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 Photons and scattering forces p photon p photon F d p photon p absorbed ns photon h c0 Pi ns c0 dt pi pf h c0 red p backscatte (per second ) 2ns photon F h 2ns sin c0 2 red p backscatte 2ns photon absorption h 2P h Pi ns i F mirror c0 h c0 I inc Fr p photon dS h S Δpphoton Ray optics (Mie) regime 6 Large particle: 2a 1 I inc x, y, z Fr p photon dS h S Fr Q ns Pi c0 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 For a sphere with 2a=5μm, the value of 2πa/λ is 30 for the 514.5nm laser 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 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 p photon h h ns c0 and the photon flux in the incident ray is Ni Pi h ns Pi ns Pi 2 n Fz R cos( 2 ) T R cos( n ) c0 c0 n 0 ns Pi Fy 0 c0 2 n R sin( 2 ) T R sin( n ) n 0 8 F = Fz + iFy nP F s i c0 i 2 2 i 1 Re T e R ei n 0 n ns Pi T 2 [cos(2 2r ) R cos(2 ) 1 R cos(2 ) Q s 2 1 R 2 R cos( 2 r ) c0 (1) Fscat nP e( F ) s i c0 Fgrad ns Pi m( F ) c0 ns Pi T 2 [sin(2 2r ) R sin(2 ) R sin(2 ) Qg 2 1 R 2 R cos(2r ) c0 (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 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/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. 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. 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 Transversal forces in 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 yaxis (transversal). The transverse force is symmetric about the center of y 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/ Daper dspot = 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 Wave-optics (Rayleigh) regime 2a Electric dipole-like (small) particle : 1 The dipole moment: p E p F ( p ) E B E 2 t 2 m2 1 3 a Dipole polarizability: n 2 m 2 Fgrad 2 s Fgrad a3 I inc Fscat np c0 Pscat Cscat I inc a 64 I inc Cscat - the scattering cross-section Theory applies for metallic/semiconducting particles as well, if dimension comparable to the skin depth. 14 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. f1=-100 f2=300 f3=+160 fobj=+1.82 2w2=6.26 R=106 2w0=1.25 2w3=6.26 2wmin=5.2μm 2w trp=0.24μm 2z trp =0.18μm Laser 632.8nm R=160 d5=160 d1=175 d2=425 d3=1500 d4=320 z=1.84 The diffraction limit in water, for an uniform irradiance, of this objective is dzero≈ 3.4μm MicroRaman Spectroscopy 15 Raman I scattered exc4Vscattering Iexc Our numbers: Focused Gaussian beam 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 2 wtrp zR Vscat I Raman scat I0 20mL 1 z / w dz 83 zR zR zR 0 2 2 w dz 2 w trp Vscat = 8π2/3 x wtrp4 /λ ≈ 3*10-8mL Imax = (w0/wtrp)2 I0 =5.5x108 I0 2 2 trp 2 4 wtrp 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 Testing and calibrating an optical trap CCD camera with absorption filters Vtrap≈ 8π2/3 x wtrp4 /λ≈ 0.02μm3 Vobject ≈ 10μm3 Vobject / Vtrap ≈ 500 f1=100 d7=310 Camera lens f=55mm f2=750 f3=+150 P=19mW 2w trp=0.27μm 2w0=0.9 LED BS Laser 632.8nm Pinhole d6=127 2z trp=0.83μm d5=160 d1=750 d2=850 d3=300 d4=310 Screen calibrated with a 300lines/inch Ronchi ruler z=1.88 18 Calibrating the screen Ronchi rulers at the object plane were used to calibrating the on-screen magnification 14μm The sample stage with white light illumination and green laser trap 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 Magnification: M=Δlscreen x 300/1” A 5μm PS tweezed bead, in a high density solution, imaged with the 100x objective For the 100X objective, the magnification in the image is 1162.5 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 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 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 Coated PSS in a 0.8mW trap in two different traps 4.88μm PSS in a 0.8mW trap 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 Estimating the trapping force 22 Slow, uniform motion in the fluid. Stokes viscosity, Brownian motion. Fdrag=kvfall vfal vth vth Fdrag=kv 1 ( 0 )d 3 g kexp 6 v fall v ksphere 3d Ga=kvfall ksphere kexp 5 108 Ns / m Fdrag k v Ftrap,|| l Ga Fmax Horizontal manipulation Free falling and thermal speeds Particle type and size in μm average Vfall(μm/s) average Vth(μm/s) ≈4μm clusters of coated PSS 9 4 4.88μm uncoated PSS 25 10 1.16 μm uncoated PSS 17 16 v meas v v Th Fmax k (vmeas vth ) 2 v fall 2 For 4.88μm PSS in water (0.8mW): ρ=1.05g/cm3; vmeas=22μm/s; η=10-3Ns/m2 Fest≈2pN The range of secure manipulation speeds and trapping forces have been investigated for water and colloid immersed microobjects 23 pN PSS = polystyrene sphere SWCN = single wall Carbon nano tube NP =nano particle Clusters size unit: μm μm/s 24 Building a confocal Ramantweezing system from scratch halogen lamp DM3000 system PMT objective & sample P4 Monochromator L curvature Video camera Imaging system BS BS subt. filters LLF P3 HeNe Laser M – Silver mirror P – Pinhole LLF - laser line filter BS – beam-splitter BP - broad-band polarization rotator BPR beam expander Experimental setup M2, M3 P1 LLF Ar+ Laser P2 M1 Spectrometer characteristics 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 [email protected]) Incident laser beam Oil immersion objective (NA=1.25) 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. Cover glass (n=1.525; t=150μm) Oil layers (n=1.515) 28 Confocal microRaman spectra Backward scattered Raman light Δz≈440μm Slide Aqueous solution of PS spheres (m=1.19) Incident laser beam Oil immersion objective (NA=1.25) Oil layer (n=1.515) Cover glass (n=1.525, t=150μm) 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.ohiostate.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 Better results than in Creely et al., Opt. Com. 245, 2005 Confocal scan 5mW in front of the objective; double coated interference BS 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 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 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 0 mM 0.15 mM 15.0 mM 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 •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 36 Some useful values for biological applications Energy 1 photon ( λ=1μm) Thermal energy KBT (room temperature) 1 ion moving across a biological membrane Force For optical trapping For breaking most protein-protein interactions For breaking a covalent bond Length 4 pNxnm 30 pNxnm 1 pN 20 pN 1000 pN Typical bacteria diameter 1 μm Typical laser wavelength for biological applications 1 μm Trap size Time 200 pNxnm Cell division 0.5 μm 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 Substance Raman line (cm-1) Present in our Raman spectra for tweezed objects for bulk samples Water 984 No 1648 No 3400 No 210 Yes 290 Yes 620 Yes 960 Yes Magnetite 676 No Maghemite 252 ? 650 ? 740 ? Silane Polystyrene 1001.4 Yes 1031.8 Yes 1602.3 Yes 3054.3 Yes 38 Stability in the trap for 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 39 Alternate trapping beams I ( , z) Hermite-Gaussian TEM00 2P e 2 w( z ) 2 2 w( z ) 2 Laguerre-Gaussian TEM01* - doughnut (with apodization or Phase Modulator) Bessel ( with a conical lens –axicon -) Il ( , , z) 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. Bessel l=1 VCSEL arrays I ( ) l 2 2 l! kt P z wc zmax I0 e 2 ( / w0 ) 2l 1 J l2 (kt , )e 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) Holographic Optical Tweezers (the hologram is reconstructed in the plane of the objective) 2 2 w2 0 2 z 2 z 2 max 40 Gaussian optics and propagation matrix I ( , z) 2P e w( z ) 2 Beam complex q-parameter 2 2 w( z ) 2 Rayleigh range beam waist At the minimum waist, the beam is a plane wave (R-> ∞) beam radius of curvature zR 2 R( z ) z 1 z r' r T ' Transfer matrix for light propagation Paraxial approximation Calculating the beam parameter based on the propagation matrix 41 Frèsnel coefficients Non-magnetic medium 1 r arcsin sin m tan2 ( r ) Rp tan2 ( r ) m nsphere nsurround θ Transmissivity Reflectivity sin 2 ( r ) Rs 2 sin ( r ) m r Ts Tp sin(2 ) sin(2r ) sin 2 ( r ) sin(2 ) sin(2r ) sin 2 ( r ) cos2 ( r ) Rp Tp 1 Rs Ts 1 “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 42 Axial forces in rayoptics 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. Gradient, scattering and total forces as a function of the distance S of the trap focus from the origin along the zaxis (axial). The stable equilibrium trap is located just above the center O of the sphere, at SE. 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 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 I anti Stokes n h / k BT e e I Stokes n 1 hc 1 ( ) k BT Dispersion and bandwidth 45 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 cos(x ) 106 nm 0.6 Dispersion[nm/mm] G F m mm BANDWIDTH = (SLIT WIDTH) X DISPERSION 63.2nm excitation laser: the resolution is 4cm-1 46 Photon counting Hamamatsu R943-02 PMT lower counting rate limit is set by the dark pulse rate: 20cps @ -20C 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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