From cells to organs with lasers

From cells to organs with lasers Halina Rubinsztein-Dunlop M. J. Friese, Friese, G. Knö Knöner, ner, T. Nieminen, Nieminen, S. Parkin , J. Ross , W. Singer , C. Talbot, N.R. Heckenberg Centre for Biophotonics and Laser Science, School of Physical Sciences University of Queensland, Brisbane, Australia

Biophotonics in Australia, February 2006

Biophotonics z

Biophotonics is the science of generating and harnessing light (photons) to image, detect and manipulate biological materials

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At the interface of physical, biological and medical sciences z The Institute for Lasers, Photonics and Biophotonics, University at Buffalo z NSF Centre for Biophotonics, Science and Technology, UC Davis z Bio-X, Stanford z Division of Biophysics and Imaging, Ontario Cancer Institute 2

z Beckman Laser Institute, Irvine

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OUR EXPERTISE Fundamental Laser Science

Laser Manipulation Eg. Laser Tweezers

Functional Biopolymer Spectroscopy Eg. Melanin spectroscopy Light activated processes

Biomedical & Clinical Applications

Biomedical Imaging

Eg. Lasers in Dentistry

Plus expertise in tissue engineering…….,through our Affiliates

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Examples of Biophotonics projects

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Hyperpolarised Gases for MRI Imaging

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Light activated processes

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Laser micromanipulation and examples of applications 4

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Hyperpolarized Gas MR Imaging Dynamic images of the human lung during inhalation and expiration of 3He The use of hyperpolarized 3He and 129Xe for imaging air spaces and certain tissues in humans. Traditional MRI techniques derive images from hydrogen. In places such as the lungs where hydrogen is not so abundant, imaging is difficult using these techniques.

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Hyperpolarized Gas MR Imaging z 3He

and 129Xe polarisation processes are both based on the spin exchange optical pumping technique Time necessary to hyperpolarize the noble gas as well as the amount of gas produced and the process used to collect it is different. 6

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Spin Exchange Optical Pumping (SEOP)

• SEOP method can be used for both noble gases • Same Rb absorption line → same laser λ = 794.6 nm • Otherwise, process is quite different for 3He and 129Xe 7

Variables for SEOP •

Gas Pressure -

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Affects the Rb absorption line Changes laser requirements

Temperature - Rb number density

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Gas mixture -

Determined by collisional cross-section

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Time in the Spin Exchange region

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T1 of the vessel Magnetic field, photon quality 8

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HP Gas Polarizer z

Pyrex cell containing 98% 3He, 2% N2, Rb

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laser

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Laser frequency narrowing apparatus

Helmholtz Coils create uniform magnetic field

Laser polarization of Rb

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collisional mixing z

Oven

5P½

mj=½

mj=-½

σ+ 5S½ z

Collisional transfer to He

1/3

2/3

Radiative decays

r B

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Laser Frequency Narrowing – why bother? Maximize 3He Polarization Rate Maximize spin exchange collisions with Rb Density of species Maximize Rb polarization Quality of the laser light

Laser power

•Polarization

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Absolute Power

•Spectrum

•Spectral

power 10

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Improving Spectral Power Density

Free running laser spectrum spans 3-5 nm Rb absorption ~0.5 nm Power (W)

Putting all the laser power in a narrow band would increase spectral power density and thus Rb polarization rate λ (nm)

Centre λ (Rb resonance, 794.7 nm) 11

3He z z

: progress

Produced HP 3He Achieved ~15% polarization on first attempt

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Images z

The cell

15 % polarisation

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Imaging

balloon

rat’s lung

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Light modified fluorophore transporters z

The interaction of light with some organisms can drive the transport of solutes across the cellular membrane.

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The transport of these solutes may be measured by z z z z

patch clamping methods by optical microscopy epifluorescent bright field microscopy imaging of calcium ions using two photon excitation at an electronic resonance and monitoring the fluorescence emission of the ion. 15

A hybrid of single-photon and multi-photon imaging z

Decoupling of the membrane transporter and the excitation of fluorescence from the fluorophore.

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The system allows simultaneous and independent activation of the light induced membrane transport and imaging of the fluorophore. Giardia

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Uptake of the fluorophore

Justin Ross 17

Confocal Microscopy, light-activated pump action of cells

More from the Confocal

5 µm Justin Ross 18

The localisation of Rhodamine 123 in Geardia trophozoites

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A hybrid of single-photon and multi-photon imaging fs pulse Exc ita tio n Light

Em ission Fiters

Fluoresc e nc e Em ission Lig ht

PMT

Ac tiva tion Light

Confoca l Sc anner Unit

Dichroic Mirror

Ti:Sapph Nd:YVG

Steering Mirrors

Objec tive lens Sa mple

Stage

Condenser lens

Hg Lamp Ac tivation Filter

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Spectral Response of light induced uptake of Rh123 by Geardia S pe ctra l Re s pons e of G. duode nalis Upta ke of Rhoda mine 123 - S tra in WB1B 90 Ga us s ian Fit of Data Rhoda mine 123 Emis s ion Rhoda mine 123 Exc ita tion

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Change in Fluores cence (µM/µJ )

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0 350

400

450

500 Wa ve le ngth (nm)

550

600

650

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Influx and Efflux

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Justin Ross

Influx and Efflux Influx and Efflux of Dye from Giardia 1.5 Dye Concentration in Cell 35

Rate of Dye Influx

1.3

Dye Concentration (uM)

0.9 31

0.7 0.5

29 0.3 27

0.1

Concentration Change (uM/s)

1.1

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-0.1 25 -0.3 23

-0.5 0

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100

150

200

250

300

350

400

Time (s)

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Optical micromanipulation - using light to make: • Laser tweezers • Laser scissors • Laser catapult • Laser screwdriver • Optical spanner (wrench) (Simpson et al, Opt Lett,1997) 23

optical torque wrench measures torque as it is applied

Typical Laser Tweezers setup

Can trap and manipulate high index particles in water Typical: 1µm radius, 1pN/mW

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Optical Trapping z

Ray optics model

• Force on a point dipole

Force results from exchange of momentum with beam

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Optical Force

nP F = Qopt c Fdrag = 6πηavmax Qtrans = 6πηvmax a

n - refractive of the trapped object, P - incident laser power Qopt - efficiency factor trap against viscous drag or movement

c nP 26

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2 mm polystyrene spheres

Gregor Knöner

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Trapping and manipulation of 2 µm polystyrene spheres

Gregor Knöner

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Trapping of a macrophage

Gregor Knöner

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Studies of the dynamic structure of individual nucleosomes (DNA organised into nucleosomes)

Stretching nucleosomal arrays with feedback-enhanced optical trap.

Brent D. Brower-Toland et al. PNAS, 2002

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Rotating Tweezers Creating torque and measuring it as it is applied

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Asymmetric particles in laser beam Special laser beams Multiple traps

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Optical Angular Momentum I Helical wavefront (‘orbital’ a.m.) •‘optical vortex’ •‘singular beam’ •‘Gauss-Laguerre’ GLpl

Gaussian beam AM = integer x ћ per photon

l=1

Phase singularity on axis, dark spot generated by laser, or phase plate or hologram 32

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Optical Angular Momentum – ‘orbital’ Helical wavefront

p pφ

l=1

r L= r x p

l=3

Associated with spatial distribution

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Laser beam with orbital a.m.

Helical wavefronts

Absorbing particle He et al., PRL (1995)

l=3

Not useful because of heating

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Optical Angular Momentum II Spin

Circularly polarised light…….ћ per photon (spin)

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Transfer of AM to a waveplate • CaCO3 particles in H2O are 3-D trapped in polarised light • They either rotate continuously or align to a particular orientation

Plane polarised laser beam λ/4 or λ/2 plate high N.A. lens Trapped calcite crystal

• In linear light, their orientation is controllable • In elliptical light, their rotation frequency is controllable. Friese et al., Nature 1998

Optical torque

Drag torque 36

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Transfer of angular momentum of light alignment

Optical axis of crystal aligns to electric field vector 37

Calcite crystal rotates in circularly polarised light

Friese et al, Nature 394, 348-350, 1998

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All optically driven micromachine elements

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Friese et al, Appl. Phys. Lett. 78, 1, 2001

Micro-rheology Circularly polarised light

h per photon

λ/4 waveplate zero

Try to maintain circular symmetry to avoid orbital40A.M.

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Making better probes Spherical CaCO3 Crystals

optical image

SEM image

Better probe particles - smooth rotation - 3D trapping

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Alexis Bishop et al, PRL 2004

Theory z z z z z z

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Where α = shear stress

µ = of stress to strain Viscosity => Viscosity – ratio γ = shear rate γ

Where ∆σ = change in polarisation Applied Torque => ατ R = or = laser power ω Where αa=Pstress shear stress We measure viscosity the µ = by applying ω = optical to frequency γµ== viscosity shear ofrate liquid fluid a => sphere circularly τ Dγ=rotated 8πµa 3Ω byWhere Dragusing Torque a = sphere’s radius The response thea fluid polarised light of with known torque Ω = frequency of rotation

∆σP

z Viscous drag =torque z Applied Torque Drag

Torque Where ∆σ = change in 3σP z viscosity of the∆ surrounding liquidpolarisation =8 a Ω

τD

τ πµ R = ω∆liquid σP Where µ = viscosity of surrounding µ = 3 a = sphere’s radius 8 π a Ωω Ω = frequency of rotation

Sur rounding Liquid

“ This sphere’s

c reating P = laser power quite a stir !” ω = optical frequency 42

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CaCO3 sphere spinning inside an artificial liposome (15µm dia)

10µm

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Alexis Bishop et al, PRL 2004

Four vaterite spheres rotating inside an artificial liposome

10µm

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Laser tweezers, scissors and wrench

Gregor Knöner

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Fluid-Coupled MechanoTransduction Flow-Coupled Shear Stress

Elliot Botvinick, Gregor Knöner et al

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Optically driven micromachines

Simon Parkin 47

Optically driven micromachines

Gregor Knöner

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Two-photon photopolimerization

Two-photon initiated photopolymerization for threedimensional fabrication

SEM image of a micro-bull sculpture. 49

Kawata, Japan 2001

Exposure Apparatus lamp

tsunami

condenser 3d piezo stage objective

millennia

attenuator

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imaging optics shutter

λ 2 λ 4 M1

Nd:YAG fiber laser

CCD

subsequent production and trapping Ti:Sapphire laser, 1 W, 80fs, 780nm for production of structure

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Two-photon photopolimerization at UQ

15 mm

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21 mm

Julien Higuet

Microhologram for Lab-on-Chip z

on axis phase hologram Æ transfer of angular momentum to linear polarized Gaussian beam

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SEM of phase hologram

8 mm

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Angular momentum transfer • polystyrene spheres, d = 2.1 µm • incident beam: Gaussian, linear polarized

linear polarized beam no angular momentum

λ/4 waveplate

circularly polarized spin angular momentum Æ transfer to anisotropic particle

TEM00, linear pol. no angular momentum

phase hologram

LG04, helical wavefront orbital angular momentum 54 Æ transfer to anisotropic particle

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Setup for Microhologram Rotation

microhologram

rotating beads

collection lens 55

Rotating beads with Microhologram • incident laser beam: linearly polarized, Gaussian

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UQ Laser tweezers team •Studnets: Gregor Knöner Simon Parkin Julien Higuet Justin Ross Cavin Talbot

•Staff: A/Prof. N. Heckenberg Dr. MArlies Friese Dr. Timo Nieminen Dr. Wolfgang Singer HRD

•Former Members: Martin Dienstbier Dr. Alexis Bishop

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Thank you for your attention !!!

Simon Parkin

Rotation using orbital AM

LG02 doughnut beam

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