Basic Principles

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A New Light on the BiosciencesKishan
DholakiaSchool of Physics and
AstronomyUniversity of St Andrews,
Scotlandwww.st-and.ac.uk/atomtrapkd1_at_st-and.ac
.uk
SFM 2007
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This lecture

• Basic Principles
• Multiple traps, novel light fields
• A new light for bioscience
• Cell sorting Photoporation Raman

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Size Scale
Human Hair 60mm (0.06mm)
Red Blood Cell 10 microns (0.01mm)
Light may interrogate, trap and separate objects
at this scale 1 micron 1 millionth of a metre!
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Transverse and Longitudinal Forces
Gradients can yield
If both are present a 3D trap may result
Refraction makes light change momentum. In
return, the particle experiences an equal but
opposite change in momentum Ashkin et al. Opt
Lett 11, 288 (1986)
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Particles from micron size to a few tens of nm
may be trapped polarisability of the object is
key
Nanoparticle trapping opens up new bioscience
studies of Raman, imaging and tagging
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Dholakia, Spalding, MacDonald, Physics World,
Oct. 2002
Holographic /interferometric/time sharing can
create 2D/3D arrays of trap sites useful for
optical sorting/guiding and other studies
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Multiple traps creating optical landscapes
Lens
Liquid Crystal Spatial Light Modulator (SLM)
Holographic Image
Acousto-Optic Deflectors (AODs) can be scanned at
hundreds of kHz
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Time sharing the light field can create multiple
traps positions. Video in collaboration with I
Poberaj group, Slovenia
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Optical Vortex Holograms
Here color represents phase, which is a periodic
variable (0 - 2p) so the horizontal line is not a
discontinuity.
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Laguerre-Gaussian modes an optical vortex

• circularly symmetric modes, characterised by
• radial mode index p
• azimuthal mode index l (determines helicity)
  this leads to helical wavefronts and orbital
  angular momentum

p 0, l 0
p 0, l 1
p 0, l 4
L. Allen et al., PRA 45, 8185 (1992)
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Laguerre-Gaussian modes
l 0
l 1
l 3
J. Courtial et al., PhysRevLett 80, 3217, 1998
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Bessel light beams
Bessel beams have an intensity cross-section that
does not change as they propagate termed
propagation-invariant. THE CENTRE DOES NOT
SPREAD.
Durnin et al, JOSA A and PRL 1986/1987
With and being the radial and
longitudinal components of the wavevector
Radial intensity profile
Zeroth order Bessel beam
intensity
The Bessel beam showing the narrow central maximum
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Experimental Bessel beam

• Finite aperture limits propagation distance of
  non-diffracting central maximum to zmax
• The axicon offers the most efficient method for
  generating a Bessel beam in the laboratory

Of increasing interest in bioscience, eg OCT,
trapping, multiphoton processes
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Bessel beams an optical rod of light
P. Fischer et al. Opt Express 13, 6657 (2005)
OPN 2006 selected as a Highlight of Optics
2006
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Optical guiding in Gaussian and Bessel
femtosecond beams
H. Little et al., Opt Express 12, 2560 (2004) K.
Dholakia et al. New J. Phys. 6, 136 (2004)
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CW guiding
femtosecond guiding visualise the beam using
fluorescent dye
H. Little et al., Opt Express 12, 2560 (2004) K.
Dholakia et al. New J. Phys. 6, 136 (2004)
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Cell sorting
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ACTIVE SORTING
Nature Biotechnology  23, 83 - 87 (2004)
Published online 19 December 2004
doi10.1038/nbt1050 Microfluidic sorting of
mammalian cells by optical force switching Mark M
Wang, Eugene Tu, Daniel E Raymond, Joon Mo Yang,
Haichuan Zhang, Norbert Hagen, Bob Dees, Elinore
M Mercer, Anita H Forster, Ilona Kariv, Philippe
J Marchand  William F Butler
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Potential Landscapes
Eggs inhabit the lowest energy positions in an
egg-box
In Plinko the path followed by a counter can be
either deterministic (----)
or statistical
(----)
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Particle placement
Like in any landscape, the lowest energy position
for a particle is a function not only of the
landscape but also of the particles size and
density.
nsphere lt nmedium
nsphere gt nmedium
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Particle Escape
How do particles get from one location in a
landscape to another?
A particle can escape unaided from a local well
due to thermal activation. e.g. Kramers rates
McCann et Al. Nature Dec. 1999
Larger particles like cells are unlikely to
escape due to thermal activation alone.
Above 5 mm particles will usually require some
form of external activation.
A common example is the Stokes drag exerted upon
a particle by a fluid flow.
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Particle Activation Fluid Flow
In a single trap the particle
escapes in line with the
drag force
gradient force
Where a second trap is
e
-
e
ò
present the trajectory of
µ
h
0
d
V

e
the particle is affected
0
viscous drag
If the traps are linked then
ph
F 6
rv

there is a preferred route
of escape
s
viscosity
v
velocity
r
radius
In the extreme case we can
consider what happens in
a line trap
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Potential Landscape Plus Driving Force
The landscape can also comprise of a set of
parallel ridges like this roof
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Sorting in a Potential Landscape
But it is more interesting when the ridges go
across the fall-line
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Nature 426, 421 (2003) Optics Letters 32, 1144
(2007) See also D K. Ladavac et al. Physical
Review E 70, 010901(R) (2004).
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Hopping Silica Spheres aim to sort without flow
5µm
2.3µm silica spheres hopping into the centre of
a Bessel beam
Video is speeded up - real process 1/40 of the
speed shown here (normal video length about 20
minutes)
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Cell sorting with a Bessel Beam
Cell types tagged with spheres (anitbody-anitgen
binding) works well for small samples L. Paterson
et al., Appl Phys Lett 87, 123901 (2005) to
appear J Biomed. Optics (2007)
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Cell transfection
Transfection The transfer of exogenous DNA into
a cell. www.nature.com
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CW - Diode Based Approach

• inexpensve approach
• Low power requirement 1mW for 40ms
• Stable colonies of antibiotic resistant RFP / GFP
  expressing cells formed.

L. Paterson et al, Optics Express 13, p595
(2005) Femtosecond laser nanosurgery Vogel et
al, Appl. Phys B vol81, 1015 (2005)
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Femtosecond approach

• First demonstrated by Tirlapur et al (Nature,
  418, p. 290, 2002) other groups have used other
  short pulsed sources. J.S. Soughayer et al.,
  Anal. Chem. 72, 1342 (2000)
• Compatible with multiphoton microscopes
• Non-linear process - high intensity. I 10 W/cm

This study D. Stevenson et al, Optics Express
14, p7125 (2006)
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Tweeze and porate with the same laser
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fs - Transfection Efficency
Study based on 4000 cells
D. Stevenson et al, Optics Express 14, p7125
(2006)
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A different approach?

• Multiphoton process requires exact placement of
  cell membrane.
• Error of a few µm can prevent transfection.

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Bessel Photoporation(The Optical Syringe)

• Non-gaussian light beams behave differently.
• The bessel beam preserves a non-diffracting
  central core over long distances.
• Can perform fs-poration over a much greater
  depth of focus.
• Beam also self-heals - is this a further
  advantage?

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Practical Implementation
Axicon
Gaussian Beam
Pseudo-Bessel Beam
(a)
(b)

• In practice use axicon to form Bessel beam.
• Beam is then telescoped to give desired core
  size.
• Aspheric lens used to focus beam on sample.
• Non-diffracting core for fixed distance.
• Smaller the core, shorter propagation length.

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Photonics solves real biological issues..
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Self-healing of the Bessel beam shows poration
beyond obstacles
(c)



Self-healing in optical traps Nature 419, 145
(2002)
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Raman spectroscopy
The scattered light carries information on the
molecular constituents of the cell identify cell
abnormalities
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Fibre optic trap
NO HIGH-N.A. OPTICS Compatible with
microfluidics
Advantages for holding and analysing large cells
lower power density, compatible with
microfluidics, no intercellular
trapping/deformation
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S, McGreehin, R Marchington, P Reece, T Krauss .
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SEM images of lasers (a) cross-section through a
laser (b) lasers facing one another across the
channel (c) close-up of facets and channel wall.
Note that the channel insulation has not yet
been added.
Photographs of channel with insulation (a) plan
view (b) cross-sectional view
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Laser
Laser
Laser
Laser
Laser
Integrated optical micromanipulation S.
Cran-McGreehin et al., Lab-on-a-chip (Aug 2006)
S. Cran-McGreehin at al., Optics Express 14,
7723-7729 (2006)
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Photographs of device (a) mounted on PCB, with
tubes leading to pump (b) fine capillaries feed
fluid beneath the glass lid, sealed with NOA-71,
and electrical power is provided via wire-bonds
from the circuit board
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Use as a Particle detector

• The method uses one laser in forward bias the
  second laser is normally forward-biased to
  maintain the trap, but is periodically switched
  to reverse bias, when it acts as a photodetector.
  
• The measured photocurrent is of the order of
  100mA, and is reduced by the presence of a
  particle in the beam path. A larger particle
  blocks more of the light, giving a larger
  reduction in the photocurrent.

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Pathway
Cell samples
In vitro models
Tissue samples
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In vitro Models

• Cell systems
• Tumour cells well-characterised cell lines
• Lymphoid cells lymphoma, leukaemia
• Epithelial cells cervical, prostate, breast
  cancer
• Normal cells
• Engineered cells representing malignant pathways
• Primary keratinocytes expressing papillomavirus
  genes
• Primary bronchial epithelial cells expressing
  telomerase, CDK4 etc

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A single human keratinocyte cell held in an
optical trap
P Jess et al. Optics Express 14, 5779 (2006)
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Microfluidic Raman on demand single cell analysis
Raman acquisition time is minutes so this is low
throughput at the moment
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Removing fluorescence?
Why might annular beams assist the Raman signal?
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Focussing of Annular beams
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Generation and Detection of Fluorescence

• Fluorescence largely generated from bulk
  glass and optical coatings

Propagation of the fluorescence generated from
one specific point in an objective lens
Propagation of excitation beam

• The detected fluorescence from this single point
  depends upon
• Amount of fluorescence generated
• ie. Larger the excitiation beam larger detected
  fluorescence signal
• Distance away from paraxial axis
• ie. Closer to the centre of the lens larger
  detected fluorescence signal
• Distance away from the focal plane
• ie. Closer to the focal plane larger detected
  fluorescence signal

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Collection and detected efficiencies
Gaussian Peak intensity in centre Majority
of fluorescence generated centre and is
efficiently collected LG and hG Peak intensity
away Majority of fluorescence generated
away from centre from centre and not
efficiently collected
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Experimental Results
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Signal to noise measurements

• The ratio between the Gaussian and
  Laguerre-Gaussian (red) / holey-Gaussian (blue)
  acquisition times for a given standard
  deviation.
• Improved acquisition rates of the annular
  beam.

• The wavenumber dependence of the standard
  deviation calculated from 125 successive spectra
  
• Annular beams generate less noise
• I Cormack et al., APL 91, 023903(2007)

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Acknowledgements Integrated Trap S McGreehin, T
Krauss, R Marchington and P Reece Nanoparticles
M Dienerowitz, V Garces-Chavez Sorting M
MacDonald, G Spalding, D Rhodes, G Milne, L
Paterson, A Riches Photoporation X Tsampoula, V
Garces-Chavez, D Stevenson, B Agate, CTA Brown, W
Sibbett Raman P Jess, I Cormack, M
Mazilu Biology/Medicine co-Investigators F
Gunn-Moore (neuroscience) A Riches (cancer
biology) CS Herrington (pathology) Visit us at
www.st-andrews.ac.uk/atomtrap