Micromanipulation techniques: Optical Tweezers and force measurements

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Micromanipulation techniques Optical Tweezers
and force measurements Force measurements and
light microscopy
Marcel Janson FOM Institute AMOLF Amsterdam,
The Netherlands

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FOM Institute AMOLF Amsterdam Physics lab with
biophysics interest
Group Bio-assembly and organization Marileen
Dogterom Astrid van der Horst Cendrine Faivre
Jacob Kerssemakers Mathilde de Dood Astrid
van der Horst Martijn van Duijn Gerbrand
Koster Eva Riemslag Marco Consentino
Lagomarsino Henk Bar

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Outlook
1. Micro manipulation with Optical Tweezers
Working principle Applications on living
cells Single molecule studies / in vitro work
2. Microtubule force generation
Microtubule growth and catastrophes Experiment
s with micro fabricated barriers Use of Optical
tweezers (work in progress)

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Optical Forces
Light momentum. Light refraction change in
momentum Force.
Comet tail points away from the sun
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Optical trapping
Light momentum. Light refraction change in
momentum Force.
Microscope objective
Unfocused beam light gradient
Focused beam
resulting force
resulting force
1 micron
Small particles can be trapped and manipulated
under the microscope
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Movie bead_trapping.mov
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Adjustments made to microscope
Lens 2
Optical Fiber
Lens 1
y
x
z
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Beam path
Illuminating light
IR light gives minimum optical damage to
biological samples A high NA-lens (1.3) is needed
for high trapping forces
condenser
specimen
objective
Lens 1
Lens 2,xyz
CCD camera
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Multiple tweezers
Time sharing of light over many locations creates
multiple traps
Illuminating light
condenser
y
specimen
objective
x
Lens 1
Lens 2,xyz
AOD y
Acoustic Optical Deflector (AOD) has the same
effect as quickly moving moving lens 2 100 to
1000 Hz
AOD x
CCD camera
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Multiple tweezers
Time sharing of light over many locations creates
multiple traps
Illuminating light
condenser
y
specimen
objective
x
Lens 1
Lens 2,xyz
AOD y
Acoustic Optical Deflector (AOD) has the same
effect as quickly moving moving lens 2 100 to
1000 Hz
AOD x
CCD camera
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Application of Forces
F k x ( 1 pN / nm) Trap stiffness k is constant
over 100 nm Maximum force 100 pN
Coverslip
Pico (10-12) Newton Forces Single molecule
forces ! 1 pN Weight of a red bloodcell 6
pN Stall Force of a single molecular motor
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Application of Forces
x
F k x ( 1 pN / nm) Trap stiffness k is constant
over 100 nm Maximum force 100 pN
F
Coverslip
Pico (10-12) Newton Forces Single molecule
forces ! 1 pN Weight of a red bloodcell 6
pN Stall Force of a single molecular motor
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Position detection
Four signals give the x and y coordinate of the
object
Light is imaged onto a Quadrant photo detector
Condensor
Coverslip
Objective
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Position detection
Four signals give the x and y coordinate of the
object
Light is imaged onto a Quadrant photo detector
Condensor
Coverslip
Objective
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Trap calibration
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1/2kBT
?r
Due to its thermal energy of 1/2kBT a trapped
particle will not always be in the energy
minimum. Detector signal can be used to find trap
constant k (pN/nm)
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Trap calibration
E
1/2kBT
?r
Due to its thermal energy of 1/2kBT a trapped
particle will not always be in the energy
minimum. Detector signal can be used to find trap
constant k (pN/nm)
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Trap calibration
E
Low intensity Floppy trap
1/2kBT
?r
Due to its thermal energy of 1/2kBT a trapped
particle will not always be in the energy
minimum. Detector signal can be used to find trap
stiffness k (pN/nm)
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Example 1 Forces generated by crawling cells
Fibronectin-coated beads are placed on cell
surface Connection to Force generating
cytoskeleton Forces different along the cell
Galbraith et al, Cell, 2001
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Example 2 Trapping of organelles
Prerequisite for trapping contrast in index of
refraction protein rich virus or
bacteria chromosomes Densely packed DNA in
nucleus (root hair) Problem low contrast low
forces
Drosophlia embryos Lipid droplets move on
microtubules Fraction that escapes trap measure
for number of motors Fraction changes during
development
2.5 ?m
welte et al, Cell, 2001

Organelle manipulation but quantitative forces
are hard to obtain
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Example 3 Forces generated by single kinesin
molecules
Visscher et al, Nature, 99
8 nm x 6 pN 48 pNnm 60 ATP
Movie kinesin milligan.mov http//www.scripps.edu
/milligan/projects.html
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Example 4 DNA packaging in bacteriophage ?29
Micro pipette as manipulation tool 6.6 micron DNA
in 42 by 54 nm capsid. Motor runs on ATP 57
pN Different speeds at different packing levels
Smith et al, Nature, 2001
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Part 2 Force generation by growing microtubules
C. rieder
Growing ends of microtubules bump into
organelles Force generation ?
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Dynamic instability of microtubules
Seed
Movie freecat no wall.mov
Seed location
Dynamic instability The switching between phases
of microtubule growth and shrinkage. GTP
hydrolysis is needed for catastrophes, not for
polymerization.
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Dogterom, M. and B. Yurke (1997), Science 278,
856-860.
Microtubule buckling
L
seed
F
2 ?m
50 ?m
Free Microtubules grow from surface connected
seeds towards barriers. Microtubule generates its
own opposing force. Broad force range can
be covered by different seed-barrier distances.
Barrier
Undercut
L ?10 ?m, F ? 7 pN
Glass
Glass
Elastic deformation used for force
measurements
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Buckling and catastrophes
Movie grbuck5.mov
Movie wallcat.mov
Long Microtubule Fast growth Catastrophes are
rare
Short Microtubule No growth, Stall
Force Catastrophe after 20 sec. (500 sec.
normally)
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Force velocity curves
Dogterom, M. and B. Yurke (1997), Science 278,
856-860.
Growth velocity goes down with increasing force.
Forces up to 30 pN at relatively slow growth
velocities. Kinesin (4-6 pN).
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Modelling
Strong lateral interaction limit Peskin, C.S.,
G.M. Odell and G.F. Oster (1993), Biophysical
Journal 65, 316-324.
No lateral interaction limit Mogilner, A. and G.
Oster (1999), European Biophysics Journal
with Biophysics Letters 28, 235-242.
Catalin Tanase
Forces predicted by theory depend on the assumed
microscopic details of the growth process
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Nuclear positioning, role for pushing forces
Microtubule
Cell cortex
Centrosome
Chromosome
Kinetochore
In Fission yeast catastrophes occur more often
while contacting the cell wall. Microtubules grow
less fast.
Tran et al, JCB, 2001
Similar effects of force on microtubule dynamics
is seen in fission yeast. Other proteins, like
tip1 probably play additional roles Brunner and
Nurse.
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Use of elastic deformations in force measurements
AFM
Crawling cells
Galbraith et al, Cell, 2001
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Optical tweezers based force measurement
Monitor microtubule dynamics at short time and
length scale. Stall force can be measured for
short microtubules
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Aster manipulation inside micro-chamber
Micro-fabricated chambers
Cendrine Faivre
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Summary
Part 1 Optical tweezers combine light microscopy
and force measurements nano meter displacements /
pico Newton forces can be observed Applications
single molecule studies / probing cellular
dynamics Part 2 Elastic deformations used to
measure polymerization forces of
microtubules Force affects growth velocity and
catastrophe time Optical tweezers give more
freedom for manipulation

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Elastic deformations of force generating
microtubules
1. Effect of force on microtubule dynamics is
twofold a. growth velocity slows down b.
catastrophe probability goes up
2. Polymerization forces exceed 10 pN !
3. Growth details determine forces.