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EmployingEmploying highhigh--resolutionresolution LCDsLCDs forfornovelnovel methodsmethods in in biomedicalbiomedical opticsoptics

Monika RITSCH-MARTE

Division for Biomedical PhysicsMedical University of Innsbruck

generatinggenerating tailoredtailoredultrasoundultrasound withwith laserslasers

optoopto--acousticacousticholographyholography

newnew methodsmethods in in microscopymicroscopy

spiral spiral phasephasecontrastcontrast

miniature LC displays, pixels individually addressable,Up to about 2000 x 1200 pixels, 10 µm per pixel)

designingdesigningmicromicro--toolstools

of lightof light

opticalopticalmicromanipulationmicromanipulation

SpatialSpatial Light Modulator (SLM)Light Modulator (SLM)

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→→ microscopymicroscopy withwith FourierFourier filtersfilters usingusing SLMsSLMs

→→ optoacoustic optoacoustic holographyholography

→→ otherother activitiesactivities in in thethe lab (lab (shortshort))

OverviewOverview::

→→ holographicholographic micromicro--toolstools of lightof light

Light Light carriescarries::

momentummomentum

energyenergy

angularangular momentummomentum

⇒⇒ heatheat oror cutcut

⇒⇒ rotaterotate oror twist twist

whichwhich givesgives youyou a handle to...a handle to...

⇒⇒ movemove oror traptrap

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RadiationRadiation pressurepressure::

No No commentcomment on on thisthis !!

RadiationRadiation pressurepressure::

notnot thisthis........

butbut contributescontributes to to thisthis........

and and latelylately doesdoes also also thisthis........

and and certainlycertainly notnot thisthis........

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MechanicalMechanical light light forcesforces::

large large powerpower ((laserlaser light)light)

smallsmall objectsobjects ((micronmicron--sizedsized to to atomicatomic scalescale))

exceptionexception: : muchmuch time...time...

sunsun sailssails ((plannedplanned))

net force acting on sphere

bright raydim ray

dielectric sphere

reflectedlight

refracted light

reaction force

intensity gradient of incident collimated laser light

total momentumbefore sphere

momentumof light

equals

total momentumbehind sphere

changed mom-entum of lightcauses a change in sphere‘s momentum

OriginOrigin of of mechanicalmechanical forcesforces in a in a laserlaser focusfocus ((opticaloptical tweezerstweezers):):

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single-modefiber

high numericalobjective

cover slip

Whole setupHybrid trap

Ytterbium fiber laser

Pmax = 5W, λ = 1064 nmclean TEM00 mode

Experimental Experimental setset--upup::

stretching of red blood cells

demonstration of the versatility of our trap

length scale : beads with d = 10 μm magnification: 200 x

magnification: 1000 x

MoviesMovies::

micromicro--abacusabacus !!

http://http://medphysik.uibk.ac.atmedphysik.uibk.ac.at

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WhatWhat biologicalbiological systemssystemscancan bebe handledhandled??

Glasgow, Brisbane, Szeged

...rotatinge.g. Cell organelles, micro-mills

...placing and bringing into contact

particles or inducing controlledcollisions (e.g. virus and

antibodies)

S. Chu,Stanford University

...stretchinge.g. DNA – molecules

S.M. Block,Rowland Institute of

Science,Princeton

...holdinge.g. motor molecules (kinesin, dynein) against motion along

microtubuli

W.D. Phillips

at

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University of California,

Irvine

...selectively destroyinge.g. fluorescence-marked organelles

and…...fusinge.g. T-cells (local activation)

Beckmann Laser Institute and Clinic

...perforatingfor inserting things into cells

(„opto-poration“)

M.W. Berns...cuttinge.g. chromosomes

studyingstudying……

OurOur biologicalbiological applicationsapplications

…… livingliving lunglung cellscells

…… phagocytoticphagocytotic retinalretinal cellscells

plannedplanned: : selectiveselective trappingtrapping ofoftumortumor cellscells in in bloodblood oror liquorliquor

ProfilProfil (30), (30), JuliJuli 20032003

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MovieMovie:

Stretching Stretching pulmonarypulmonary surfactantsurfactant

alveolar alveolar typetype II II cellscells releasereleasepulmonarypulmonarysurfactantsurfactant

W. Singer et al.: Biophys. J. 84, 1344-1351, 2003

Calcein and FM 1-43

HolographicHolographic OpticalOptical TweezersTweezers

latest developement

D. Grier (Chicago), K. Dholakia (St. Andrews)and other groups

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SLM(transmissive)

f1

L2

L1

f1

f2

f2

Collimated laserbeam

SLM(reflective)

Objective

Microscope focal plane

Rear objectiveaperture

SetSet--upup withwith SLM:SLM:

SLM

[ ]πϕ 2,mod),( ykyx y ⋅=[ ]πϕ 2,mod),( xkyx x ⋅= ⎥⎦

⎤⎢⎣

⎡+

⋅= π

λπϕ 2),(mod),( 22 yx

fyx

DynamicalDynamical 3D 3D beambeam controlcontrol::

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• Laguerre-Gauss-Modes (TEMpl*) with index p = 0

l

wr

wrrI

2

2

2 2exp~),( ⎟⎟⎠

⎞⎜⎜⎝

⎛ ⋅⋅⎥⎦

⎤⎢⎣

⎡−ϕ

….“topological charge“

• each photon carries an (orbital) angularmomentum

hr

lL =pr

pr

l

5=ll

DoughnutDoughnut modesmodes & orbital & orbital angularangular momentummomentum::

• orbital angular momentum as transversecomponent of photon momentum

)sin(

)sin(

θ

θ

∝⇒

=

trans

trans

p

kkr

rr

kr

transkr

θ

propagationdirection

DoughnutDoughnut modesmodes & orbital & orbital angularangular momentummomentum::

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generate doughnut by phase front control

[ ]πθθϕ 2,mod),( Lr ⋅=

helical phase

DoughnutDoughnut modesmodes as as phasephase singularitiessingularities::

5=l

30=l

up to l = 20010=l

Transfer Transfer angularangular momentummomentum ((induceinduce rotation):rotation):

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example: micromicro--pumppumpsstatattionionaarryy(!) (!) llight ight iinntteennssiittyy;; oorrbbiittaall aanngulgulaarr momemomenntumtum

AssembleAssemble moremore complexcomplex opticaloptical micromicro--tooltoolss::

advantageadvantage: : imagingimaging of of severalseveral adjacentadjacenthologramsholograms intointo microscopemicroscope apertureaperture

f f

Rear objective aperture

SLM

Incident laser beam

zeroth order (reflected beam)

Holo #1

Holo #2

ModifiedModified setset--upup ((offoff--axisaxis Fresnel Fresnel setset--upup): ):

A. Jesacher et al: Diffractive optical tweezers in the Fresnel regime, Optics Express 12, 2243-2250, 2004

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screen display corresponding opticaltraps (in microscopeobject plane)

ModifiedModified setset--upup for for severalseveral adjacentadjacent hologramsholograms: :

screen display trap position

further advantage: real-time movement of laser traps by "mouse-dragging" the corresponding hologramwindows across the display :

DirectDirect dynamicaldynamical controlcontrol: :

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Real time Real time steeringsteering: :

..\Movies\Fresnelvideo.swf

spot doughnut l=5 sup. doughnuts l=+5

spot spot, defocussed

spot

Independent Independent controlcontrol of of indepenentindepenent hologramsholograms: :

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spot

spot,defocussed

spot

sup. doughnutmodes l=5

doughnutl=5

spot

79µm x 59µm

……and and resultingresulting complexcomplex multiple traps: multiple traps:

…… and and trappedtrapped particlesparticles

5µm7µm3µm

7µm7µm 7µm

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InteractiveInteractive microscopemicroscope sortingsorting devicedevice

• dynamical 3-dimensional steering

• numerous traps simultaneously

• arbitrary light fields programmable(including optical fields with angularmomentum!)

Advantages of Advantages of holographicholographic approachapproach::

WhatWhat for ? for ?

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15 µm bead

experimental trapping condition: DDonut > 0.55 DBead

A. Jesacher, S. Fürhapter, S. Bernet and M. Ritsch-Marte, "Size-selctivetrapping with optical 'cogwheel'-modes", Opt. Express 12, 4129-4135, (2004)

SizeSize--selectiveselective trappingtrapping

like „mesh-size in fishing net“ fishing for micro-metastases?

0 2 4 6 8 10 12 14 160

1

2

3

4

5

6

7

8

9

bead diameter in μm

max

. dounut

dia

met

er

SizeSize--selectiveselective trappingtrapping withwith excentricexcentric dounutsdounuts

bead „traffic“

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• back aperture plane imaged onto SLM• SLM acts as phase filter• the 1st diffraction order is the filtered image

F

F= reflective phase filter

superposeddiffraction grating

0th1st

UseUse thethe SLM as image SLM as image filterfilter::

filter functionsgrayscales phase shifts

imaging results

brightfield darkfield phase contrast

mica fragment in immersion oil, 20x magnification, field of view: Ø = 350µm

Emulation of Emulation of variousvarious methodsmethods byby thethe SLMSLM

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• used for creation of optical vortices(„doughnut modes“)

• here: used for imaging!

+ =

on-axis filter:„pure“ vortex blazed grating

off-axis filter:grating with branch cut

Phase Phase vortexvortex filterfilter

phaseobject

amplitudeobject

brightfield spiral contrast

„Spiral phase contrast imaging in microscopy“S.Fürhapter, A.Jesacher, S.Bernet, and M.Ritsch-Marte Opt. Expr. 13 (3) (2005)

For For thinthin objectsobjects: : isotropicisotropic edgeedge enhancementenhancement

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the „problem“ of the central pixel:• finite pixel size ⇒ broken symmetry

„Shadow Effects in Spiral Phase Contrast Microscopy“A.Jesacher, S.Fürhapter, S.Bernet, and M.Ritsch-Marte PRL (94), 2005

ShadowShadow effectseffects

filter center replaced byarea of constant phase shift

pseudo-relief emerges(similar to Nomarski)

• modified spiral filter amplifies anisotropy

)exp( 1ϕi×

)exp( 3ϕi×

)exp( 2ϕi× +

FurtherFurther contrastcontrast enhancementenhancement

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phase contrast filter & coherent illuminationinterferometer!

zeroth order phase shifted (π)

higher orders pass

temporal and spatial coherentillumination deformation in

glue-strip

A.Y.M.Ng, C.W.See, and M.G.Somekh J. of Microscopy 214 (2003)

For For thickthick objectsobjects: : selfself--referencedreferenced interferometryinterferometry

What will happen?

?

• vortex filter also leads to interferometric patterns• setup identical to „edge enhancment“ assembly

SelfSelf--referencedreferenced interferometry interferometry withwith a a vortexvortex filterfilter

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„classical“interferogram

spiralinterferogram

ObjectObject of of varyingvarying ((opticaloptical) ) thicknessthickness::

⇒ one-image-demodulation

A) Spiral interferogram

OneOne--imageimage--demodulationdemodulation

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αtang

µmµm

h

B) Calculate center-lineC) Add hight-information: tan~αhD) Interpolation final form

OneOne--imageimage--demodulationdemodulation

ComparisonComparison

emulateemulate severalseveral contrastcontrast techniquestechniques withwith SLMSLM((oiloil dropletsdroplets in in waterwater))

brightfieldbrightfield darkfielddarkfield

spiralspiral interferenceinterferencecontrastcontrast

phasephase contrastcontrast interferenceinterferencecontrastcontrast

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Remote ultrasound diagnostics ?

Laser

EO polarizationrotator (~1MHz)

LCDscreen

BeamExpander

Projectionzoom optics

Polarizer

ProjectedImage Aquarium

Absorbingsurface

Ultrasoundsensor

Optoacoustic holography:

temporal modulation

optoacoustics u.s. detection

A. Meyer et al., J. Appl. Phys. 96, 5886-5891, 2004S. Gspan et al., Acoust. Soc. Am. 115, 1142-1146, 2004

spatialmodulation: holography

LCD now acts as polarization shifter, rather than phase shifter

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Complete spatio-temporal control

of of laserlaser--inducedinduced ultrasoundultrasound amplitudeamplitude::

spatialspatial ((fieldfield patternspatterns):):

doughnutdoughnut

Bessel Bessel beambeam

Complete spatio-temporal control

of of laserlaser--inducedinduced ultrasoundultrasound amplitudeamplitude::

spatialspatial ((fieldfield patternspatterns):): temporaltemporal ((beambeam steeringsteering):):

doughnutdoughnut

Bessel Bessel beambeam

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CARS-imaging

momentum:

Conservation of

kL kL

energy:

V

ωL

ωL

Ei

nonlinearnonlinear opticsoptics for for functionalfunctionalmicroscopymicroscopy::

⇒⇒ imagingimaging of of distributiondistribution of of selectableselectablebiomoleculesbiomolecules((mapmap of of chemicalchemicalconcentrationsconcentrations))

sample area

condenser

pump beam from fiber

BSstokes beamfrom fiber

BS

filter

CARS signal todetector

Heinrich et al., Appl. Phys. Lett. 84, 816 (2004)

Coherent Anti-Stokes-Raman Scattering

dropletsdroplets of of linoleiclinoleic acidacidfromfrom splitsplit sunflowersunflower seedseed

resonantresonant signalsignal@ 2875 @ 2875 wavenumberswavenumbers

detuneddetuned byby175 175 wavenumberswavenumbers

200 200 pulsespulses

single 3 ns pulse !

first non-scanning

CARS-images

Latest results

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biological sample:oiloil dropletsdroplets fromfrom a a sunflowersunflower seedseed in Din D22OO

z=2µm

Tuned to linoleic acid at 2870cm-1

50µm

Demonstration of optical sectioning

z=4µm

Tuned to linoleic acid at 2870cm-1

50µm

biological sample:oiloil dropletsdroplets fromfrom a a sunflowersunflower seedseed in Din D22OO

Demonstration of optical sectioning

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z=6µm

Tuned to linoleic acid at 2870cm-1

50µm

biological sample:oiloil dropletsdroplets fromfrom a a sunflowersunflower seedseed in Din D22OO

Demonstration of optical sectioning

TheThe BiomedicalBiomedical PhysicsPhysics Team:Team:

Stefan BERNET, MRMTweezersWolfgang SINGER (now UQ, Brisbane)Severin FÜRHAPTERAlexander JESACHERChristian MAURER

CARSChristoph HEINRICHClara MEUSBURGER

OptoakustikStefan GSPANMatthias ZANGERL

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Thank you for your attention!

http://medphysik.uibk.ac.at