1. Optical Tweezers Sarah Nichols March 17, 2004

2. Outline Motivations Operation Layout Successes and Difficulties Future Directions

3. Why use optical tweezers? Can manipulate living cells and organelles Can manipulate living cells and organelles Can measure stretching of large molecules such as DNA Can measure stretching of large molecules such as DNA Measure and manipulate mechanoenzymes and other physically acting molecules Measure and manipulate mechanoenzymes and other physically acting molecules Relatively cheap and easy to build a simple setup Relatively cheap and easy to build a simple setup Useful tool for learning various optics procedures and terms Useful tool for learning various optics procedures and terms

4. How it works d object >>d beam -> ray optics approximation is valid d object >>d beam -> ray optics approximation is valid Reflection and refraction create opposing forces on a dielectric object Reflection and refraction create opposing forces on a dielectric object If the beam is strong enough, the gradient force can overcome the scattering and gravitational forces If the beam is strong enough, the gradient force can overcome the scattering and gravitational forces Rays from edge of lens are deflected most, so they have the biggest trapping impact Rays from edge of lens are deflected most, so they have the biggest trapping impact Amendola, 2001

5. Components 20 mW diode laser 20 mW diode laser 2 cylindrical lenses for shaping 2 cylindrical lenses for shaping Mirrors for redirection Mirrors for redirection 2 spherical lenses for resizing 2 spherical lenses for resizing Periscope to raise the beam Periscope to raise the beam Dichroic mirror to direct beam into microscope Dichroic mirror to direct beam into microscope Microscope with 100x oil immersion objective, eyepiece removed Microscope with 100x oil immersion objective, eyepiece removed Camera connected to TV screen for viewing Camera connected to TV screen for viewing Laser Cylindrical lenses M1 Spherical lenses M2 Periscope Microscope Infinity-correcting lens

6. Layout

7. Layout Camera Filter Dichroic mirror Beam block Microscope

8. Optimization Beam must be aligned to travel exactly vertically through microscope Beam must be aligned to travel exactly vertically through microscope For best trapping, beam should be spherical For best trapping, beam should be spherical Optimal beam size overfills the objective slightly FWHM 6 mm for our setup (Amendola, 2001) Optimal beam size overfills the objective slightly FWHM 6 mm for our setup (Amendola, 2001) Microscope objective does not collect light from infinity, so a lens should focus light 160 mm behind the objective Microscope objective does not collect light from infinity, so a lens should focus light 160 mm behind the objective Trapping most easily achieved with moving objects; surfactants used in bead solutions Trapping most easily achieved with moving objects; surfactants used in bead solutions

9. Results Despite numerous alignments, beam was weak until very recently Despite numerous alignments, beam was weak until very recently Objective replacement improved beam strength Objective replacement improved beam strength Weak trapping demonstrated for yeast cells (~ 5 um) as well as 5 and 10 um polystyrene spheres Weak trapping demonstrated for yeast cells (~ 5 um) as well as 5 and 10 um polystyrene spheres With small yeast cells, can drag horizontally and vertically With small yeast cells, can drag horizontally and vertically

10. Results Untrapped 5 um spheresTrapped sphere

11. Questions Why is it easier to trap with yeast cells for a given size? (they exhibit more Brownian motion, but why?) Why is it easier to trap with yeast cells for a given size? (they exhibit more Brownian motion, but why?) Why does overfilling rule not hold? “Optimal” FWHM size: 6 mm. Measured size: 6-6.5 mm. Trapping size: 4 mm. Why does overfilling rule not hold? “Optimal” FWHM size: 6 mm. Measured size: 6-6.5 mm. Trapping size: 4 mm. Why does non-infinity correction make trapping harder? Why does non-infinity correction make trapping harder?

12. Suggested Improvements Clean lenses, replace mirrors Clean lenses, replace mirrors Replace diode laser for a less elliptical beam Replace diode laser for a less elliptical beam Determine reason for cell/sphere distinctions Determine reason for cell/sphere distinctions Mount a lens on a translation stage for beam steering with less intensity loss Mount a lens on a translation stage for beam steering with less intensity loss Determine reason for failure to agree with predictions - pure intensity loss or other reasons? Determine reason for failure to agree with predictions - pure intensity loss or other reasons? Upgrade camera for digitized still and motion pictures Upgrade camera for digitized still and motion pictures

13. Acknowledgements Thanks to Dr. John Noé and Yiyi Deng for their help in adjusting to the vagaries of the system. Thanks also to Duke Scientific for their donation of microspheres to the Laser Teaching Center for testing. References P. Amendola. “Design and Construction of an Optimized Optical Tweezers.” Intel Science Talent Search Report (2001). Y. Deng, “Optical Tweezers”, http://laser.physics.sunysb.edu/~yiyi (2003). S. Smith et al., Am. J. Phys. 67, 26 (1999). Z. Ulanowski and I.K. Ludlow, Meas. Sci. Technol. 11, 1778 (2000).

14. Slide Setup Deng (2003)