Passive Fractionation of Colloids and Cells Using Optofluidics

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

2. Cell or colloidal fractionation - rationale

3. Possible tools that can be used

4. Optical fractionation

5. Active vs. Passive sorting (Why passive fractionation?)

6. 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. 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?

8. 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)

9. An animation to demonstrate the principle of optical chromatography Laser Flow Foptical Fdrag T. Imasaka, Y. Kawabata, T. Kaneta, and Y. Ishidzu, "Optical Chromatography," Anal. Chem. 67, 1763-1765 (1995).

10. 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, 3221-3225 (2006). **

11. 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

12. Implementation of Optical chromatography using a photonic crystal fiber

13. 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. 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

15. 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

16. 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

17. Optical chromatography using a photonic crystal fiber 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 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.

19. Basic principle Optical Landscape Buffer 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

20. 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).

21. 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. 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.

23. 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)

24. 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.

25. Thank you  Praveen C. Ashok Prof. Kishan Dholakia