Optical tweezers application to single molecule manipulation

Nathalie Westbrook Laboratoire Charles Fabry de l’Institut d’Optique

nathalie.westbrook@institutoptique.fr

Optical tweezer: dielectric objects can be trapped at the focus of a laser beam

works for atoms, molecules, micron-size beads, viruses, bacteria, living cells, organelles…

trapping a yeast cell (43 s)

Well suited to the study of forces involved in biological processes: mechanical properties of DNA, cellular motility, DNA transcription, ribosome translocation, … 1

Principle of optical trapping

Orders of magnitude, optimal trapping conditions

Trap displacement and multiple traps

Direct trapping of biological objects: cell sorting, cell membrane elasticity, …

Application to single molecule manipulation: forces are applied on handles (spherical beads), and precise calibration is required

Outline

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Optical Tweezers : the first experiments

Ashkin 1970 –! Levitation of dielectric beads (!~µm) +

acceleration

–! 3D trap using 2 laser beams

(applied to atom trapping)

Ashkin 1987: manipulation of biological objects (bacteria) Ashkin PRL,

1970

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Principle of trapping with optical tweezers Rayleigh regime (small particles<λ)

F. Gallet, cours école prédoctorale Les Houches 2003

Dielectric object with polarisability α$

Gradient force

Laser with intensity I Force proportional to E2

∝  intensity I if E corresponds to an electromagnetic wave

The force traps the object at the point of maximum intensity (object with index higher than surrounding medium)

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Interpretation in the ray optics regime (Mie scattering: particle size > λ)

Lateral trapping Longitudinal trapping

Momentum transfer due to refraction

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From Physics World 2008

Radiation pressure force

Interpretation of this force in the Rayleigh regime (bead diameter a<<λ)$Elastic diffusion (absorption/spontaneous emission in the case of an atom) The incident/absorbed photons are always in the direction of the laser, the diffused/emitted photons are scattered in all directions.

The bead is also submitted to a force that pushes it in the direction of propagation of the laser beam: it opposes the (longitudinal) trapping

Interpretation of this force in the ray optics regime (Mie scattering a>> λ)

Mechanical effect of the light reflected on the dielectric bead

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How to make a stable trap with one beam?

The bead can stay trapped if:

Fgradient > Fradiation pressure

We thus need a strong gradient: & sharply focused laser

Microscope objective with high numerical aperture (water or oil immersion)

Fscatt

Fgrad

NA>1,25

Other option: two counterpropagating laser beams

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Optical tweezers: typical orders of magnitude

Force measurement: 1 to 100pN for micron size objects

Coupling with position measurements with a few nm accuracy

Well suited tool for quantitative manipulation at the subcellular or molecular scale

(relatively) Non invasive method

There are other methods of micromanipulation : magnetic tweezers, atomic force microscopy,… (torque, larger forces) See Neuman&Nagy review in Nature Methods, 2008

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Optimal trapping conditions

Bead size a ~ λ ~ 1 µm Rayleigh regime a<<λ: Fgrad increases as a3

Mie regime a>> λ: Fgrad independent of a

Choice of wavelength to minimize absorption (heating/destruction of the biological sample) near infrared: NdYAG 1,06µm or laser diode ~ 0,8µm Power depends on the required forces: typ 10pN for 100mW incident power

Immersion microscope objective: NA>1,25 for maximum gradient good transmission in the IR water immersion easier (no spherical aberration introduced by the microscope slide when the trap is away from the surface)

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Mirror on galvo or acoustooptic

modulator

Displacement of the trap

Entrance pupil

Afocal telescope

F. Gallet, cours école prédoctorale Les Houches 2003

Trap position

dichroic mirror

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Displacement of the trap

Entrance pupil

The afocal telescope

- expands the beam so that it covers the entrance pupil

- images the moving mirror onto the entrance pupil 11

Multiple traps

You can also use an hologram to create any distribution of multiple traps (you project the image of that hologram on the entrance pupil of the microscope objective)

2 !m

Visscher et al, 1996

Multiple trap with AOD

Mirror or AOD

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A few videos of optical trapping Different objects were trapped in the student and research labs at Institut d’Optique:

-! dielectric beads (diameter 1 µm) : two laser powers and a second bead enters the trap 1’15’’

-! Live bacteria around buccal cells (typ size 1 µm) 29’’

-! Moving around yeast cells (typ size 5 µm) 25’’, another video 43’’

-! Deformation of red blood cells (typ size 8µm) 48’’

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Direct trapping of biological objects Using the deformability of cells as a measure of their malignancy

The « optical stretcher » J. Guck et al, Univ. of Cambridge, UK

Using multiple holographic traps for cell sorting

Dholakia et al, Univ. of St Andrews, UK

Application to single molecule manipulation

•! Mechanical studies on biopolymers (DNA, RNA) to which a dielectric bead is attached:

!! stretching of DNA !! unzipping the double helix (« mechanical » sequencing)

•! Study of molecular motors : myosin on actin (muscle contraction), kinesin on microtubule (cellular cargo), RNA polymerase (transcription), ribosome motion (translation), …

Requires a precise calibration of the force applied to the bead

•! calibration of the position of the bead relative to the center of the trap (in nm)

•! calibration of the stiffness of the trap (in N/nm) 15

Force measurement From the position calibration and the stiffness calibration Distance x from center when force is applied

Ftrap=-'x

mRNA

Ftrap

FRNA

hairpin

x

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4 quadrants photodiode (in the condenser back focal plane)

Condenser

Objective

Back focal plane

Detecting the position of the bead with nanometer resolution

Imaging of the bead through videomicroscopy can be accurate (centroid calculated with a few nm accuracy) but slow.

Method preferred: back focal plane interferometry Interference in the far field between the direct laser light and the light forward scattered by the bead

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Understanding back focal plane interferometry: effect of a displacement of the bead

Laser focus Trapped bead Condenser

Detection in the back focal plane of

the condenser

Displacement of both bead and trap: no signal

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Calibration of the position of the bead with respect to the center of the trap

Objective: calibrate the position detector in volts/nm

Three possibilities to move the bead by a known amount: -  bead stuck on the microscope slide by lowering down the

trap, known displacement using a piezoelectric microscope stage

-  Trap moved rapidly by a known amount, so that the bead initially has no time to move

-  One separate laser for the detection: the bead can be moved using the trap by a known amount

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Using two separate lasers for trapping and position detection

Lang et al, Biophys J 2002

The displacement of the bead by moving the trap laser can be calibrated

using the position detection laser

The two lasers are superimposed but differ both in polarization and wavelength, so only the position detection laser is detected on the quadrant photodiode

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Observing the Brownian motion of the bead in the optical trap

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Fluctuation of motion of a bead in an harmonic trap with stiffness ' under the influence of collisions with the fluid

Educational simulation about optical tweezers and applications at University of Colorado, Boulder:

http://phet.colorado.edu/en/simulation/optical-tweezers

Demo of brownian motion

Calibration of the trap stiffness Bead brownian motion due to gradient force + viscous drag 1st method: Power spectrum of the bead motion with cut off frequency fc

fc= κ/2πβ κ : trap stiffness β : viscous drag β=6πηa η : viscosity a : bead radius

2nd method: equipartition theorem

x: position of the bead in the trap

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Simultaneous calibration of bead displacement and trap stiffness

Antoine Le Gall, PhD thesis at IOGS 2011 Optics Letters dec 2010

Using an AOM, the trap laser is moved rapidly by a known amount: the bead initially has no time to move and then we measure how fast it comes back

Height of peak calibrates the position of the bead

Decay time calibrates the stiffness

Motion of kinesin along microtubules

Displacement of kinesin by 8nm steps

Using the force clamp mode: servo loop on the laser trap position to maintain a constant force during the motion of the kinesin

Lang et al, Biophys J 2002

Steven Block’s group at Stanford Univ

Bead displacement

trap displacement

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Reaching high resolution using a dual trap arrangement Study of the RNA polymerase (RNAP) motion along DNA to transcribe it into RNA

Abbondanzieri et al, Nature 2005 (Steven Block’s group at Stanford)

Record resolution of 0.35nm (single base pair)

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Following translation by single ribosomes one codon at a time Wen, Lancaster, Hodges, Zeri,Yoshimura, Noller, Bustamante & Tinoco Jr (Berkeley)

Nature 2008

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Combining fluorescence techniques and optical tweezers

Ishijima et al, Cell 1998

Yanagida group at Osaka Univ

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Example of displacement that coincides with Cy3-ADP release

Combining single molecule fluorescence and optical tweezers: avoiding photobleaching induced by the

trap laser

Alternating trap and fluorescence to reduce photobleaching

Brau et al, Biophys J 2006

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Examples of commercial systems

JPK Nanotracker (dual beam trap + 3D tracking with high resolution)

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Thorlabs modular kit

Zeiss (combined OT&microdissection)

A few references •  Recent advances in optical tweezers, Moffitt, Chemla, Smith & Bustamante, Annu. Rev. Biochem. 2008, vol 77

•  Single molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force spectroscopy Neuman & Nagy, Nature Methods, june 2008

• Light forces the pace: optical manipulation for biophotonics Stevenson, Gunn-More & Dholakia, J. of Biomedical Optics, 2010

• Photoniques, N° de juillet-août 2013: la pince optique (principes et applications par JP Galaup + guide d’achat)

•  High-resolution, long-term characterization of bacterial motility using optical tweezers, TL Min et al, Nature Methods 2009 (Univ. Of Illinois at Urbana-Champaign)

•  Recent advances in laser tweezers Raman spectroscopy for label-free analysis of single cells, JW Chan, J. Biophotonics 2013 (Univ of California, Davis)

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1)  Numerical experiment Educational simulation about optical tweezers and applications at

University of Colorado, Boulder:

http://phet.colorado.edu/en/simulation/optical-tweezers

Demo

2) Live experiment in the student lab Trapping 1micron silica beads with a 20mW red diode laser

3) Visit the OT experiment in our lab Single Molecule Biophysics Team (Biophotonics group) at Institut d’Optique – Flavie Gillant (phD), Karen Perronet

-  new calibration methods

- OT on cell membrane to understand transduction to the nucleus (atherosclerosis)

Experiment optical tweezers yourself