Transcript Optical tweezers - Niels Bohr Institutet

Optical Tweezers
status 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-how
2 force-scope optical
tweezers:
3D spatial resolution ~1nm
Temporal resolution ~ MHz
Single particle tracking:
3D spatial resolution ~5nm
Temporal 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 powerloss
Silicon is transparant to infrared light.
Makes diode a 1st order filter.


2


Pout ( f )
1 a
2
 a 

2
Pin ( f ) 
(diode) 
1

f
f


3dB


(diode)
f3dB

8 kHz and depends on laser intensity.
a: fraction of incoming power that is correctly detected in
depletion layer.
Berg-Sørensen, Oddershede, Florin, and Flyvbjerg, J.Appl. Physics. vol. 93 p.3167 (2003)
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 3D
Mie 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 with
diameters ranging from 8nm to 44nm and aspect rations between
1.7 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.
I m  r  s  2 r s sin 
2
2
r: field reflectivity (background)
s: scattered field with phase f.
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
The obtained spring constant, , carries information about the interaction
between the EM field and the Qdot. Using the following relations, the
polarization of an individual Qdot can be found:
Fgrad 
a
2
2I
2
E 
c
I  I0 e

P

( E 2 )
where
I : intensity
P: total laser power delivered at the sample
x,y: directions orthogonal to the light propagation
: width of diffraction limited laser spot.
 ( x 2  y 2 ) / 2 2
Idxdy  I 0 2 2

Fgrad   x
2 c 4
a 

P
Normalized by vacuum permittivity, we obtain
a = 2.8 x 107 Å3.
Literature very sparse, only one value reported for
Qdots with radius=2nm: a ~ 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 living
organisms, 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 maintain
a 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
pHi (t  0)  pH ex
pH  t =τ½  
2
GFP, anaerobic
(weak signal)
Listeria
CFDA, aerobic
CFDA, aerobic
monocytogenes
innocua
CFDA, anaerobic
CFDA, anaerobic
Physiological damage depends on growth
conditions and bacterial species
Fluorophore
Laser
Power
(mW)
Initial pHi
+
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
Species
Aerobic
growth
E. coli
M.B. Rasmussen, L. Oddershede, and H. Siegumfeldt,
Applied and Environmental Microbiology, vol. 74 p.598 (2008)
τ½a
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:
Varians: <x2(t) > = 2Dta
Varians: <x2(t) > = 2Dt
2
P ( f )  x~ ( f ) 
Begrænset
a=0
2
~
F( f )
2
P ( f )  x~ ( f ) 
2 2 f 2
Subdiffusion
0<a<1
1.
2
~
F( f )
2 2 f (a 1)
Normal diffusion Super diffusion
a=1
a>1
Polymer netværk
(a = 0.75)
1. Molekylære motorer
2. Polymerisering
2.
Membraner
(a = 0.66)
3. Cytoplasmatisk strømning
How do the lipid granules move?
mostly bý subdiffusion, a=0.75
 2
r ( t )  t a
Normal diffusion
Super diffusion
Plateau pga. endelig
celle størrelse
Optical
tweezers
Single particle tracking
Nano-mekanics of cell division
S. pombe strains expressing green fluorescent protein (GFP):
IDEA: Wish to manipulate organelles expressing GFP and use nano
particles as handles for the optical tweezers.
S. pombe perfect model system.
Mikroinjection of nano partikler
Problem:
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
Future
Single molecule studies of nanotoxicology
Force measurements in vivo
Viral infections
Combination with other techniques
Check out; www.nbi.dk/~tweezer