Presentation on theme: "Trapping for Biophotonics Kishan Dholakia SUPA, School of Physics and Astronomy University of St Andrews, Scotland"— Presentation transcript:
Trapping for Biophotonics Kishan Dholakia SUPA, School of Physics and Astronomy University of St Andrews, Scotland
This talk Overview of “state-of-the art” for complex optics (light beam shaping) for cell manipulation, sorting and cell transfection (nanosurgery) Emphasis on topics relevant for the interdisciplinary community Industrial Physicist 1999
F=? Optical forces: small but significant Very small!! But for a microscopic sphere use Newton’s laws…..
The ORIGINAL TRAP developed by Ashkin in 1970 Potentially easy to align/trap, No high Numerical Aperture optics Interesting for studies in microfluidics, high throughput, Raman Ashkin, Phys Rev. Lett. 24, 156 (1970); Constable et al. Opt. Lett (1993) video: M Ritsch-Marte Group, Innsbruck, Austria Two counter-propagating beams (fibre optic) trap
Light forces may probe cancer: optical stretching J. Guck et al., Biophys. J. 88:5 (2005) B. Lincoln et al., Cytometry 59A (2004) Even metastatic cancer cells (modMCF7) can be distinguished from less aggressive cancer cells (MCF7) and from normal cells (MCF10) 144
Elasticity-based Flow Cytometry Heterogeneous Sample from Tumor Optical Stretcher Normal and Background Cells Primary Tumor Cells Metastatically Competent Cells B. Lincoln et al., Cytometry 59A:203, 2004 Neuronal precursors Glial precursors Others k Primary culture 143 Inherent cell marker obviates the need for preparation No contamination with fluorescent markers or magnetic beads No immune response from the use of antibodies Leaves cells unaltered and uncontaminated for therapeutic use (e.g., selecting out stem cells for regenerative medicine) Matching of mechanical properties to delivery mechanism and mechanics of host tissue Optical deformability of cells = tightly regulated cell marker for stem cell sorting, cancer diagnosis, etc.
A Ashkin et al, Opt Lett 11, 288 (1986) Quake Group: Phys. Rev. Lett. 91, (2003) Optical forces: subtle but important!
Laser Pick frequency to minimize absorption (no “optocution”).: eg 780nm-1100nm Power depends entirely upon application Beam Quality: M 2 < 1.1, TEM 00 typical Pointing stability: critical for high-res. work Objective Lens Magnification doesn’t matter, aberrations do A numerical aperture (N.A.) > 1.2 is essential if 3D traps are required At the heart of a basic trap:
(for small displacements) A Parabolic Potential Energy “Well” For a given laser power and particle size, trapped matter experiences Eric Dufresne, Ph.D. thesis,
With no damping (e.g., in vacuum) the result would be a resonant frequency as follows: Mass of object: 5 x kg Typical trap stiffness: -- where is the elastic constant or stiffness of the optical trap and is the damping parameter. A Classical Oscillator A parabolic “well” implies a linear relationship between force and displacement, as with a mass on a spring.
In typical biological applications, the stiffness of the optical tweezers is around 0.05 pN/Nm and the trapped objects are around diameter 1 micron, corresponding to a mass of kg.. Hence, the resonant frequency would be around 50kHz. However, because biological experiments must be performed in an aqueous medium, significant damping force arises. For a particle of radius r, moving in a fluid of viscosity, the Stoke’s drag constant For typical biological application we find that the roll-off frequency well below 1 kHz. Since this is much lower than the resonant frequency, the motion is very over- damped. In fact, it means that inertial and gravitational forces can be ignored altogether
PicoNewton forces 1 picoNewton (pN, N) is roughly equal to… … the gravitational attraction between you and a book at arm’s length … the radiation pressure on a penny from a flashlight 1 yard away … 1 millionth the weight of a grain of salt MotorsAntibody-antigenb-avidinFibroblasts FORCE (pN) ,000 Optical Tweezers Atomic Force Microscope
Tethering or Clamping of Single Molecules From S. Block lab: Single-Molecule Biology: study of molecular motors (“rowers and porters”) requires: we INDIRECTLY hold the molecule with tweezers by attaching it to a glass bead
All living cells contain a wide variety of molecular motors that take chemical energy and convert this to work. Functions that are essential to life: eg DNA replication, RNA transcription and protein synthesis to cell division, vesicle trafficking, cell locomotion, endocytosis. Two types of motor: “Rotary motors” are embedded in membranes and are driven by the flow of ions across transmembrane electrochemical gradients; eg the bacterial flagellar motor. “Linear motors” work in an isotropic chemical environment: energy from chemical reactions, usually the hydrolysis of the chemical, adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and phosphate.
Force :required to: rupture a covalent bond = 1000pN; convert DNA from a double helix to a ladder =50pN; break most protein-protein interactions =20pN ; force produced by most motor proteins = 5pN. Length: diameter of a bacterium and optimal size for beads held in optical tweezers =1 micron resolution of light microscope = 300nm; diameter of eukaryotic cell organelles = 100nm; large protein assemblies and virus particles =30nm; work stroke produced by motor protein =5nm; Adapted from Molloy and Padgett, Contemp. Phys (2002) What can we measure?
From sphere displacements and trap stiffness, infer macromolecule forces/dynamics: e.g also Berg-Sorenson and Flyvbjerg Rev Sci Instrum (2004) Force measurements in OT 55
Use of a quadrant photodiode provides higher capture rate than CCDs while retaining nanometer-scale position detection (“centre of gravity”) Force is proportional to Displacement Project a magnified image of the trapped sphere onto a quadrant photodiode. The position of the sphere is defined by differential signals from the quadrants. AB C D
20 Use of a quadrant photodiode provides higher capture rate than CCDs while retaining nanometer-scale position detection (“centre of gravity”) Force is proportional to Displacement Christoph Schmidt group: Use dark field, phase contrast or interferometric methods. AB CD Project a magnified image of the trapped sphere onto a quadrant photodiode. The position of the sphere is defined by differential signals from the quadrants. 54
21 From sphere displacements and trap stiffness, infer macromolecule forces/dynamics: From Molloy and Padgett, Contemp. Phys 43, 241 (2002): see also Berg-Sorenson and Flyvbjerg Rev Sci Instrum (2004) Force measurements in OT 55
23 Mechanical recording made from a single processive kinesin, a molecular porter that walks along the microtubule track taking steps that are commensurate with the microtubule 8nm lattice repeat. In the experiment a single (double headed) kinesin molecule was attached to a latex microsphere held in optical tweezers. The position of the microsphere was monitored using a 4-quadrant detector, as the kinesin walked along a fixed microtubule track (cartoon, upper). Note that the staircase structure to the position data (graph, lower) is a direct result of the single kinesin pausing in between individual ATP cycles. This record was kindly provided by Drs. N J Carter and R A Cross, Molecular Motors Group, Marie Curie Research Institute, Oxted Rh8 0TL Surrey. For further information please see:
24 Recent work has achieved angstrom resolution Nature, Nov 2005
Tying a knot in DNA… The enzymology of topoisomerases at the single molecule level. Such polymeric topological constraints arise naturally in cells during DNA Replication. Xiaoyan R. Bao, Heun Jin Lee, and Stephen R. Quake, Phys Rev Lett 91, (Dec 2003)
Why shape your light field (need for complex optics)? -Gaussian beam limiting: depth of focus -selective excitation (STED) -Novel beam shapes: trapping low index particles, mixing droplets -Rotation: studies of angular momentum and microrheology -Multiplexed studies in biology: enhanced depth of focus -Cell and colloid organisation or sorting in 2D/3D - Multi-particle interactions -Creation of optical potential energy landscapes -Chemical reactions: single droplet control
Liquid Crystal “Spatial Light Modulator” (SLM) Lens Holographic Image Acousto-Optic Deflectors (AODs) can be scanned at hundreds of kHz: Multiple traps: creating complex optics (advanced beam shaping)
Time sharing the light field can create multiple traps positions. (here achieved with an AOD) (This video in collaboration with I Poberaj group).
If the laser is elsewhere (at other traps, or travelling between trap sites), then a bead will diffuse, ti meaway from its nominal trap site, a distance: For a 1-micron diameter bead in water, the diffusion coefficient is: So, if the laser is absent for 25 microseconds, the bead is expected to diffuse 5nm. This represents a maximum limit to the accuracy to which the spheres can be positioned when using time-shared trapping. When the laser’s away, the beads will stray!
31 “Steering” with a Phase-Only Optic is equivalent to …Beam steering:a phase retardation:
32 Liquid Crystal Display Technology Liquid Crystal “Spatial Light Modulator” (SLM) Lens Holographic Image Dynamic control is possible through SLM technology also allows for easy creation of beams with novel characteristics: 71
Diagram on left from D. G. Grier and Y. Roichman, Holographic optical trapping, Applied Optics 45, (2006). The phase modulating optic (a “Diffracting Optical Element”) is effectively positioned at the entrance of the objective lens, by making the DOE/SLM conjugate to the back aperture. Spatial Light Modulator
Optical micrographs showing 2D microarrays of P. aeruginosa bacteria. (a) A transmission micrograph of a 21 × 21 2D microarray of P. aeruginosa formed with a 100×-, 1.25-NA oil immersion (Zeiss Plan-Apo) objective at λ = 900 nm using <2 mW per trap. (b) A false-color isosurfaces were generated from volumetric data obtained from deconvolved confocal images of a 5 × 5 microarray of P. aeruginosa assembled with a 100×-, 1.3-NA oil immersion (Zeiss Plan-Apo) objective at λ = 514 nm using <2 mW per trap, and embedded in hydrogel. The average center-to-center distance is 1.52 ± 0.06 μm and the average space between each bacterium is 354 ± 134 nm. (c) A 3D representation of (b). Taken from Akselrod et al., Biophysical Journal 9, 3465 (2006) Combining the SLM and AOD to pattern cells at will..
Examples of artificial arrangements of live mouse stem cells created with the optical tweezers. In addition to showing a range of different patterns of cells it also illustrates control of distance relative to another cell, ranging from direct physical contact to separation of distances of 1–2 mm (top left panel) to several mm (panels showing faces of a dice). Patterning stem cells with holographic traps Manipulation of live mouse embryonic stem cells using holographic optical tweezers Jonathan Leach et al. J Mod Opt 56, 448 (2009)
Introductory Reference: Dholakia, Spalding, MacDonald, Physics World, Oct Beams used in optical traps need not be Gaussian. (a) Laguerre-Gaussian (LG) optical modes have helical wavefronts (b), which - in addition to polarization - control the angular momentum transmitted to a trapped particle. Trap low index particles. (c, d) Bessel beams have a number of special properties useful in particle manipulation. Novel Beams Extend the Optical “Toolkit”
Vortices in Nature Credit: NASA Langley Research Center (NASA-LaRC). Wake vortex study at Wallops Island Optical Vortices: useful for manipulating droplets/cells Complex Optics: creating and using optical vortices
Schematic and images that illustrate the positioning and subsequent repulsion of two aqueous droplets in the dual vortex trap. Simulated and measured spatial intensity profiles for the optical vortex beam during the displacement of the hologram, which caused a lateral shift in embedded phase singularity or dark core Fusing droplets with optical vortices Lorenz et al. Anal. Chem. 2007, 79,
“Non-Diffracting light”: Bessel beams White light Bessel modes: P. Fischer et al. Opt Express 13, 6657 (2005) Reformation or self- healing Durnin et al, JOSA A and PRL 1986/1987 S Tatarkova et al. Phys Rev Lett (2003); L Paterson et al. Appl. Phys. Lett (2005); J Biomed Opt (2007) - stem cell sorting
40 Flow-free optical sorting: selection by BB or motion on a BB pattern red/white blood cell sorting in a Bessel beam Appl Phys Lett 87, (2005); J Biomed Opt 12, (2007)
Acknowledgements Praveen Ashok Rob Marchington Patience Mthunzi Xanthi Tsampoula M MacDonald* V Garces-Chavez* Tomas Cizmar David Stevenson Joerg Baumgartl Michael Mazilu Tom Brown Wilson Sibbett Thomas Krauss School of Biology Frank Gunn-Moore School of Medicine Simon Herrington Andrew Riches