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Raman Spectra of Optically Trapped Microobjects

Raman Spectra of Optically Trapped Microobjects. Emanuela Ene. Diffraction rings of trapped objects. Content. Background : Optical Tweezing Confocal Raman Spectroscopy. Testing and calibrating an Optical Trap. Building a Confocal Raman-Tweezing System Experimental spectra.

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Raman Spectra of Optically Trapped Microobjects

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

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

  3. Laser trapping 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; FA is the resulting force due to the refracted photons’ momentum change. The image of the red fluorescence makes the beam geometry visible.

  4. Optical trapping The refraction of a typical pair of rays “a” and ”b” of the trapping beam gives the forces “Fa” and “Fb” 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

  5. pi  Δpphoton pf Photons and scattering forces

  6. Large particle: 30 for the 514.5nm laser For a sphere with 2a=5μm, the value of 2πa/λis 25 for the 632.8nm laser Pi=1mW; ns=4/3 (water); Qmax=0.3 (immersion objective, glass sphere with m=1.2) Fr,max=1.3pN Ray optics (Mie) regime The radiation force has an axial (scattering) and a gradient (transversal) component. • Pi 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) Some numbers

  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; PT2Rn is an infinite set of refracted rays r As before, for one photon the momentum is a and the photon flux in the incident ray is

  8. F = Fz + iFy (1) (2) 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. Computational modeling uses vector equations. The beam is resolved in an angular distribution of plane waves. Modeling in this regime ignores diffraction effects.

  9. 0.5μm-radius silica sphere (m=1.09) for different laser spots The best trapping is for the smaller waist and the focus outside the sphere. Axial forcesin 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/ns) 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. Silica spheres (m=1.09) with different radii when the minimum beam waist is 0.4μm The best trapping is for the bigger sphere and the focus outside the sphere.

  10. y Transversal forcesin ray-optics regime as calculated by A. Ashkin, ( Biophys. J 61, 1992) 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). 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.

  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 Cells modeled as transparent spheres Reflective Mie particles: • 2D trapped with a TEM00 only when the focus is located near their bottom • trapped inside the doughnut of a TEM01* beam, or in the dark region for Bessel or array beams

  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= Dbeam/ Daperdspot = 2wtrap = K(τ)*λ*f/# dAiry= dzero (τ >2) = 2.44*λ*f/# τ=1: the Gaussian beam is truncated to the (1/e2)-diameter; the spot profile is a hybrid between an Airy and a Gaussian distribution τ<1/2 : untruncated Gaussian beam

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

  14. f1=-100 f2=300 f3=+160 fobj=+1.82 2w2=6.26 2w3=6.26 R=106 2w0=1.25 2wmin=5.2μm 2w trp=0.24μm 2z trp =0.18μm Laser 632.8nm R=160 d5=160 z=1.84 d1=175 d2=425 d3=1500 d4=320 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. The diffraction limit in water, for an uniform irradiance, of this objective is dzero≈ 3.4μm

  15. Our numbers: w0 =3.76mm λ=632.8nm w trp =0.16μm Z trp =0.17μm Vscat- scattering volume Imax /2 Imax Imax /2 2wtrp zR zR MicroRaman Spectroscopy Focused Gaussian beam Vscat = 8π2/3 x wtrp4 /λ ≈ 3*10-8mL Imax = (w0/wtrp)2 I0=5.5x108 I0

  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. CCD camera with absorption filters d7=310 Camera lens f=55mm f1=100 f2=750 f3=+150 2w trp=0.27μm LED P=19mW 2w0=0.9 Laser 632.8nm BS 2z trp=0.83μm d6=127 Pinhole d5=160 d1=750 d2=850 d3=300 d4=310 z=1.88 Testing and calibrating an optical trap Vtrap≈ 8π2/3 x wtrp4 /λ≈ 0.02μm3 Vobject ≈ 10μm3 Vobject / Vtrap ≈ 500 Screen calibrated with a 300lines/inch Ronchi ruler

  18. Calibrating the screen Ronchi rulers at the object plane were used to calibrating the on-screen magnification 14μm 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 The sample stage with white light illumination and green laser trap Magnification: M=Δlscreen x 300/1” For the 100X objective, the magnification in the image is 1162.5 A 5μm PS tweezed bead, in a high density solution, imaged with the 100x objective

  19. Cell “stuck” near a 0.8µm PMMA sphere with 6nm gold nanoparticles coating Water immersed complex microobjects have been optically manipulated 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 SFM image of a cluster of 0.18μm PS “spheres” coated with 110nm SWCN. Scanning range: 4.56μm PMMA = polymethylmethacrylate

  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 timeand 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. 1.16μm PSS horizontally moved in two different traps 1.16μm PSS in a 0.8mW trap Coated PSS in a 0.8mW trap 4.88μm PSS in a 0.8mW trap Speeds distributionsfor uncoated and coated polystyrene spheresand 632.8nm laser; optical manipulation in aqueous solution and in golden colloid • 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

  22. Horizontal manipulation Fdrag=kvfall Fmax vth Fdrag=kv vfall vth v Ga=kvfall Ga Estimating the trapping force Slow, uniform motion in the fluid. Stokes viscosity, Brownian motion. Free falling and thermal speeds For 4.88μm PSS in water (0.8mW): ρ=1.05g/cm3; vmeas=22μm/s; η=10-3Ns/m2 Fest≈2pN

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

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

  25. Detecting Raman lines • 180o 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. 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. Axial resolution Backward scattered Raman light Silicon wafer (n=3.88 δ=3μm@633nm) Cover glass (n=1.525; t=150μm) Incident laser beam Oil immersion objective (NA=1.25) Oil layers (n=1.515) 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.

  28. Confocal microRaman spectra Δz≈440μm Backward scattered Raman light Slide Aqueous solution of PS spheres (m=1.19) Incident laser beam Cover glass (n=1.525, t=150μm) Oil layer (n=1.515) Oil immersion objective (NA=1.25) 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.html

  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, 2005

  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. 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 yeastcells (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. 0 mM 0.15 mM 15.0 mM 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 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. Pisanici II, T.R. et al , Nanotoxicity of iron oxide nanoparticle internalization in growing neurons, Biomaterials , 2007, 28( 16), 2572-2581

  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. Summary • aworking 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

  36. Some useful values for biological applications

  37. Stability in the trapfor wave regime • 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 Surface (creeping) wave generates a gradient force 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

  38. Alternate trapping beams Hermite-Gaussian TEM00 Laguerre-Gaussian TEM01* - doughnut (with apodization or Phase Modulator) Bessel ( with a conical lens –axicon -) 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. kt =k sinγ (γ is the wedge angle of the axicon); k=wave number P = total power of the beam wc= asymptotic width of a certain ring zmax=diffraction-free propagation range ( consequence of finite aperture) Bessel l=1 VCSEL arrays Holographic Optical Tweezers (the hologram is reconstructed in the plane of the objective)

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

  40. r m θ Transmissivity Frèsnel coefficients Non-magnetic medium Reflectivity “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

  41. 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 SE. Axial forcesin ray-optics regime as calculated by A. Ashkin, ( Biophys. J 61, 1992) 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.

  42. 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

  43. 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

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

  45. Photon counting Hamamatsu R943-02 PMT lower counting ratelimit 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

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