1. Praveen C. Ashok and Kishan Dholakia Passive Fractionation of Colloids and Cells Using Optofluidics Optical Manipulation Group, University of St Andrews, Scotland

2. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Cell or colloidal fractionation - rationale Mixture of cells or colloids Sorting, Analysis Specific species of cells or colloids Portable Sterile High throughput Efficient Sensitive Cheap Desirable Features

3. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Possible tools that can be used Portable Cheap Minimal sample volume sterile Microfluidics Efficient Sterile Passive & Active Non-invasive Optical micromanipulation Sensitive Versatile Imaging/Spectroscopy

4. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Optical fractionation ActivePassive Mixture of cells/colloids Hydrodynamic focussed single file sample flow Optical analysis of the sample Deflect the sample based on feedback from analysis Mixture of cells/colloids Focussed or non- focussed sample flow Optical interrogation region – Due to optical radiation force, sample gets selectively deflected based on size, shape or refractive index

5. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Active vs. Passive sorting (Why passive fractionation?) Active sortingPassive 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

6. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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.

7. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited 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? Plan of this lecture

8. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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. Optical Chromatography 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)

9. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture An animation to demonstrate the principle of optical chromatography F optical F drag Flow Laser T. Imasaka, Y. Kawabata, T. Kaneta, and Y. Ishidzu, "Optical Chromatography," Anal. Chem. 67, 1763-1765 (1995).

10. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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 Technique of optical chromatography – progress over last decade and challenges 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, 3221- 3225 (2006). ** * 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).

11. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Bulk optics systems –Optical alignment is critical Challenges and solution Solution....... On-chip light delivery using waveguide Choice of waveguide TEM 00 mode Mildly focused (Low numerical aperture Large mode area Photonic Crystal Fiber (LMA – PCF) –Low divergence compared to normal single mode fiber –TEM 00 mode output –Endlessly single mode operation in wavelength

12. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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

13. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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).

14. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Size driven and refractive index driven separation of colloids Retention distance calculated theoretically (line) and experimentally (points) for different sizes and refractive indices of particles 1070 nm Yb-fiber laser was used to achieve fractionation in these experiments

15. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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 Simultaneous Fractionation & on-chip fluorescent excitation A photograph of the chip while 532nm laser was used for on-chip fluorescent excitation along with 1070nm laser for fractionation

16. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Fractionated Human Embryonic Kidney (HEK) cells, which have engulfed colloidal particle through phagocytocis from those who haven’t engulfed the particles. Enhance the refractive index contrast using phagocytotically inserted colloids 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

17. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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

18. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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 landscape based optofluidic sorting

19. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Basic principle Optical Landscape Buffer Sample By tuning several parameters, one of the species can be selectively locked into the periodic optical landscape thereby allowing it to be selectively deflected

20. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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. State of the art 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).

21. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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

22. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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. 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. BUT

23. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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) Possible solutions

24. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture 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. Conclusion

25. Optical Manipulation Group, University of St AndrewsSFM-11 Internet invited lecture Thank you Praveen C. AshokProf. Kishan Dholakia