Monika RITSCH-MARTE. Division for Biomedical Physics Medical University of Innsbruck

Transcription

1 Employing high-resolution LCDs for novel methods in biomedical optics Monika RITSCH-MARTE Division for Biomedical Physics Medical University of Innsbruck Spatial Light Modulator (SLM) miniature LC displays, pixels individually addressable, Up to about 2000 x 1200 pixels, 10 µm per pixel) optical micromanipulation designing micro-tools of light generating tailored ultrasound with lasers opto-acoustic holography spiral phase contrast new methods in microscopy 1

2 Overview: holographic micro-tools of light microscopy with Fourier filters using SLMs optoacoustic holography other activities in the lab (short( short) Light carries: which gives you a handle to... energy heat or cut momentum move or trap angular momentum rotate or twist 2

3 Radiation pressure: No comment on this! Radiation pressure: and certainly not this... not this... but contributes to this... and lately does also this... 3

4 Mechanical light forces: large power (laser light) small objects (micron-sized to atomic scale) exception: much time... sun sails (planned) Origin of mechanical forces in a laser focus (optical tweezers): dim ray reflected light intensity gradient of incident collimated laser light bright ray total momentum before sphere momentum of light equals reaction force refracted light dielectric sphere total momentum behind sphere changed momentum of light causes a change in sphere s momentum net force acting on sphere 4

5 Experimental set-up up: Hybrid trap Whole setup Ytterbium fiber laser single-mode fiber cover slip high numerical objective P max = 5W, λ = 1064 nm clean TEM 00 mode Movies: demonstration of the versatility of our trap stretching of red blood cells length scale : beads with d = 10 μm magnification: 200 x micro-abacus! magnification: 1000 x 5

6 What biological systems can be handled?...holding e.g. motor molecules (kinesin, dynein) against motion along microtubuli S.M. Block, Rowland Institute of Science, Princeton...stretching e.g. DNA molecules S. Chu, Stanford University...placing and bringing into contact particles or inducing controlled collisions (e.g. virus and antibodies) W.D. Phillips at...rotating e.g. Cell organelles, micro-mills Glasgow, Brisbane, Szeged 6

7 ...cutting e.g. chromosomes M.W. Berns...perforating for inserting things into cells ( opto-poration ) Beckmann Laser Institute and Clinic...fusing e.g. T-cells (local activation) and...selectively destroying e.g. fluorescence-marked organelles University of California, Irvine Our biological applications Profil (30), Juli 2003 studying living lung cells phagocytotic retinal cells planned: selective trapping of tumor cells in blood or liquor 7

8 Stretching pulmonary surfactant Calcein and FM 1-43 alveolar type II cells release pulmonary surfactant Movie: W. Singer et al.: Biophys. J. 84, , 2003 Holographic Optical Tweezers latest developement D. Grier (Chicago), K. Dholakia (St. Andrews) and other groups 8

9 Set-up with SLM: Microscope focal plane Objective f 1 Rear objective aperture L1 f 1 f 2 L2 Collimated laser beam SLM (reflective) f 2 SLM (transmissive) Dynamical 3D beam control: [ k, ] ϕ( x, y) = mod[ k π 2 2 x x, 2π ] ϕ( x, y) = mod y y 2π ϕ( x, y) = mod ( x + y ), 2π λ f SLM 9

10 Doughnut modes & orbital angular momentum: Laguerre-Gauss-Modes (TEM pl* ) with index p = 0 r I( r, ϕ) ~ exp 2 w l. topological charge 2 r 2 l w each photon carries an (orbital) angular momentum p r p r 2l l =5 r L =lh Doughnut modes & orbital angular momentum: orbital angular momentum as transverse component of photon momentum propagation direction k r trans k r θ r r ktrans = k sin( θ) r p sin( θ) trans 10

11 Doughnut modes as phase singularities: generate doughnut by phase front control [ θ, π ] ϕ( r, θ) = mod L 2 l = 30 helical phase l = 10 5 up to l = 200 Transfer angular momentum (induce rotation): 11

12 Assemble more complex optical micro-tool tools: example: micro-pump stationary(!) light intensity; orbital angular momentum Modified set-up (off-axis Fresnel set-up up): advantage: imaging of several adjacent holograms into microscope aperture zeroth order (reflected beam) Holo #1 f f Rear objective aperture Holo #2 SLM Incident laser beam A. Jesacher et al: Diffractive optical tweezers in the Fresnel regime, Optics Express 12, ,

13 Modified set-up for several adjacent holograms: screen display corresponding optical traps (in microscope object plane) Direct dynamical control: further advantage: real-time movement of laser traps by "mouse-dragging" the corresponding hologram windowsacrossthedisplay: screen display trap position 13

14 Real time steering:..\movies\fresnelvideo.swf Independent control of indepenent holograms: spot doughnut l=5 sup. doughnuts l=+5 spot spot, defocussed spot 14

15 and resulting complex multiple traps: spot doughnut l=5 sup. doughnut modes l=5 spot spot, defocussed spot 79µm x 59µm and trapped particles 7µm 7µm 7µm 3µm 7µm 5µm 15

16 Interactive microscope sorting device Advantages of holographic approach: dynamical 3-dimensional steering numerous traps simultaneously arbitrary light fields programmable (including optical fields with angular momentum!) What for? 16

17 Size-selective trapping 9 8 max. dounut diameter µm bead bead diameter in μm experimental trapping condition: D Donut > 0.55 D Bead like mesh-size in fishing net fishing for micro-metastases? A. Jesacher, S. Fürhapter, S. Bernet and M. Ritsch-Marte, "Size-selctive trapping with optical 'cogwheel'-modes", Opt. Express 12, , (2004) Size-selective trapping with excentric dounuts bead traffic 17

18 Use the SLM as image filter: back aperture plane imaged onto SLM SLM acts as phase filter the 1st diffraction order is the filtered image F 0th 1st F superposed diffraction grating = reflective phase filter Emulation of various methods by the SLM mica fragment in immersion oil, 20x magnification, field of view: Ø = 350µm brightfield darkfield phase contrast filter functions grayscales phase shifts imaging results 18

19 Phase vortex filter on-axis filter: pure vortex blazed grating off-axis filter: grating with branch cut + = used for creation of optical vortices ( doughnut modes ) here: used for imaging! For thin objects: isotropic edge enhancement brightfield spiral contrast phase object amplitude object Spiral phase contrast imaging in microscopy S.Fürhapter, A.Jesacher, S.Bernet, and M.Ritsch-Marte Opt. Expr. 13 (3) (2005) 19

20 Shadow effects the problem of the central pixel: finite pixel size broken symmetry modified spiral filter amplifies anisotropy filter center replaced by area of constant phase shift pseudo-relief emerges (similar to Nomarski) Shadow Effects in Spiral Phase Contrast Microscopy A.Jesacher, S.Fürhapter, S.Bernet, and M.Ritsch-Marte PRL (94), 2005 Further contrast enhancement exp( iϕ 1 ) exp( iϕ ) 2 + exp( iϕ ) 3 20

21 For thick objects: self-referenced interferometry phase contrast filter & coherent illumination interferometer! higher orders pass temporal and spatial coherent illumination zeroth order phase shifted (π) deformation in glue-strip A.Y.M.Ng, C.W.See, and M.G.Somekh J. of Microscopy 214 (2003) Self-referenced interferometry with a vortex filter vortex filter also leads to interferometric patterns setup identical to edge enhancment assembly? What will happen? 21

22 Object of varying (optical) thickness: classical interferogram spiral interferogram one-image-demodulation One-image image-demodulation A) Spiral interferogram 22

23 One-image image-demodulation h α tang µm µm h ~ α B) C) D) Calculate Add Interpolation hight-information: center-line final form tan Comparison emulate several contrast techniques with SLM (oil droplets in water) brightfield darkfield phase contrast interference contrast spiral interference contrast 23

24 Remote ultrasound diagnostics? Optoacoustic holography: LCD now acts as polarization shifter, rather than phase shifter temporal modulation Laser spatial modulation: holography LCD screen Beam Expander Projected Image Projection zoom optics optoacoustics u.s. detection Aquarium EO polarization rotator (~1MHz) Polarizer Absorbing surface Ultrasound sensor A. Meyer et al., J. Appl. Phys. 96, , 2004 S. Gspan et al., Acoust. Soc. Am. 115, ,

25 Complete spatio-temporal control of laser-induced ultrasound amplitude: spatial (field patterns): doughnut Bessel beam Complete spatio-temporal control of laser-induced ultrasound amplitude: spatial (field patterns): temporal (beam steering): doughnut Bessel beam 25

26 CARS-imaging Coherent Anti-Stokes- Raman Scattering nonlinear optics for functional microscopy: Heinrich et al., Appl. Phys. Lett. 84, 816 (2004) Conservation pump of beam from fiber momentum: k L k L condenser sample area imaging of distribution of selectable biomolecules (map of chemical concentrations) stokes beamenergy: BS from fiber V ω L ωl E i CARS signal to detector filter Latest results droplets of linoleic acid from split sunflower seed resonant 2875 wavenumbers 200 pulses detuned first by nonscanning 175 wavenumbers CARS-images single 3 ns pulse! 26

27 Demonstration of optical sectioning biological sample: oil droplets from a sunflower seed in D 2 O Tuned to linoleic acid at 2870cm -1 z=2µm 50µm Demonstration of optical sectioning biological sample: oil droplets from a sunflower seed in D 2 O Tuned to linoleic acid at 2870cm -1 z=4µm 50µm 27

28 Demonstration of optical sectioning biological sample: oil droplets from a sunflower seed in D 2 O Tuned to linoleic acid at 2870cm -1 z=6µm 50µm The Biomedical Physics Team: Stefan BERNET, MRM Tweezers Wolfgang SINGER (now UQ, Brisbane) Severin FÜRHAPTER Alexander JESACHER Christian MAURER CARS Christoph HEINRICH Clara MEUSBURGER Optoakustik Stefan GSPAN Matthias ZANGERL 28

29 Thank you for your attention! 29