Passive Fractionation of Colloids and
Cells Using Optofluidics
Praveen C. Ashok and Kishan Dholakia
Optical Manipulation Group,
University of St Andrews, Scotland
Cell or colloidal fractionation - rationale
Mixture of cells
or colloids
Portable
Sterile
Sorting, Analysis
High
throughput
Desirable
Features
Efficient
Specific species
of cells or colloids
Sensitive
Cheap
Optical Manipulation Group, University of St Andrews
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Possible tools that can be used
Microfluidics
Optical
micromanipulation
Imaging/Spectroscopy
• Portable
• Cheap
• Minimal sample volume
• sterile
• Efficient
• Sterile
• Passive & Active
• Non-invasive
• Sensitive
• Versatile
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Optical fractionation
Optical
fractionation
Active
Passive
Mixture of
cells/colloids
Hydrodynamic
focussed single
file sample flow
Deflect the
sample based on
feedback from
analysis
Optical analysis
of the sample
Optical Manipulation Group, University of St Andrews
Mixture of
cells/colloids
Focussed or nonfocussed sample
flow
Optical interrogation region – Due to optical
radiation force, sample gets selectively
deflected based on size, shape or refractive
index
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Active vs. Passive sorting (Why passive fractionation?)
Active sorting
Passive sorting
High throughput with fluorescence
detection
Relatively low throughput
Requires external tagging for
fluorescence based fractionation
External tagging not necessary. Rely
upon inherent properties of sample –
size, shape or refractive index
Techniques like Fluorescence Activated
Cell Sorting (FACS) is an already
established technique
Proof of principle level demonstrations.
Compatible to be implemented in
microfluidic platform
Compatible to be implemented in
microfluidic platform
Optical Manipulation Group, University of St Andrews
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Why passive fractionation?
• Passive sorting opens up opportunities to achieve
sorting of various biological samples which cannot
be sorted otherwise using conventional techniques
like FACS
• Technology still at its infancy.
• Further exploration is required to implement passive
fractionation systems for specific applications.
Optical Manipulation Group, University of St Andrews
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Plan of this lecture
• Optical chromatography
– Implementation of an optofluidic chip for optical
chromatography using embedded fibers for on-chip laser
beam delivery
• Passive optical fractionation using optical potential
energy landscape (brief description)
– Challenges
– What we should learn from the colloidal sorting
experiments to implement this technique for cell sorting?
Optical Manipulation Group, University of St Andrews
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Optical Chromatography
• A passive optical fractionation technique for cells and colloids.
• Uses the the interplay between microfluidic viscous drag force
and the optical radiation force to achieve spatial separation of
microparticles.
When a particle in a microfluidic flow encounters a gently focused laser beam
propagating coaxially in the opposite direction to the flow, the particle
experiences a force against the flow due to the radiation pressure of the laser beam.
The particle comes to a rest point where the optical forces are balanced by the
Stokes’ viscous drag force. The distance of the rest point from the focus of the laser
beam is referred to as the retention distance. This rest point of particle depends on
the size, shape or refractive index of the particle.
(An animation is followed on the next slide to demonstrate this principle)
Optical Manipulation Group, University of St Andrews
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An animation to demonstrate the principle of optical
chromatography
Flow
Foptical
Fdrag
Laser
T. Imasaka, Y. Kawabata, T. Kaneta, and Y. Ishidzu, "Optical Chromatography," Anal. Chem. 67, 1763-1765 (1995).
Optical Manipulation Group, University of St Andrews
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Technique of optical chromatography –
progress over last decade and challenges
• Progress in the microfluidic chip designs for specific
applications*
• Applied for fractionation of a variety of biological samples**
• No progress in the implementation of optics
– Free space light delivery
– Needs careful optical alignment
*
A. Terray, S. J. Hart, K. L. Kuhn, and J. Arnold, "Optical chromatography in a PDMS microfluidic environment,"
Optical Trapping and Optical Micromanipulation 5514, 695-703 (2004).
S. J. Hart, A. Terray, J. Arnold, and T. A. Leski, "Preparative optical chromatography with external collection and
analysis," Opt. Express 16, 18782-18789 (2008).
**
S. J. Hart, A. Terray, K. L. Kuhn, J. Arnold, and T. A. Leski, "Optical chromatography of biological particles," Am. Lab.
36, 13-17 (2004).
S. J. Hart, A. Terray, T. A. Leski, J. Arnold, and R. Stroud, "Discovery of a Significant Optical Chromatographic
Difference between Spores of Bacillus anthracis and Its Close Relative, Bacillus thuringiensis," Anal. Chem. 78, 32213225 (2006).
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Challenges and solution
• Bulk optics systems
– Optical alignment is critical
• Solution.......
•
On-chip light delivery using waveguide
• Choice of waveguide
•
•
TEM00 mode
Mildly focused (Low numerical aperture
• Large mode area Photonic Crystal Fiber (LMA – PCF)
– Low divergence compared to normal single mode fiber
– TEM00 mode output
– Endlessly single mode operation in wavelength
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Implementation of Optical chromatography
using a photonic crystal fiber
Chip
Designed a microfluidic chip with pre-defined fiber insertion channel to embed the fiber
into the chip.
PDMS
Chip was fabricated in polydimethylsiloxane (PDMS) using soft lithography
Alignment
The chip designed ensuring that the fiber insertion channel was pre-aligned to the flow
channel (precise co-axial alignment of the beam and the flow)
Leak-proof
embedding
In order to avoid aberrations of the beam delivered into the fluidic channel, fiber was
directly embedded into the microfluidic channel (without any wall separating fiber insertion
channel and flow channel.)
Capping
In order to avoid liquid getting into the holes of PCF due to capillary action a simple capping
method
Optical Manipulation Group, University of St Andrews
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Optical chromatography using a photonic crystal fiber
Design of the microfluidic chip embedded with LMA PCF
P. C. Ashok, R. F. Marchington, P. Mthunzi, T. F. Krauss, and K. Dholakia, "Optical chromatography using a
photonic crystal fiber with on-chip fluorescence excitation," Opt. Express 18, 6396-6407 (2010).
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Size driven and refractive index driven
separation of colloids
1070 nm Yb-fiber laser was used to
achieve fractionation in these
experiments
Retention distance calculated theoretically (line) and
experimentally (points) for different sizes and refractive
indices of particles
Optical Manipulation Group, University of St Andrews
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Simultaneous Fractionation & on-chip
fluorescent excitation
•
LMA-PCFs are endlessly single mode
– Hence can couple multiple wavelength into it simultaneously
– Used this principle to achieve simultaneous fractionation (1070 nm laser) and
fluorescence excitation (532 nm) in this chip.
– Makes it possible to check the purity of the sample while fractionation if one of the
species is fluorescing
A photograph of the chip while 532nm laser was used for on-chip
fluorescent excitation along with 1070nm laser for fractionation
Optical Manipulation Group, University of St Andrews
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Enhance the refractive index contrast using
phagocytotically inserted colloids
•
Fractionated Human Embryonic Kidney (HEK) cells, which have engulfed colloidal
particle through phagocytocis from those who haven’t engulfed the particles.
Separation of cells with and without spheres, and
analysis of the separated and concentrated sample with
on-chip fluorescence excitation
• Possible to selectively attach functionalized colloids to specific types of
cells and achieve subsequent fractionation
Optical Manipulation Group, University of St Andrews
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Optical chromatography using a photonic crystal fiber
Features
• On chip light delivery using PCF
• Simultaneous On chip fluorescent
excitation and fractionation
• Separation of colloids and cells
• Alignment-free
The prospectus of using embedded waveguide for optical beam delivery and
optical signal collection in microfluidic chips opens up opportunities to develop
alignment-free optofluidic devices that are more desirable for field applications
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Optical landscape based optofluidic sorting
• Another optical fractionation method.
• Unlike optical chromatography, which is a static
optical separation method, this offers possibility to
perform dynamic sorting of colloids and cells.
Optical Manipulation Group, University of St Andrews
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Basic principle
Buffer
Optical Landscape
Sample
Buffer
• By tuning several parameters, one of the species can be selectively locked
into the periodic optical landscape thereby allowing it to be selectively
deflected
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State of the art
• Can create optical potential energy landscape by time or spatially
multiplexing optical trapping beams
• Possible to perform sensitive size driven colloidal sorting.
• Theory is developed to explain the fractionation of spherical colloidal
particles .
• This theory can be used to choose the right set of parameters (tunable
parameters) for fractionating a specific colloidal sample.
K. Xiao, and D. G. Grier, "Multidimensional Optical Fractionation of Colloidal Particles with Holographic Verification," Phys. Rev. Lett. 104
(2010).
K. Ladavac, K. Kasza, and D. G. Grier, "Sorting mesoscopic objects with periodic potential landscapes: Optical fractionation," Physical
Review E 70 (2004).
Optical Manipulation Group, University of St Andrews
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Challenges in realizing cell sorting
• The tunable parameters
– Optical power per trap
– Distance between traps in the periodic landscape
– Angle of orientation of the line trap with respect to the
flow direction
– Flow speed
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Challenges in realizing cell sorting
• The right sets of these tunable parameters can be determined for
fractionating a specific sample, if the sample is spherical and the
size and refractive index of the sample are known along with the
viscosity of the buffer medium used.
BUT
• For cells, it is not easy to determine absolute values of size
and refractive index
– Need to determine viscosity of the buffer medium as well.
• These unknown parameters makes it difficult to optimize the
tunable parameters theoretically or empirically.
Optical Manipulation Group, University of St Andrews
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Possible solutions
• Need to develop a method with which a cell species
can be correlated to a spherical colloidal particle so
that existing theory can be used to determine the
right set of tunable parameters.
• Modify the theory to determine the tunable
parameters without the knowledge of the unknown
parameters in the case of cells
– (A less probable option)
Optical Manipulation Group, University of St Andrews
SFM-11 Internet invited lecture
Conclusion
• Passive fractionation can open up new opportunities to
achieve fractionation of cell species that are not possible to
be fractionated with already established active sorting
methods.
• Embedding waveguides in a microfluidic chip opens up new
opportunities to develop portable optofluidic devices with
sensing and manipulation functionalities.
• Although optical landscape based optical sorting technique is
a promising passive optical sorting technique, further studies
are necessary to develop this as a technique suitable for
fractionation of cells.
Optical Manipulation Group, University of St Andrews
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Thank you 
Praveen C. Ashok
Optical Manipulation Group, University of St Andrews
Prof. Kishan Dholakia
SFM-11 Internet invited lecture