Optical Tweezersstatus and biophysical applications

Lene Oddershede, Lektor, PhD,Niels Bohr Institutet

Københavns Universitet

Nano Toolbox

Atomic force microscopy (nano-Newton, nano-m):Unfolding of proteins, binding strengths

Optical tweezers (pico-Newton, nano-m):Single molecule motors, biopolymers

Fluorescense (nano-m toÅngstrøm):Single molekyle reaktions, visualization

Genetic manipulation

Elektron mikroscopy

Future use:’drug delivery’, nano-robots, nano-elektronics ...

Techical know-how2 force-scope optical tweezers:3D spatial resolution ~1nmTemporal resolution ~ MHz

Single particle tracking:3D spatial resolution ~5nmTemporal resolution ~ 25 Hz

Micropipettes

Fluorescens microscopy

Genetic manipulations

Oddershede, Grego, Nørrelykke, Berg-Sørensen, Probe Microscopy vol. 2, p129 (2001)K. Berg-Sørensen and H. Flyvbjerg, Rev. Sci. Ins. 75, p.594 (2004)Dreyer, Berg-Sørensen, Oddershede, Applied Optics vol. 43, p1991 (2004)

Characteristic photodiode powerlossSilicon is transparant to infrared light.Makes diode a 1st order filter.

8 kHz and depends on laser intensity. : fraction of incoming power that is correctly detected indepletion layer.

(diode)

3dBf

Berg-Sørensen, Oddershede, Florin, and Flyvbjerg, J.Appl. Physics. vol. 93 p.3167 (2003)

2(diode)

3dB

22

1

1

)(

)(

fffP

fP

in

out

What can be trapped? Gold nano-particles

Succesful optical trapping of 18 nm - 254nm gold particles

Literature: 36.4 nm and 40 nm trapped in 3DMie particles could not be trapped in 3D

P.M. Hansen, V.K. Bhatia, N. Harrit, L. Oddershede, Nano Letters, vol.5, p.1937 (2005)

Gold nano rods – nano rotators

We have succesfully optically trapped in 3D gold nanorods withdiameters ranging from 8nm to 44nm and aspect rations between1.7 and 5.6 (lenghts up to 85 nm).

The rods align with the E-field, can be used as nano-rotators. The optical forces correlate with polarizability of rod.

Selhuber-Unkel, Zins, Shubert, Sönnichsen, Oddershede, Nano Letters vol. 8 p.2998 (2008).

Silver nano-particles

Change in contrast with size. Has been seen for Au previously, first time for Ag.2 2 2 sinmI r s r s

r: field reflectivity (background)s: scattered field with phase .the last term is proportional to d3 and dominates for small particles.

We optically trap Ag particles with diameters 20nm – 275 nm Spring constants have similar behavior as for Au.

Ag nano-particles enhance fluorescense (Au quenche)

L. Bosanac, T. Aabo, P.M. Bendix, L.B. Oddershede, Nano Letters vol.8 p.1937 (2008)

Quantum Dots • Quantum dots are the super nova of the nano world.• Very broad absorption band but narrow emission, characterized by blinking. • Superior bleaching properties.• Made of semiconductor material• Ours are of CdSe with a core diameter of ~10 nm, zink-sulfide shell, and with an emission wavelength of 655 nm. We have proven possible 3D optical trapping of individual quantum dots.This makes possible simultaneous manipulation and visualisation.

Jauffred, Richardson, Oddershede, Nano Letters vol.8 p.3376 (2008).

Polarizability of an individual Qdot

2 2 2

2

2

( ) / 20

20

4

( )2

2

2

2

grad

x y

grad

F E

IE

c

I I e

P Idxdy I

F x

c

P

����������������������������

����������������������������

The obtained spring constant, , carries information about the interactionbetween the EM field and the Qdot. Using the following relations, thepolarization of an individual Qdot can be found:

whereI : intensity P: total laser power delivered at the samplex,y: directions orthogonal to the light propagation: width of diffraction limited laser spot.

Normalized by vacuum permittivity, we obtain

= 2.8 x 107 Å3.

Literature very sparse, only one value reported forQdots with radius=2nm: ~ 104 Å3.

Jauffred, Richardson, Oddershede, Nano Letters vol.8 p.3376 (2008).

Physiological damage?• It is important to consider the possible physiological damage done by the optical trap on the trapped cell. • One key issue is to choose a wavelength which is not absorbed by water(would create heating) and not absorbed by biological specimen either.• Infra-red lasers fulfill these criteria.

To address possible physiological damage by a 1064 nm laser on livingorganisms, different bifferent bacterial types were optically trapped. • Simultaneously, the emission from a pH sensitive fluorophore (CFDA, GFP) inside the organism was monitored. • The capability of a cell to maintain a pH gradient across the cell wall is a measure of its physiological condition. Healthy cells are able to maintaina gradient, comprised cells not to the same extend.

Physiological damage of E. coli

E. coli expressing GFP

Trapped with 6 mW

Trapped with 18 mW

i ex½

pH ( 0) pHpH =τ

2

tt

ListeriaGFP, anaerobic(weak signal)

CFDA, aerobic

CFDA, anaerobic

CFDA, aerobic

CFDA, anaerobic

monocytogenes

innocua

Physiological damage depends on growth conditions and bacterial species

Species Aerobic growth

FluorophoreLaser Power(mW)

Initial pHi τ½a

E. coli + GFP 6 7.8 » 60 min

E. coli + GFP 18 7.8 30 min

L. monocytogenes + GFP 6 7.3 » 60 min

L. monocytogenes + CFDA 6 7.7 » 60 min

L. monocytogenes - CFDA 6 7.6 57 min

L. innocua + CFDA 6 7.6 » 60 min

L. innocua - CFDA 6 7.4 10 min

B. subtilis + CFDA 6 7.2 21 min

M.B. Rasmussen, L. Oddershede, and H. Siegumfeldt, Applied and Environmental Microbiology, vol. 74 p.598 (2008)

Optical tweezers can trap cytoplasmatic organelles without perturbing the cellular membrane

Viscoelasticity of yeast cell cytoplasm

•Fedt kugler i levende celler kan benyttes som håndtag for OP•De bevæger sig subdiffusivt pga. microtubulus and aktin netværk•Mere aktin ved cellens ender gør, at lipid kuglerne bevæger sig mindre frit der.

Brownsk bevægelse: Anormal diffusion:

Super diffusion > 1

Begrænset = 0

Normal diffusion = 1

1. Molekylære motorer

2. Polymerisering

Subdiffusion0 < < 1

1. Polymer netværk( = 0.75)

2. Membraner( = 0.66)

3. Cytoplasmatisk strømning

Varians: <x2(t) > = 2Dt

22

2

2

2

)(~

)(~)(f

fFfxfP

Varians: <x2(t) > = 2Dt

)1(2

2

2

2

)(~

)(~)(

f

fFfxfP

How do the lipid granules move?

ttr 2)(

Plateau pga. endeligcelle størrelse

Super diffusion

Normal diffusion

Opticaltweezers

Single particle tracking

mostly bý subdiffusion, =0.75

S. pombe strains expressing green fluorescent protein (GFP):

IDEA: Wish to manipulate organelles expressing GFP and use nanoparticles as handles for the optical tweezers. S. pombe perfect model system.

Nano-mekanics of cell division

Mikroinjection of nano partiklerProblem:S. pombe has an extremely stiff cell wall.Solution:Enzymatic breakdown of outer parts, microinjection of protoplast, followed by cell regeneration.

Conclusions - Perspectives

Technical issues• Possible to trap individual nano-particles, e.g. gold and silver spheres, gold rods,• Non-invasive, provided correct wavelenght, small energy deposit.

Examples of biological applications• Molecular motors: polymerase, kinesin, ribosome, virus.• Mechanical strength of mRNA hairpins, pseudoknuder, • Non-equilibrium nano-scale systems. • In vivo studies, viscoelasticity of cellular cytoplasm. • Nano-mechanics of cell division

FutureSingle molecule studies of nanotoxicologyForce measurements in vivoViral infectionsCombination with other techniques

Check out; www.nbi.dk/~tweezer