Optical tweezers application to single molecule manipulation

Transcription

1 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

2 Outline 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 2

3 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,

4 Principle of trapping with optical tweezers Rayleigh regime (small particles<λ) Dielectric object with polarisability α$ Gradient force Force proportional to E 2 intensity I if E corresponds to an electromagnetic wave Laser with intensity I The force traps the object at the point of maximum intensity (object with index higher than surrounding medium) F. Gallet, cours école prédoctorale Les Houches

5 Interpretation in the ray optics regime (Mie scattering: particle size > λ) Momentum transfer due to refraction Lateral trapping Longitudinal trapping From Physics World

6 Radiation pressure force 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 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. Interpretation of this force in the ray optics regime (Mie scattering a>> λ) Mechanical effect of the light reflected on the dielectric bead 6

7 How to make a stable trap with one beam? The bead can stay trapped if: F scatt F gradient > F radiation pressure We thus need a strong gradient: & sharply focused laser Microscope objective with high numerical aperture (water or oil immersion) F grad NA>1,25 Other option: two counterpropagating laser beams 7

8 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,

9 Optimal trapping conditions Bead size a ~ λ ~ 1 µm Rayleigh regime a<<λ: F grad increases as a 3 Mie regime a>> λ: F grad 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) 9

10 Displacement of the trap Trap position Entrance pupil dichroic mirror Afocal telescope Mirror on galvo or acoustooptic modulator 10 F. Gallet, cours école prédoctorale Les Houches 2003

11 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

12 Multiple traps Visscher et al, 1996 Multiple trap with AOD 2!m Mirror or AOD 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) 12

13 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 !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 13

14 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 14

15 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

16 Force measurement From the position calibration and the stiffness calibration Distance x from center when force is applied F trap =-'x x hairpin mrna F RNA F trap 16

17 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 Back focal plane Condenser Objective 4 quadrants photodiode (in the condenser back focal plane) 17

18 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 18

19 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 19

20 Using two separate lasers for trapping and position detection The two lasers are superimposed but differ both in polarization and wavelength, so only the position detection laser is detected on the quadrant photodiode The displacement of the bead by moving the trap laser can be calibrated using the position detection laser Lang et al, Biophys J

21 Observing the Brownian motion of the bead in the optical trap 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: Demo of brownian motion 21

22 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 f c f c = 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 22

23 Simultaneous calibration of bead displacement and trap stiffness 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 Antoine Le Gall, PhD thesis at IOGS 2011 Optics Letters dec 2010 Decay time calibrates the stiffness 23

24 Motion of kinesin along microtubules Displacement of kinesin by 8nm steps trap displace ment Using the force clamp mode: servo loop on the laser trap position to maintain a constant force during the motion of the kinesin Bead displace ment Lang et al, Biophys J 2002 Steven Block s group at Stanford Univ 24

25 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) 25

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

27 Combining fluorescence techniques and optical tweezers Ishijima et al, Cell 1998 Yanagida group at Osaka Univ Example of displacement that coincides with Cy3-ADP release 27

28 Combining single molecule fluorescence and optical tweezers: avoiding photobleaching induced by the trap laser Brau et al, Biophys J 2006 Alternating trap and fluorescence to reduce photobleaching 28

29 Examples of commercial systems JPK Nanotracker (dual beam trap + 3D tracking with high resolution) Thorlabs modular kit Zeiss (combined OT&microdissection) 29

30 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 (Univ of California, Davis)

31 1) Numerical experiment Educational simulation about optical tweezers and applications at University of Colorado, Boulder: Demo Experiment optical tweezers yourself 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) 31