Lasers in Medicine and Life Sciences Summer School, Szeged · 2016-07-12 · Lasers in Medicine and...

Lasers in Medicine and Life SciencesSummer School, Szeged

July 2016

Optical manipulation

Pál Ormos

Biological Research Centre, Hung.Acad.Sci.

Szeged

Lasers in Medicine and Life Sciences Summer School

July 2016

Optical manipulationPál Ormos

Biological Research Centre, Hung.Acad.Sci.

Szeged

Content

1. Optical trapping basics

2. Rotation in optical traps

3. Trapping objects of special shape

4. Sample applications

Light-momenum

Momentum change = force(magnitude, direction)

During refraction,

diffraction, absorption,

reflection

there is force

Absorption, reflection: light pressure

Sun-Earth – several tons

Car in the sun – several hundredths of a gram

Light intensities

Sunshine on the Earth surface: 0,1 W/cm2

focussed laser light: 108 W/cm2 (1W, 1m2)

Mechanical effects of light


Negligible in our macroscopic world.

Significant in the case of very large and very small sizes

Photon rocket

Small sizes

Transparent bead pushed by light


Friction on a sphere: Stokes formula:

vrF 6

smr

Fv /105

6

4

r = 2 x 10-6 m,

= 9 x 10-4 Pa

Historical events

Comet tail points away from the Sun

Kepler (1609)

Historical events

J. C. Maxwell: “In a medium in which waves are propagated there is

a pressure in the direction normal to the wave, and

numerically equal to the energy contained in unit of

volume”

(1873)

Historical events

P N LebedevMeasured the pressure of light (1901).

He also showed that the pressure is twice

as great for reflecting surfaces as for

absorbing surfaces.

Historical events

The experimental apparatus of Lebedev for

the measuring of light pressure.

The light rod R with a set of flat metallic

sheets with very small mass is inside a

vacuum chamber G. The light comes from

a voltaic arc B and passing through a

system of lenses and mirrors (C, D, K, W)

is focused on the sheets. By shifting the

double mirror it is possible to direct

the light on the front or back side of the

sheet and thus to reverse the direction of

the torque on the rod. The part sends a

part of the light beam on the thermocouple

T which measures the amount of impinging

energy.

Optical tweezers

lens

a

pa

fO

b

b’

a’

pb’

pb

pa’

Fa

Fb

F

Laser beam

Arthur Ashkin, 1974

Mechanical effects on a sphere

Mechanical effects on a sphere

Highly focussed beam

Optical tweezers

1974

A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm

and S. Chu. 1986.

"Observation of a Single-Beam Gradient

Force Optical Trap for Dielectric Particles."

Opt. Lett. 11 (5) 288-290.

Optical tweezers

• Forces

– Gradient force traps

– Scattering force pushes

High NA lens is needed

There is an optimal n for trapping

Trapping force: ~pN just right

Optical tweezers

Wavelength considerations

Problem: heating - the object

- the water

Infrared light is mostly used

Force measurement in an optical trap

Dx

Fext Ftrap=kDx

Myosin walking on actin

Finer et al., Nature 368 (1994)

II. Trap non spherical objects

Fresnel-formulae:b

aaI R·I

T·I

1. The polarization and incidence planes are perpendicular:

2. The polarization and incidence planes are parallel

3. General case ( is the angle between polarization and incidence plane):

)(sin

2sin2sin,

)(sin

)(sinR

22

2

ba

ba

ba

ba

T

)(cos)(sin

2sin2sinT,

)(tg

)(tgR

222

2

baba

ba

ba

ba llll

22

ll

22sinTcosTTsinRcosRR , ll

Orientation of a flat particle in an optical trap

Torque of liearly polarized light

Polarization dependent light refraction (described by the Fresnel-formulae)

Largest extension in the direction

perpendicular to the light propagation

Orientation of a flat particle in trap

formed by polarized light

Largest extension

polarization

laser beam

Optical axis

Trapped particle

Orientation of different particles in laser tweezers

polarizáció

chloroplast:

chromosome

trapped free

polarisation laser beam

cross shaped particle

laser beam

laser beam

polarisation

polarizáció

Garab, et al. Eur.Biophys. J. 2005

Produce particles of arbitrary shape

by two-photon photopolymerization

yx

z



Start: I 2R·

R· M RM ·

Growth: RMn · + M RMn+1 ·

Stop: 2 ·RMn RM2nR

Two photon processes are scaled by the square of the intensity better spatial

resolution

This brings smaller half width for a gaussian beam thus better spatial resolution

The chemical process:

Rotor to characterize the system

3 m

Laser beam adjustment

/2 plate

/4 plate

Rotation of trapped particle

Measure the connection between torque and orientiation

/2 plate

Microscope

objective

Trapped object

Laser beam

polarization

w/2

w

polarization

Mlight

Mmedium

w

Viscous drag

For a cylinder:u

: viscosity, u: speed, R: radius, l: length, : density, C: Euler constant

In balance:

4ln

2

1

4

RuC

ulFdrag

lightdrag MM

Torque as a function of phase dirrerence

experiment vs. ray tracing simulation

Galajda and Ormos, Opt. Exp. (2003)

Good properties of the method

• Torque can be applied and measured continuously

(statically or dynamically)

• Torque can be adjusted independently from grabbing force

- to a large extent

Twist a single DNA molecule

Oroszi et al. Phys.Rev.Lett. (2006)

polarization polarization polarization

untwisted-50 turns +40 turns



optical axis

plastic surface

DNA

M

EQ

P

aL

aM

Determination of torsional modulus from

Brownian fluctuations

Torsional elasticity of DNA: 430 pNnm2

From the dependece of torsional elasticity upon elongation, we could choose

between models describing twist-storing polymers

Possibilities

• Measure torque

• Apply torque

Typical magnitude: 10-19 Nm: just right

- Unlimited applications in biology

torsional properties of biomolecules

protein-DNA interactions

rotating systems

Rotation in optical tweezers

Basic mechanisms:

1. Light carries the angular momentum

2. Light carries no angular momentum, the

grabbed object has helicity

1.Circularly polarised light acting on

birefringent particle(: ellipticity)

w

w

2sin])([cos12

2sin2cos)](sin[2

20

20

eo

eo

nnkdE

nnkdEm

torque on unit surface of a birefringent material of thickness d:

Electric field strength:

1.Circularly polarised light acting on birefringent

particle

Optical alignment and spinning of laser-trappedmicroscopic Particles

M. E. J. Friese, T. A. Nieminen, N. R. Heckenberg & H. Rubinsztein-Dunlop

Nature 394, 348–350 (1998)

Angular trapping with birefringent particle

Michelle Wang http://wanglab.lassp.cornell.edu

2. Light driven rotation: propeller

propeller

Light driven propeller


Propeller

M. objective

Galajda and Ormos, Appl. Phys. Lett. (2001)

Complex optomechanical machines

Reverse rotational direction

Galajda and Ormos, Appl. Phys. Lett. (2002)

Study of light induced rotation:

controlling the direction

Equation of a logarithmic spiral in a polar

coordinate system:

a

kea actgk

Reverse rotational direction



-10 -5 0 5 10 15 20

-4

-2

0

2

4

6

Ro

tati

on

ra

te[s

-1]

Position of the objective [m]

Control of optical traps

• Moving the trap

• Creating more traps

• Trap arrays

Moving the trap

M1

L1L2

M2

O

f1 f1 f2 f2

Technologies for moving the trap

Advantage Disadvantage

Galvo-mirrorLarge dynamic range No axial shift

Limited multiplexing

Acusto-optic

modulator

Precise shift

Fast operation

Limited dynamic range

No axial shift

SLMAxial, lateral shift Slow

Limited range

DLPFast No real beam

adjustment

Technology

Multiplexing the traps

• Multiply the beam – trivial

• Move (jump) the beam between positionsHow fast should this happen?

Diffusion velocity of the trapped particle:

r

TkD B

6 for water and r=1m :

s

mD

213102

Dtx 2t= 1 ms, x=1.4x10-8 m

t= 100 ms x=1.4x10-7 m

These values illustrate how fast the trap should jump between multiplex positions

Galvo mirror

Acusto-optic modulator

Standing waves in a vibrating quartz generate a diffraction grating

SLM- the device

A pixelated mirror. In each pixel the phase can be changed fom 0 to 2π.

The phase front can be arbitrarily modified.

SLM: Spatial Light Modulator

Phase holograms

Blazed grating

Phase holograms

Fresnel lens

Phase holograms

Phase vortex

Laguerre-Gaussian beam

Function of the SLM

Complex light pattern Generating hologram

Function of the SLM

Phaseshift pattern Intensity distribution

Photopolymerisation with SLM

• Two-photon photopolymerization by direct

laser writing

microscope objective

photopolymer drop

coverslip

hardened photopolymer

x y

z

Galajda and Ormos: App.Phys. Lett., 78, 249-251 (2001)

Kelemen et al.: Opt. Exp: 15, 14488-14497 (2007)

Vizsnyiczai et al: Opt. Exp.: Vol. 22 pp. 24217-24223 (2014)

Photopolymerisation with SLM

• Two-photon photopolymerization by direct

laser writing

DLP

The device is an array of movable mirrors (~30m size, up to HDTV resolution)

– bistable positions (Texas Instruments)

Manipulator

Manipulator

Probing a cell

Manipulator applications

• Microspectroscopy

• Manipulation of live cells

Concept of microspectroscopy

Realise local excitation with optically

manipulated microtools

Microspectroscopy 1

Optical waveguide pointer (in collaboration with the

Glückstad group)

Selective excitationof fluorescent beads

Flu

ore

scent

beads

Palima et al. Opt. Expr., 20: 2004-2014 (2012)

Microspectroscopy 2

Cell

Enhanced fluorescence/ Raman signal

Gold NP-coated tip

Nanoparticle assisted excitation

Microtool pointing accuracy

Pointing stability is 50nm in x and 80nmin Y direction

Displacement at 16.7mW/beam

tip displacement X [nm]

tip

dis

pla

ce

me

nt Y

[n

m]

Fluctuation of the tip position at various trapping powers

Functionalization of TPP

structuresVolumetric or surface-targeted

Our approach:

• Metal NP coating (enhanced Raman, fluorescence)

• Protein coating (biotin - STA - biotin bridge)

Fluorescence enhancement by a NP-coated TPP structure

excitation emission

fluorescent

layer

Protein-coating: attachment of a cell to a TPP structure

Functionalization with gold NPs

Strategy for the epoxy-based SU-8 photopolymer

SU-8

Acid-treatment:

epoxy ring

opening

SU-8

PEG-diamine:

creating –NH2

groups

SU-8

PEG-diamine:

homo-bifunctional

crosslinker

SU-8

Au NP treatment:

electrostatic

binding

Au NP: 15-100

nm

citrate reduction

negatively

charged

500 nm

Microstructur

e coated

with

80 nm Au

NPs5 m

Covering SU-8 with gold NP

Particle density

Akebote et al., Eur. Polymer.J. 48, 745–1754 (2012)

http://www.sciencedirect.com/science/journal/00143057/48/10

Intensity enhancement

Characterization with test structures

Akebote et al., Opt. Mat. 38, 301-309 (2014)



Tipped structures



Rounded structures

Enhanced fluorescence observation

Large-area enhancement

• Enhancement factor: > 6

• Structured enhancement

• Enhancement at NP-

fluorophore distances > 1m

Cannot be explained with only plasmonic enhancement!

Confocal

fluorescent image

Enhancement map

En

ha

nce

me

nt

facto

r

Distance [m]


0500

10001500200025003000350040004500

0 10 20 30 40Position [m]

~3.5 x

• Enhancement: ~ 3.5 times

• Inhomogeneous background

Bright-field Confocal

Large-area enhancement on an endothelial cell layer


Incoming

excitation

Fluorescen

t layer

x

z

Reflected

excitation

Δx = 990 ± 67 nm

n = 1.33

𝜃 = 15o

= 658.5 ± 44.7 nm

Wavelength around

excitation/emission

Origin of enhancement:• Standing wave formation due to the reflection from

the gold NP layer

• Enhanced fluorescence in the intensity maxima

Manipulation of live cells

Problems of direct optical manipulation of live cells:

• Laser light is harmful to the cells

• The position in the trap is not well defined

Cell survival vs. Laser

irradiation

Concept

Solution

• Use optically manipulated microtool

– The shape is optimized for efficiency and

stability

– Trapping light does not reach the cell

Components of the technique

• Fabricate microtool by two-photon-

polymerization

• Attach the cells to the microtool

• Realise 6D manipulaton with holographic

optical tweezers

Attachment of the cell

• Functionalisation of the surfaces (both the

structure and the cell)

• Avidin-biotin bonding is used

-NH2 groups

BiotinilationSU8 surface

covered with

streptavidine

NH2 NH2 NH2

Acid and PEG-

diamine treatment

Epoxid

surfac

e

10 m

Desired system

We have the HOT with the necessary properties:

real-time motion control of the approrpiately positioned traps

Realised system

Aekbote et al.: Biomed. Opt. Exp.: 7 pp: 45-56 (2016)

Attachment of the cell to the

manipulator

Successful attachment of the

cell to the tool

Translation of an indirectly trapped K562

cell in its medium with a four-spheroid

manipulator along the three coordinate

axes: (a-c) dragging along the x axis, (e)-(f)

along the y axis and (h)-(j) along the z

(optical) axis. (v = 20 m/s) The histograms

on panel (d) show the displacement of two

spheroids during x-drag and that on (g)

shows the tilt during y-drag. The two lines

on panel (e) indicate the maximum tilt

angle. Scale bar: 10 m.

Manipulation in the

Holographic Optical Trap

Forgatás az Z tengely körül

Example application:

Improve fluorescence imaging

• Due to the elongated point-spread-function

of microscope objectives, lateral resolution

is far better that that in the axial direction.

• It is possible to eliminate this problem by

combining images taken from different

directions.

Resolution enhancement

• The resolution of a microscope is limited by the Point-

Spread-Function (PSF)

• The image of a point like object is an elongated spot,

anellipsoid, with size depending on the

Point-

like

object

X

Z

PSFXZ

X

Y

PSFXY

The process of resolution

improvement

Observe the sample from different directions.

Strategy:

1.Z-scan in normal fluorescent microscope from

different directions

2.Eliminate light coming from outside the focal plane by

deconvolution

3.Unify the images taken from different directions.

The principle of resolution

improvement

• By combining the images taken from

different directions we obtain an isotropic

resolution of PSFXY

X

Z

PSFXZ

X

Z

PSFXZ

Z-scan from different

directions• The sample is moved by 250 nm steps

• Image in every position

• Rotate with a given angle

• Image again

Observation

plane

Typical Z-scan

Validation of the procedure

• The resolution was characterized with 300

nm fluorescent beads attached to the cell.

Resolution improvement

2D projections of the 3D fluorescence intensity data arrays recorded on a trapped cell that

was labelled with 300nm fluorescent beads (A-D). Image of an unprocessed data array (A),

the same array after deconvolution (B), aligned data arrays of 8 various directions (C) and

the final combined array after selecting the minimum pixel intensities (D). Intensity traces

along the three axis in the original, deconvolved and combined data arrays (E-G) of the

spot marked with the red arrow on panels B and D.

Resolution improvement

True isotropic resolution is achieved

Reconstructed images of a

double stained cell

A: the reconstructed image of the cell nucleus only. B: the reconstructed image of the

mitochondrion only. C: combined image of the nucleus and the mitochondrion. Cross

sections of the combined 3D images of the stained cell at the planes marked by the

yellow lines (D-F).

Final demonstration

Aqdditional groups

active in the field• Jesper Glückstad, Copenhagen

• Steven Phillis, Bristol

In the case of microscopic objects moving

at similar velocities hydrodynamic

interactions may cause synchronisation.

The effect is believed to be important in

the swimming of microorganisms.

Example: Cilia

http://www.youtube.com/watch?v=BbI47l2nbDQ&feature=related

Example: Cilia

http://www.youtube.com/watch?v=Fc70Uk1fj

Tw

Example: Flagellae

E. Coli swimming with flagellae

Howard Berg, Harvard

Synchronisation due to hydrodynamic

interactions has been suggested and

discussed but without experimental

verification

Fv

Hydrodynamic interaction

Hydrodynamics synchronisation

experiment

Two light driven propellers are held and

rotated

Movie of the experiment

Scheme of the experiment

The rotors are rotated with similar and

continuously varied rateR

ota

tional velo

city

Time

1. rotor

2. rotor

The phase lag in the dependence of

applied torque

Hydrodynamic synchronization is demonstrated

Details of the effect

Mod 360 Histogram

Phase d

iffe

rence (

degre

e)

Phase difference(degree)frames

frames

Histogram of relative angles

At changing torque difference

Distance of the rotors: 6m

2000 – 12000 frames 35000 – 44000 framesAll frames

Distance of the rotors: 7m

2000 – 12000 frames 60000 – 80000 framesAll frames

Simulation

The simulation system

Simulation

• Fluid flow is described by the Stokes equation

• Hydrodynamic interaction is described by stokeslets

(Oseen-tensor)

• Brownian fluctuation is introduced by a force term:

trTKF beadBBrown

D

16

2

pv 2 0v

)()()( jj rFrrGrv

Fv

Simulated motion

Application examples

Biology

Photonic force microscope

http://www.embl.de/~tischer/Tischer_GIT_2002.pdf

Photonic force microscope

Reversible application of stress upon single cells

Yeast cell area monitored while moving between a

neutral environment and a saline environment (0.5M NaCl).The microfluidic/optical tweezers system.

Emma Erikson, Göteborg University

Carlos Bustamante

http://alice.berkeley.edu/

Counter propagating tweezers

Stretching a single DNA molecule

with optical tweezers

Smith et al. Science 271:795-799.(1994)

Basic experiment

Viruses must package their genomes for delivery to other host cells.

Bacteriophage F29 packages its 6.6-mm long, double-strand DNA into a 42x54 nm

capsid.

Bacteriophage motor

The bacteriophage 29 portal motor can package DNA

against a large internal force(D. E. Smith, S. J. Tans, S. B. Smith, S. Grimes, D. L. Anderson, C. Bustamante

NATURE, 413, 748 (2001))

Stalled, partly prepackaged complexes are attached to a polysterene microsphere by means of the unpackaged end of the DNA. This microsphere is captured in the optical trap and brought in into contact with a second bead that is held by a pipette. This bead is coated with antibodies

against the phage, so a stable tether is formed between the two beads.

In the absence of ATP, the tether displays the elasticity expected for a single DNA molecule. Shortly after adittion of ATP, the two microsphere move closer together, indicating packege activity.

150

Studies of DNA Packaging by Single f29 Bacteriophage Particles

Pippete position was adjusted

by feedback to maintain DNA

tension at a preset value of

5 pN

Packaging is highly efficient: it takes 5.5 min

on average to package a length of DNA.

Pauses in movement of variable duration can

be seen: on average there are 3.1 pauses per

micrometre of DNA packaged and these pauses

have a mean duration of 4 s.

Wen, J-D., Lancaster, L., Hodges, C., Zeri, A-C., Yoshimura, S-H., Noller, H.F., Bustamante, C., and I. Tinoco, Jr.,

Following translation by single ribosomes

Nature 452, 598-603 (3 April 2008)

The results

Elongation at constant force pullingDistribution of step sizes

(avg: 2.7 nm = 6 basedistance=2 codon)

Details of one step Transition times

Conclusion

• One codon is translated at one time

• There are three substeps

Thank you for your attention!

Lasers in Medicine and Life Sciences Summer School, Szeged · 2016-07-12 · Lasers in Medicine and...

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