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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

- Future plans

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 m1.2 FA is the resulting force

due to the refracted photons momentum change.

The image of the red fluorescence makes the beam

geometry visible.

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

Photons and scattering forces

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

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

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.

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.

Silica spheres (m1.09) 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.

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 NA1.25 water immersion

objective (?max70) and traps a m1.2,

polystyrene, sphere. Sr/a and Q are

dimensionless parameters (aradius of the sphere

rdistance 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.

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

Modeling optical tweezing in ray optics (Mie)

regime

- For trap stability,

Fgrad gtgtFscat - 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 t Dbeam/ Daper

dspot 2wtrap K(t)?f/ -

dAiry dzero (t gt2) 2.44?f/ - t1 the Gaussian beam is truncated to the

(1/e2)-diameter - the spot profile is a hybrid between an Airy and

a Gaussian distribution - tlt1/2 untruncated Gaussian beam

Wave-optics (Rayleigh) regime

Theory applies for metallic/semiconducting

particles as well, if dimension comparable to

the skin depth.

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

MicroRaman Spectroscopy

Focused Gaussian beam

Vscat 8p2/3 x wtrp4 /? 310-8mL Imax

(w0/wtrp)2 I0 5.5x108 I0

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).

Testing and calibrating an optical trap

Vtrap 8p2/3 x wtrp4 /? 0.02µm3 Vobject

10µm3 Vobject / Vtrap 500

Screen calibrated with a 300lines/inch Ronchi

ruler

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?lscreen 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

Water immersed complex microobjects have been

optically manipulated

Diffraction rings of trapped objects. Sub-micromet

er 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

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.

Speeds distributions for 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

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)

?1.05g/cm3 vmeas22µm/s ?10-3Ns/m2

Fest2pN

The range of secure manipulation speeds and

trapping forces have been investigated for water

and colloid immersed microobjects

Building a confocal Raman-tweezing system from

scratch

halogen lamp

PMT

objective sample

DM3000 system

P4

Monochromator

L curvature

BS

Video camera

Imaging system

BS

beam expander

P3

LLF

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

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

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.

Axial resolution

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.

Confocal microRaman spectra

?z440µm

Backward scattered Raman light

Slide

Aqueous solution of PS spheres (m1.19)

Incident laser beam

Cover glass (n1.525, t150µm)

Oil layer (n1.515)

Oil immersion objective (NA1.25)

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

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

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).

Future plansmonitoring 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)

Future plansusing optical tweezing both for

displacing magnetic micro- or nano-particles

through the cells 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

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

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

- Appendices

Some useful values for biological

applications

(No Transcript)

Stability in the trap for wave regime

- Fgrad/ Fscat a-3 gtgt1
- 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

- 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 sin? (? is the wedge angle of the axicon)

kwave number P total power of the beam wc

asymptotic width of a certain ring zmaxdiffractio

n-free propagation range ( consequence of finite

aperture)

Bessel l1

VCSEL arrays

- Holographic Optical Tweezers
- (the hologram is reconstructed
- in the plane of the objective)

Gaussian optics and propagation matrix

beam radius of curvature

Paraxial approximation

Calculating the beam parameter based on the

propagation matrix

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

Axial 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 NA1.25 immersion

objective ?max70 and traps a m1.2 PS sphere.

Sr/a and Q are dimensionless parameters.

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

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

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 the resolution is 4cm-1

Photon counting

- Hamamatsu R943-02 PMT
- lower counting rate limit is set by the dark

pulse rate -

20cps _at_ -20?C - 15 quantum efficiency _at_( 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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