1. Oklahoma State University 1 Raman Spectra of Optically Trapped Microobjects Emanuela Ene Diffraction rings of trapped objects

2. 2 Content Building a Confocal Raman-Tweezing System Experimental spectra Future plans Testing and calibrating an Optical Trap Background: Optical Tweezing Confocal Raman Spectroscopy

3. 3 Ashkin: first experiment Acceleration and trapping of particles by radiation pressure, Phys Rev Lett, 1970, Vol.24(4), p.156 Ashkin et al. Observation of a single-beam gradient force optical trap for dielectric particles, Opt Lett, 1986, Vol. 11(5), p.288 Spatially filtered 514.5nm, ~100mW, beam incident upon a N.A. 1.25 water-immersion microscope objective traps a 10μm glass sphere (Mie size regime ) with m=1.2; F A is the resulting force due to the refracted photons’ momentum change. The image of the red fluorescence makes the beam geometry visible. Laser trapping

4. 4 The refraction of a typical pair of rays “a” and ”b” of the trapping beam gives the forces “F a ” and “F b ” whose vector sum “F” is always restoring for axial and transverse displacements of the sphere from the trap focus f. Typically, the “spring” constant (trap stiffness) is 0.1pN/nm.This makes the optical tweezing particularly useful for studying biological systems. A. Ashkin, Biophys. J. 61, 1992 Optical trapping

5. 5 Photons and scattering forces  pipi pfpf Δ p photon

6. 6 Ray optics (Mie) regime The radiation force has an axial (scattering) and a gradient (transversal) component. P i affected by losses on the overfilled aperture and by spherical aberrations Q- trapping efficiency (depends on the geometry of the particle, relative refractive index “m”, wavelength, radial distribution of the beam) Large particle: P i =1mW; n s =4/3 (water); Q max =0.3 (immersion objective, glass sphere with m=1.2) F r,max =1.3pN For a sphere with 2a=5μm, the value of 2πa/ λ is 25 for the 632.8nm laser 30 for the 514.5nm laser Some numbers

7. 7 Light forces in the ray optics regime A single incident ray of power P scattered by a dielectric sphere; PR is the reflected ray; PT 2 R n is an infinite set of refracted rays As before, for one photon the momentum is and the photon flux in the incident ray is r a

8. 8 F = Fz + iFy These sums (1) and (2), as given by Roosen and co-workers (Phys Lett 59A, 1976), are exact. They are independent of particle radius “a”. The scattering and the gradient forces of the highly convergent incident beam are the vector sums of the axial and transversal force contribution of the individual rays of the beam. T (transmitivity) and R (reflectivity) are polarization dependend, thus the trapping forces depend on the beam polarization. (1) (2) Computational modeling uses vector equations. The beam is resolved in an angular distribution of plane waves. Modeling in this regime ignores diffraction effects.

9. 9 Axial forces in ray-optics regime as calculated by Wright and co-workers (Appl. Opt. 33(9), 1994) Vector-summation of the contributions of all the rays with angles from 0 to arcsin(NA/n s ) for a Gaussian profile on the objective aperture with a beam waist-to-aperture ratio of 1. Linearly polarized laser of 1.06μm assumed. On the abscise: the location of the sphere center with respect to the beam focus. The best trapping is for the bigger sphere and the focus outside the sphere. The best trapping is for the smaller waist and the focus outside the sphere. 0.5μm-radius silica sphere (m=1.09) for different laser spots Silica spheres (m=1.09) with different radii when the minimum beam waist is 0.4μm

10. 10 y Gradient, scattering and total forces as a function of the distance S’ of the trap focus from the origin along the y- axis (transversal). The transverse force is symmetric about the center of the sphere, O. The gradient force Qs is negative, attractive, while the scattering force Qs is positive, repulsive. The value of the total efficiency, Qt, is the sum of two perpendicular forces. An axially-symmetric beam, circularly polarized, fills the aperture of a NA=1.25 water immersion objective (  max =70°) and traps a m=1.2, polystyrene, sphere. S’=r/a and Q are dimensionless parameters (a=radius of the sphere; r=distance from the beam axis). Transversal forces in ray-optics regime as calculated by A. Ashkin, ( Biophys. J 61, 1992)

11. 11 Gaussian profile on the objective aperture Transparent Mie spheres: Both transversal and axial maximum trapping forces are exerted very close to the edge of the transparent sphere Trap performances decrease when the laser spot is smaller than the objective aperture The best trapping is for the smaller waist and bigger particle radius Reflective Mie particles: 2D trapped with a TEM 00 only when the focus is located near their bottom trapped inside the doughnut of a TEM 01 * beam, or in the dark region for Bessel or array beams Cells modeled as transparent spheres

12. 12 Modeling optical tweezing in ray optics (Mie) regime For trap stability, Fgrad >>Fscat the objective lens filled by the incident beam high convergence angle for the trapping beam Usually a Gaussian TEMoo beam is assumed for calculations. But Gaussian beam propagation formula is valid only for paraxial beams (small  )! Truncation: τ= D beam / D aper d spot = 2w trap = K(τ)* λ*f/# d Airy = d zero (τ >2) = 2.44* λ*f/# τ =1: the Gaussian beam is truncated to the (1/e 2 )-diameter; the spot profile is a hybrid between an Airy and a Gaussian distribution τ<1/2 : untruncated Gaussian beam

13. 13 Wave-optics (Rayleigh) regime Electric dipole-like (small) particle : Theory applies for metallic/semiconducting particles as well, if dimension comparable to the skin depth. Dipole polarizability: F grad - the scattering cross-section The dipole moment:

14. 14 The diffraction limit in water, for an uniform irradiance, of this objective is d zero ≈ 3.4μm Our modeling for Gaussian beam propagation uses ray matrices The values for the trap parameters are estimated: the beam is truncated and no more paraxial after passing the microscope objective. Distances are in millimeters unless stated otherwise. 2z trp =0.18μm 2w trp =0.24μm 2w 0 =1.25 d 4 =320d 1 =175d 2 =425d 3 =1500 z=1.84 f 1 =-100f 2 =300f 3 =+160 Laser 632.8nm d 5 =160 f obj =+1.82 R=160 R=10 6 2w 2 =6.26 2w 3 =6.26 2w min =5.2μm

15. 15 MicroRaman Spectroscopy V scat = 8π 2 /3 x w trp 4 /λ ≈ 3*10 -8 mL I max = (w 0 /w trp ) 2 I 0 =5.5x10 8 I 0 w 0 =3.76mm λ =632.8nm w trp =0.16μm Z trp =0.17μm Our numbers : Focused Gaussian beam zRzR zRzR 2w trp I max I max /2 V scat - scattering volume I max /2

16. 16 Confocal microRaman Spectroscopy Background fluorescence and light coming from different planes is mostly suppressed by the pinhole; signal-to-noise-ratio (SNR) increases; scans from different layers and depths may be recorded separately. In vivo Raman scanning of transparent tissues (eyes).

17. 17 Testing and calibrating an optical trap Screen calibrated with a 300lines/inch Ronchi ruler V trap ≈ 8π 2 /3 x w trp 4 /λ≈ 0.02μm 3 V object ≈ 10μm 3 V object / V trap ≈ 500 2z trp =0.83μm LED 2w 0 =0.9 d 4 =310d 1 =750d 2 =850d 3 =300z=1.88 f 1 =100f 2 =750 f 3 =+150 2w trp =0.27μm P=19mW Laser 632.8nm Pinhole CCD camera with absorption filters BS d 5 =160 d 6 =127 d 7 =310 Camera lens f=55mm

18. 18 Calibrating the screen A 5μm PS tweezed bead, in a high density solution, imaged with the 100x objective The sample stage with white light illumination and green laser trap Ronchi rulers at the object plane were used to calibrating the on-screen magnification Imaging through a 50X objective: a) a 300lines/inch target in white light transmission; b) the 632.8nm laser beam focused and scattered on a photonic crystal For the 100X objective, the magnification in the image is 1162.5 Magnification: M=Δl screen x 300/1” 14μm

19. 19 Water immersed complex microobjects have been optically manipulated Cell “stuck” near a 0.8µm PMMA sphere with 6nm gold nanoparticles coating SFM image of a cluster of 0.18μm PS “spheres” coated with 110nm SWCN. Scanning range: 4.56μm Diffraction rings of trapped objects. Sub-micrometer coated clusters were optically manipulated near plant cells; both of the objects stayed in the trap for several hours PMMA = polymethylmethacrylate

20. 20 Optical manipulation in aqueous solution and in golden colloid The particle is held in the trap while the 3D sample-stage is moving uniformly. The estimated errors: 0.2s for time and 4μm for distance. Purpose: identifying the range of the manipulation speeds and estimate (within an order of magnitude) the trapping force; a large statistics for each trapped particle has been used.

21. 21 The polystyrene spheres are manipulated easier if they are rather smaller than bigger uncoated than coated immersed in water than in metallic colloids at higher trapping power Speeds distributions for uncoated and coated polystyrene spheres and 632.8nm laser; optical manipulation in aqueous solution and in golden colloid 1.16μm PSS in a 0.8mW trap 1.16μm PSS horizontally moved in two different traps Coated PSS in a 0.8mW trap 4.88μm PSS in a 0.8mW trap

22. 22 Estimating the trapping force Particle type and size in μm average Vfall(μm/s) average Vth(μm/s) ≈4μm clusters of coated PSS 94 4.88μm uncoated PSS 2510 1.16 μm uncoated PSS 1716 Slow, uniform motion in the fluid. Stokes viscosity, Brownian motion. Free falling and thermal speeds F drag =kv F max G a =kv fall v th v Horizontal manipulation For 4.88μm PSS in water (0.8mW): ρ=1.05g/cm 3 ; v meas =22μm/s; η=10 -3 Ns/m 2 F est ≈2pN GaGa v th v fal l F drag =kv fall

23. 23 The range of secure manipulation speeds and trapping forces have been investigated for water and colloid immersed microobjects pN μm/s Clusters size unit: μm PSS = polystyrene sphere SWCN = single wall Carbon nano tube NP =nano particle

24. 24 Building a confocal Raman- tweezing system from scratch M – Silver mirror P – Pinhole LLF - laser line filter BS – beam-splitter BP - broad-band polarization rotator Spectrometer characteristics Experimental setup L curvature halogen lamp PMT objective & sample DM3000 system beam expander P4 BS Monochromator Video camera Imaging system subt. filters P1 HeNe Laser Ar+ Laser M1 M2, M3 P3 P2 BPR LLF

25. 25 Detecting Raman lines 180 o scattering geometry chosen excitation laser beam is separated from the million times weaker scattered Raman beam, using an interference band pass filter matching the beam in the SPEX 1404 double grating monochromator (photon counting detection, R 943-02 Hammatsu) multiple laser excitation, different wavelengths, polarizations, powers alignment with Si wafer confocal pinhole positioned using a silicon wafer calibration for trap and optics with 5μm PS beads (Bangs Labs) metallic enclosure tested

26. 26 Calibrating the spectrometer with a Quartz crystal The (x-y) -scattering plane is perpendicular on the z-optical axis; the excitation beam polarization is “z” (V); the Raman scattered light is unanalyzed (any). 465cm -1 is the major A1 (total symmetric, vibrations only in x-y plane) mode for quartz.

27. 27 Silicon wafer (n=3.88 δ=3μm@633nm) Oil immersion objective (NA=1.25) Cover glass (n=1.525; t=150μm) Oil layers (n=1.515) Backward scattered Raman light Incident laser beam The calibration of our confocal setting was done with a strong Raman scatterer. Confocal spectra have been collected when axially moving the Si wafer in steps of 2μm. Axial resolution

28. 28 Confocal microRaman spectra Slide with 1.5mm depression, filled with 5μm PS spheres in water. Focus may move ≈ 440 μm from the cover glass. Results identical as in www.chemistry.ohio- state.edu/~rmccreer/freqcorr/images/poly.htmlwww.chemistry.ohio- state.edu/~rmccreer/freqcorr/images/poly.html Cover glass (n=1.525, t=150μm) Aqueous solution of PS spheres (m=1.19) Slide Oil layer (n=1.515) Oil immersion objective (NA=1.25) Backward scattered Raman light Incident laser beam Δz≈440μm

29. 29 An optimal alignment and range of powers for collecting a confocal Raman signal from single trapped microobjects has been identified 5.0μm PS sphere (Bangs Labs) trapped 10mW in front of the objective; broad-band BS 80/20, no pinhole Confocal scan 5mW in front of the objective; double coated interference BS Better results than in Creely et al., Opt. Com. 245, 2005Creely et al., Opt. Com. 245, 2005

30. 30 Confocal Raman-Tweezing Spectra from magnetic particles 1.16μm-sized iron oxide clusters (BioMag 546, Bangs Labs) Silane (SiHx) coating The BioMags in the same Ar+ trap were blinking alternatively. We attributed this behavior to an optical binding between the particles in the tweezed cluster (redistribution of the optical trapping forces among the microparticles).

31. 31 Future plans: monitoring plant and animal trapped living cells in real time; analyze the changes in their Raman spectra induced by the presence of embedded nanoparticles (a) Near-infrared Raman spectra of single live yeast cells (curve A) and dead yeast cells (curve B) in a batch culture. The acquisition time was 20s with a laser power of ~17mw at 785 nm. Tyr, tyrosine; phe, phenylalanine; def, deformed. (b) Image of the sorted yeast cells in the collection chamber. Top row, dead yeast cells; bottom row, live yeast cells. (c) Image of the sorted yeast cells stained with 2% eosin solution. (Xie, C et al, Opt. Lett., 2002)

32. 32 Future plans: using optical tweezing both for displacing magnetic micro- or nano-particles through the cell’s membrane and for immobilizing the complex for hours of consecutive collections of Raman spectra Pisanici II, T.R. et al, Nanotoxicity of iron oxide nanoparticle internalization in growing neurons, Biomaterials, 2007, 28( 16), 2572-2581 PC12 cells ( a line derived from neuronal rat cells) were exposed to no (left), low (center), or high (right) concentrations of iron oxide nanoparticles (MNP) in the presence of nerve growth factor (NGF), which normally stimulates these neuronal cells to form thread-like extensions called neurites. Fluorescent microscopy images, 6 days after MNP exposure and 5 days after NGF exposure. 0 mM0.15 mM15.0 mM

33. 33 Future plans: using optical manipulation for displacing microcomplexes and cells in the proximity of certain substrates that are expected to give SERS effect Klarite SERS substrate (Mesophotonics) and micro Raman spectrum for a milliMolar glucose solution with 785nm excitation laser, dried sample, 40X objective

34. 34 Summary a working Confocal Raman-Tweezing System has been built from scratch a large range of water immersed microobjects have been optically manipulated sub-micrometer objects were trapped and moved near plant cells an optimal alignment and range of powers for collecting a confocal Raman signal from single trapped microobjects has been identified our experimental Confocal Raman-Tweezing scans for calibration reproduce standard spectra from literature Raman spectra from superparamagnetic microclusters have been investigated a future development towards a nanotoxicity application is proposed

35. Appendices 35

36. 36 Some useful values for biological applications Energy 1 photon ( λ=1μm) 200 pNxnm Thermal energy K B T (room temperature) 4 pNxnm 1 ion moving across a biological membrane 30 pNxnm Force For optical trapping 1 pN For breaking most protein-protein interactions 20 pN For breaking a covalent bond 1000 pN Length Typical bacteria diameter 1 μm Typical laser wavelength for biological applications 1 μm Trap size 0.5 μm Time Cell division 1 min Cycle time for many biological processes 1ms to 1s Scanning time for a Raman spectrum (CCD camera detection) 0.2s to 10s

37. 37 SubstanceRaman line (cm-1) for bulk samples Present in our Raman spectra for tweezed objects Water984No 1648No 3400No Silane210Yes 290Yes 620Yes 960Yes Magnetite676No Maghemite252? 650? 740? Polystyrene1001.4Yes 1031.8Yes 1602.3Yes 3054.3Yes

38. 38 Fgrad/ Fscat ~ a -3 >>1 The time to pull a particle into the trap is much less than the time diffusion out of the trap because of Brownian motion Equilibrium for the metallic particle near the laser focus ( 0.5-3.0μm sized gold particles ) H. Furukawa et al, Opt. Lett. 23(3), 1998 Stability in the trap for wave regime Surface (creeping) wave generates a gradient force

39. 39 VCSEL arrays Alternate trapping beams Hermite-Gaussian TEM 00 TEM 01* - doughnut (with apodization or Phase Modulator) Bessel ( with a conical lens –axicon -) Holographic Optical Tweezers (the hologram is reconstructed in the plane of the objective) Laguerre-Gaussian k t =k sinγ (γ is the wedge angle of the axicon); k=wave number P = total power of the beam w c = asymptotic width of a certain ring z max =diffraction-free propagation range ( consequence of finite aperture) Bessel l=1 A Bessel beam can be represented by a superposition of plane waves, with wave vectors belonging to a conical surface constituting a fixed angle with the cone axis.

40. 40 Gaussian optics and propagation matrix Beam complex q-parameter At the minimum waist, the beam is a plane wave (R-> ∞ ) beam waist Rayleigh range beam radius of curvature Paraxial approximation Transfer matrix for light propagation Calculating the beam parameter based on the propagation matrix

41. 41 Frèsnel coefficients Transmissivity Non-magnetic medium “p” stands for the wave with the electric field vector parallel with the incidence plane “s” stands for the wave with the electric field vector perpendicular on the incidence plane Reflectivity θ m r

42. 42 Gradient, scattering and total forces as a function of the distance S of the trap focus from the origin along the z- axis (axial). The stable equilibrium trap is located just above the center O of the sphere, at S E. An axially-symmetric beam, circularly polarized, fills the aperture of a NA=1.25 immersion objective:  max =70° and traps a m=1.2 PS sphere. S’=r/a and Q are dimensionless parameters. Axial forces in ray- optics regime as calculated by A. Ashkin, ( Biophys. J 61, 1992)

43. 43 Optical binding Basic physics: Michael M. Burns, Jean-Marc Fournier, and Jene A. Golovchenko, Phys. Rev. Lett. 63, 1233 (1989) interference between the scattered and the incident light for each microparticle fringes acting as potential wells for the dipole-like particles changing phase shift of the scattered partial waves because diffusion which modifies the position of the wells

44. 44 Scattered intensities, theoretically:  (n +1), for the First Order Raman, Stokes branch  n, for the First Order Raman, anti-Stokes branch  (n +1) 2, for the Second Order Raman, Stokes branch

45. 45 Dispersion and bandwidth Grating rotation angle:  [deg] = Wavelength [nm] G = Groove Frequency [grooves/mm = 1800mm -1 m = Grating Order =1, for Spex1404 x = Half Angle: 13.1 o F= Focal Distance: 850mm BANDWIDTH = (SLIT WIDTH) X DISPERSION linear dispersion is how far apart two wavelengths are, in the focal plane: D L = dx /d 63.2nm excitation laser: the resolution is 4cm -1

46. 46 Photon counting Hamamatsu R943-02 PMT lower counting rate limit is set by the dark pulse rate: 20cps @ -20  C 15% quantum efficiency @( 650 to 850nm) incident 1333photons/s signal (3.79x 10 -16 W): minimum count rate should be 200counts/s for 10 S/N ratio