into account NL polarizations created at various locations in the focal volume + coherent superposition in the detector plane

( )int int ΘΦΘpropmax

0

2

0

22(det) sinα π

RREFFddPDetected power

Field near focus phase distribution

Phase anomaly of focused beams (Gouy phase)LG Gouy CR Acad Sciences Paris 1890

Intensity near focus

minusπ (Gouy shift)

0Phase near focus

Cal

culN

Oliv

ier (

LOB

)

Approximation of tightly focused field for modeling nonlinear interactions GaussianGaussian amplitude and reduced axial wave vector

( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus

+minusminusasymp zki

wz

wyxiEzyxE

zrωωω ξ

wr wz 1e radii of the focal ellipse

2221 rwkωξ minusasymp

CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685

For a linearly x-polarized beam near focus

z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction

)(cos 1 ξθ minus=slope prop (ξminus1)

Normalized intensitysquared

SHG from a labeled membrane Gouy shift deflects emission off-axis

θ

k2ω

ξ kω ξ kω

(ξlt1)

Mertz Moreaux et al (2001) Biophys J

Sample and field structure govern emission diagrams

emax asymp 06w0

Exemple of coherence effectobservation of SHG laquo hot spots raquo whenlabeled vesicules are in close proximity

Possible application sub-microm distance

measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ

N number of radiating molecules

Θ parameter describing signal reduction due to the sub-micrometer arrangement of the emitters within the focal volume (typ 10-2 for SHG)

222 INP PEFPEF σprop

Signal level in 2PEFmHG imaging

mmHGmHG INP σ2 Θprop

~ 10-4

(pour SHG)

rarr mHG signal strongly depends on the density of emitters

(SHG THG)

Intrinsic 2PEFSHG signals in cells and tissues

SHG = coherent second-ordernonlinear signal

no centrosymmetrySHG prop (density)2

rArr Dense and orderedmacromolecular structures

Zipfel et al (2003) PNAS 100 7075 (2003)

ldquoLive tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and SHGrdquo

- collagen fibrils

- sarcomeres

- astroglial fibers

- polarized microT bundles

cellulose starch hellip

Myofilaments = endogenous SHG source

Myocytes(Frog heart)

Boulesteix et al (2004) Opt Lett

Plot

niko

vet

al (

2006

) Bio

phys

J

SHG imaging of muscle structures

23 s acquisition

Applications sarcomere contraction measurements study of myogenesis

4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable

Saxitoxin (8 nM) 214 plusmn 002 microm

Control 231 plusmn 002 microm

rArr contracture at rest 7

N = C

R -O

H R

Possible molecularorigin the dense (crystalline) packingof peptide bonds such as amide group

Collagen triple-helix

Fibrils

Fibers

Macromolecular Organization

SHG is obtained from fibrillar collagen

SHGis observed

No SHG

Collagen I II III V XI hellip form fibrils

Collagen IV VIII X hellip form networks

rarr SHG = probe of macromolecular structuration

50 nm

Combined 2PEFSHG imaging of intact tissue

2PEFelastin

lamellae

SHGcollagenfibrils

Unstained carotid artery (Rat)λexc= 860nm

Boulesteix et al (2006) Cytometry A 69

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia

Endothelial cellsInternal elasticlamella

External elasticlamella

Fibroblast

Smoothmuscle cell

( ) ( ) ( )( )244sin0 uuuI prop

Point spread function (PSF) of single lens

( ) ( ) ( ) ( ) PSFdiuvJPvuI =primeprimeprimeprimeprime= int21

0

20 2exp2 ρρρρρ

v equiv r middot NA middot 2π λu equiv z middot NA2 middot 2π nλJ0 = 0th order Bessel functionρrsquo = θ middot n NA (radial coordinate in pupil)P(ρrsquo) = pupil function

Intensity distribution in the focal region of an objective with numerical aperture NA=n sin(α)

( )int=cstu

dvvuI is constant along zNote

NAnr

2sin2λ

αλδ =asymp

αn

αsinnNA =

Numerical aperture (NA)

( ) ( ) 2120 vvJvI prop

Born amp Wolf (1980) Principles of optics Muumlller (2006) Introd to confocal fluorescence microscopy

Wilson amp Sheppard (1984) Theory and Practice of Scanning Optical Microscopy

Simplified expression (within Kirchhoff Debye paraxial and scalar approximations)

lateral width

~λ2NA

axialwidth

~2nλNA2

Assuming P(ρrsquo) = 1

bull Lateral resolution ~λ2NA (related to width of PSF amp OTF)bull No true axial resolutionMissing cone of spatial frequencies the axial position of a thinfluorescent plane (laterally uniform) can not be determined

Standard fluorescence microscope (wide field)

( ) ( ) ( )

( ) ( )xyxPSFzyxO

zdydxdzzyyxxPSFzyxOZYXI

otimes=

primeprimeprimeprimeminusprimeminusprimeminus= intintintinfin

Image formation process

Can also be described in spatial frequency spaceby means of the optical transfer function (OTF)

( ) )( vuPSFFOTF vu =ΩΩ

Muumll

ler

Intro

d to

con

foca

lflu

or m

icro

sc (

2006

)

Lateral cut-off frequency (nλ)

Axi

al c

ut-o

ff fre

quen

cy(n

λ)

laquo missing cone raquo

OTF

Lamp

camera

F

Laser

spatial filter

detector


3D imaging by (linear) confocal microscopy

488

nm

~500

nm

z

2det excexcconf PSFPSFPSFPSF asympsdot=

( ) ( ) ( )( )444sin0 uuuI prop

( )int=cstu

dvvuI peaks for upropz=0

PSFexc PSFconf

OTF wide field OTF confocal

λ=500nmNA=13

n=15log scale

Dia

spro

et a

l Bi

omed

Eng

Onl

ine

(200

6)M

uumllle

r In

trod

to c

onfo

calf

luor

esce

nce

mic

rosc

opy

(200

6)

rarrTrue axial resolution

Laser

spatial filter

detector


3D imaging by (linear) confocal microscopy[+] Optical sectioning

⎩⎨⎧

propΔpropΔ

2NAnzNAr

λλ

[+] Many available fluorophores for biology

αsinnNA =

Wide field Confocal

Resolution

488

nm

~500

nm

z Excitation is not confined rarr photobleaching phototoxicityVery sensitive to scattering of visible light in tissues rarr limited penetration (~100microm)

4det

minuspropsdot= zPSFPSFPSF excconf

αn



scattered photons

ballistic photons


z

N0

N0 exp(-zLs) tissue


Ls



focal fluorescence



ExcitationFluoresc

Bbackground

(surface)

S

z

log (S+B)


z





fluorescence


fluorescence


exci

tatio

n(v

isib

le)

exci

tatio

n(IR

)







Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2


PSF =


02 04 06 08 10 12 14 16 18 2001

1

10

100

Abs

orpt

ion

coef

ficie

nt (c

m-1)

Wavelength (microm)



http

om

lco

gie

du

04 05 06 07 08 09 10 11 120

200

400

600

800

1000

1200

1400

Scat

terin

g co

effic

ient

(cm

-1)

Wavelength (microm)

confocal

2PEF SHGTHG



Ls


θθcos=g




F prop I


fluorescence

exci

tatio

n(v

isib

le)

Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2


( )222 vuIPSF =


2PEF SHG prop I2


fluorescence

SHGsecond-harmonic

generation

THG prop I3

THGthird-harmonic

generation


scattering

ωP ωSex

cita

tion

(IR)

CARS prop IP2 IS




2PEF penetration

100-600 microm



g

e

g

e

Eeg





1-photon 2-photon



22PEF2

12

2PEF21

p IτTσI

τTσ

TτF

TτF ⎟

⎠⎞

⎜⎝⎛=⎟

⎠⎞

⎜⎝⎛==

pI


Averagefluorescence


TiSaph laser





time









wr

wz



202PEF2

10 IσF τ= T

20

0π

P2Iw

=

θω

ω

sinnλ032

=rw

)cos-(1nλ530

z θ=

ω

ωw

wr wz radius at 1e

NA2λ

asymp

2NAλn13

asymp

zr ww22

3

2πV ⎟

⎠⎞

⎜⎝⎛=

PSF (point spread

function)


diffraction theory





sphotons105asymp






CVγIσF 202PEF2

1τ= T



pulsed excitation


700-1200 nm~80 MHz~100 fs

source


LASER

Beam scanning

Laser in

Laser

out

Point Scanning

to microscope

Δt



http

pa

rker

lab

bio

ucie

du

128 micros

sinusoidalx-scan


y-scan

frame sync66 ms

laser

y-galvo

32-sidedpolygon

obj

telescope1

tele

scop

e2



at video rates



cases

http

ce

llser

vm

edy

ale

edu

imag

ing




10 ns

100 fs ltlt 1 ns





impulsion


M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)




104 GM



10-3 - 10-1 GM


100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)









2PEF prop 1τ





PSFz PSFz






( )2ΔΦ





Immunology





laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell



scattered photons

ballistic photons


z

N0

N0 exp(-zLs) tissue


Ls



focal fluorescence



ExcitationFluoresc

Bbackground

(surface)

S

z

log (S+B)


z





fluorescence


fluorescence


exci

tatio

n(v

isib

le)

exci

tatio

n(IR

)







Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2


PSF =


02 04 06 08 10 12 14 16 18 2001

1

10

100

Abs

orpt

ion

coef

ficie

nt (c

m-1)

Wavelength (microm)



http

om

lco

gie

du

04 05 06 07 08 09 10 11 120

200

400

600

800

1000

1200

1400

Scat

terin

g co

effic

ient

(cm

-1)

Wavelength (microm)

confocal

2PEF SHGTHG



Ls


θθcos=g




F prop I


fluorescence

exci

tatio

n(v

isib

le)

Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2


( )222 vuIPSF =


2PEF SHG prop I2


fluorescence

SHGsecond-harmonic

generation

THG prop I3

THGthird-harmonic

generation


scattering

ωP ωSex

cita

tion

(IR)

CARS prop IP2 IS




2PEF penetration

100-600 microm



g

e

g

e

Eeg





1-photon 2-photon



22PEF2

12

2PEF21

p IτTσI

τTσ

TτF

TτF ⎟

⎠⎞

⎜⎝⎛=⎟

⎠⎞

⎜⎝⎛==

pI


Averagefluorescence


TiSaph laser





time









wr

wz



202PEF2

10 IσF τ= T

20

0π

P2Iw

=

θω

ω

sinnλ032

=rw

)cos-(1nλ530

z θ=

ω

ωw

wr wz radius at 1e

NA2λ

asymp

2NAλn13

asymp

zr ww22

3

2πV ⎟

⎠⎞

⎜⎝⎛=

PSF (point spread

function)


diffraction theory





sphotons105asymp






CVγIσF 202PEF2

1τ= T



pulsed excitation


700-1200 nm~80 MHz~100 fs

source


LASER

Beam scanning

Laser in

Laser

out

Point Scanning

to microscope

Δt



http

pa

rker

lab

bio

ucie

du

128 micros

sinusoidalx-scan


y-scan

frame sync66 ms

laser

y-galvo

32-sidedpolygon

obj

telescope1

tele

scop

e2



at video rates



cases

http

ce

llser

vm

edy

ale

edu

imag

ing




10 ns

100 fs ltlt 1 ns





impulsion


M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)




104 GM



10-3 - 10-1 GM


100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)









2PEF prop 1τ





PSFz PSFz






( )2ΔΦ





Immunology





laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell

F prop I


fluorescence

exci

tatio

n(v

isib

le)

Zipf

elamp

Web

b (C

orne

ll U

niv)

linear nonlinear


S prop I prop 1z2


( )222 vuIPSF =


2PEF SHG prop I2


fluorescence

SHGsecond-harmonic

generation

THG prop I3

THGthird-harmonic

generation


scattering

ωP ωSex

cita

tion

(IR)

CARS prop IP2 IS




2PEF penetration

100-600 microm



g

e

g

e

Eeg





1-photon 2-photon



22PEF2

12

2PEF21

p IτTσI

τTσ

TτF

TτF ⎟

⎠⎞

⎜⎝⎛=⎟

⎠⎞

⎜⎝⎛==

pI


Averagefluorescence


TiSaph laser





time









wr

wz



202PEF2

10 IσF τ= T

20

0π

P2Iw

=

θω

ω

sinnλ032

=rw

)cos-(1nλ530

z θ=

ω

ωw

wr wz radius at 1e

NA2λ

asymp

2NAλn13

asymp

zr ww22

3

2πV ⎟

⎠⎞

⎜⎝⎛=

PSF (point spread

function)


diffraction theory





sphotons105asymp






CVγIσF 202PEF2

1τ= T



pulsed excitation


700-1200 nm~80 MHz~100 fs

source


LASER

Beam scanning

Laser in

Laser

out

Point Scanning

to microscope

Δt



http

pa

rker

lab

bio

ucie

du

128 micros

sinusoidalx-scan


y-scan

frame sync66 ms

laser

y-galvo

32-sidedpolygon

obj

telescope1

tele

scop

e2



at video rates



cases

http

ce

llser

vm

edy

ale

edu

imag

ing




10 ns

100 fs ltlt 1 ns





impulsion


M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)




104 GM



10-3 - 10-1 GM


100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)









2PEF prop 1τ





PSFz PSFz






( )2ΔΦ





Immunology





laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell


700-1200 nm~80 MHz~100 fs

source


LASER

Beam scanning

Laser in

Laser

out

Point Scanning

to microscope

Δt



http

pa

rker

lab

bio

ucie

du

128 micros

sinusoidalx-scan


y-scan

frame sync66 ms

laser

y-galvo

32-sidedpolygon

obj

telescope1

tele

scop

e2



at video rates



cases

http

ce

llser

vm

edy

ale

edu

imag

ing




10 ns

100 fs ltlt 1 ns





impulsion


M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)




104 GM



10-3 - 10-1 GM


100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)









2PEF prop 1τ





PSFz PSFz






( )2ΔΦ





Immunology





laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell

M B

lanc

hard

-Des

ce(U

niv

Ren

nes I

)




104 GM



10-3 - 10-1 GM


100-1000 GMZi

pfel

Web

b (C

orne

llU

niv

)









2PEF prop 1τ





PSFz PSFz






( )2ΔΦ





Immunology





laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell

laserTiSaphir

rarr 400 μm

50 microm


50 microm

S C

harp

ak e

t al

PNA

S 98

123

0 (2

001)



[Ca2+]i imaging

S C

harp

ak e

t al

Ner

urop

hysi

olog

ieet

nou

velle

s mic

rosc

opie

s (I

NSE

RM

-Par

is V

)

Nat

Met

h 2

932

200

5

100microm Chl

omel

eon

Prot

ein

(sen

stiv

eto

chl

orid

e io

ns)

mou

se n

eoco

rtex

Hel

mch

enet

al

(200

5) N

at M

eth

2 9

32-9

40




GFP

~ns

700-

1000

nm

350-

600+

nm





100 microm

yolk

vitelline membrane






2PEF λ=920 nm





z




Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell



Bou

lest

eix

et a

l (2

006)

C

ytom

etry

69A

~ns

700-

1000

nm

350-

600+

nm



Mouse ear skin

(fresh)




Rad

osev

itch

Hill

man

et a

l (2

008)

O

pt L

ett

33 2

164


zmax

P0


z


2)(0 expτ

Signal







( )τα )(ln max)(

max TzPLz exs Φ=





PNAS 98 1230 (2001)

( )τα )(ln max)(

max TzPLz exs Φ=



zmax

( )( )221 cos1

NA

NA

α

α

prop

minus=Φ

αNA


This workshellip

Field-of-view (FOV)



extended source








700-1200 nm~80 MHz~100 fs

source


B

A

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell

TiSapph

Reg Amp

z rarr

z rarr

T τ ~ 1times105

T τ ~ 4times107

fmax rTS

fmax rRA


rTS = 80 MHz

rRA = 200 kHz

log F




loss


( )2balscat II +




Zmax reached when


balfocusofout

balscat III 22



( )τα )(ln max)(

max TzPLz exs Φ=


zmax












Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell




Φ-Φ

Φ




reference





( )2ΔΦ


LOB

-X



Reference

Sample


CCD

( ) ( ) ⎟⎠⎞⎜

⎝⎛ ΔΔminus++=



νΔprop 1




obj



δϕ

τΔ


(2006) PNAS 103 17137


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell


2PEF SHG prop I2

2PEF SHG

3PEF THG prop I3

excitation(IR)

F prop I


3P excitation

3PEF

CARS prop IP2 IS

THG CARS





E(ω) P(ω)

E(ω)

P(ω)

P(3ω)

SHG

THG

P(2ω)





avec P(n)(nω) =





2PEFsignal

SHGsignal

Osc

illato

r

THGsignal


(SHG)










ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell







ϕ1 = 0

ϕ2 = 0

ϕ1 = 0

ϕ2 = π

SHG

SHG

Excitation2PEF


depend on symmetry


membrane imaging


Mor

eaux

Mer

tz e

t al

Bio

phys

J 8

0 1

568

(200

1)

SHG 2PEF

Wavelength (nm)400 450 500 550 600 650

Pow

er (a

u)

00

05

10

430 435 440 445 450

SH

G P

ower

(au

)

00

05

10

fluorescence

SHG



zLc










such as THG




kω = 2π nω λ







Example for THG



0

2

0

22(det) sinα π






0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell





0Phase near focus

Cal

culN

Oliv

ier (

LOB

)


( ) xexp 2

2

2

22

⎟⎟⎠

⎞⎜⎜⎝

⎛minusminus


wz

wyxiEzyxE

zrωωω ξ



CfM

orea

ux S

andr

e M

ertz

(200

0) J

OSA

B 1

7 1

685


z (nm)

z

ωξ k

2 Iξ

ωω πω nk 2=

880nm NA=09

ω 2ω

0

-πPha

se a

nom

aly

Axial direction




θ

k2ω

ξ kω ξ kω

(ξlt1)



emax asymp 06w0



measurement


⎟⎟⎠

⎞⎜⎜⎝

⎛Θasymp

PEF

mHG

PEF

mHG NPP

22 σσ






~ 10-4

(pour SHG)


(SHG THG)





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell





Zipfel et al (2003) PNAS 100 7075 (2003)


- collagen fibrils

- sarcomeres

- astroglial fibers






Plot

niko

vet

al (

2006

) Bio

phys

J


23 s acquisition


4 6 80

20

40

60

SHG

sig

nal (

phot

ons)

Length (microm)

sarcomere

constant variable




N = C

R -O

H R



Fibrils

Fibers



SHGis observed

No SHG




50 nm


2PEFelastin

lamellae

SHGcollagenfibrils



yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

yz

x

x

y

x

y

Lumen

Media

Intima

Adventitia



Fibroblast

Smoothmuscle cell

Focal field distribution - RESEAU FEMTOreseau-femto.cnrs.fr/IMG/pdf/Houches2009_cours15.pdf ·...

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Transcript of Focal field distribution - RESEAU FEMTOreseau-femto.cnrs.fr/IMG/pdf/Houches2009_cours15.pdf ·...