1. 9/14/2015PHYS 5123 Optical Design Project 1 Fast Optical Scanning for Confocal Raman Tweezing Spectroscopy Emanuela Ene

2. 9/14/2015PHYS 5123 Optical Design Project 2 Abstract The Confocal Raman Tweezing Spectroscopy (CRTS) has the ability to provide precise characterization of a living cell without physical or chemical contact. In our nanotoxicity study, CRTS will be employed for monitoring in real time the chemical and functional changes in nanoparticle-embedded cells.

3. 9/14/2015PHYS 5123 Optical Design Project 3 For a CRTS study a very stable optical trap is essential, so that extra cell instability is not induced. Repeatability and stability of the collected Raman spectra during optical trapping may be achieved with automatic laser beam steering. A two-axis acousto-optic deflector (AOD) and a piezo positioner are designed to be included in our existing Confocal Raman Tweezing Spectrometer (CRTS) in order to achieve fast and precise laser trap displacements.

4. 9/14/2015PHYS 5123 Optical Design Project 4 A perfect lens OSLO simulation is run for our Gaussian beam based CRTS. Beam steering OSLO computations, in both transversal and axial directions, demonstrate the range for scan angles and for linear translation. For a truncated Gaussian beam, employed in optical tweezing, we expect optical aberrations even for a perfect lens-like focusing objective.

5. 9/14/2015PHYS 5123 Optical Design Project 5 Images of a trapped budding yeast cell immediately after the trapping event (b), after 2 s (c) and 5 s (d). a) A single beam traps that part of a living cell with the highest refractive index. The trapped cell can have different orientations inside the trap. http ://www.uni - Mainz.de/FB/Medizin/Anatomie/L eube/images/ogolivingcell/jpg

6. 9/14/2015PHYS 5123 Optical Design Project 6 Background – Our CRTS application Experimental setup Axial resolution Confocal microRaman spectra Outline The Confocal Raman Tweezing Spectroscopy has the ability to provide precise characterization of a living cell without physical or chemical contact. The CRTS allows the analysis of single cells in wet samples, in contrast with the classical micro Raman spectroscopy which utilizes dried samples. In a confocal setting, the collected signal comes just from a minimum volume around the trapped- excited object. In our nanotoxicity study, CRTS is used to monitor the chemical and functional changes in nanoparticles-embedded cells in real time.

7. 9/14/2015PHYS 5123 Optical Design Project 7 Our Confocal Raman-tweezing system M – Silver mirror P – Pinhole LLF - laser line filter BS – beam-splitter BP - broad-band polarization rotator Experimental setup L curvature halogen lamp PMT objective & sample DM3000 system beam expander P4 BS imaging BS Raman L collect L focus Monochromator Video camera Imaging system subt. filters P1 HeNe Laser Ar+ Laser M1 M2, M3 P3 P2 BPR LLF

8. 9/14/2015PHYS 5123 Optical Design Project 8 Proposed solution The problem addressed is monitoring living cells, via the CRTS technique for nanotoxicity studies. Both stability of the trap, for around eight hours of successive spectra collection, and repeatability are required. Optical trapping and manipulation can be realized using mechanical microstages or electric nanopositioning. The latter method is not only far more precise, but also assures stability and repeatability. Nanopositioning systems currently used for CRTS are: galvanic mirrors, piezo-controllers, and AODs. The automatic fast laser beam steering will allow moving the beam focus in 3D to “chase” the cell that will be trapped and analyzed. Thus we will eliminate any mechanical displacement, proven to be a source of misalignments, instabilities, and irreversible changes. A two-axis acousto-optic deflector (AOD) and a piezo-positioner are designed to be included in our existing Confocal Raman Tweezing Spectrometer (CRTS) in order to achieve fast and precise laser trap displacements.

9. 9/14/2015PHYS 5123 Optical Design Project 9 The advantage of choosing to fast steering the trap only in the x-y plan simplifies the confocal pinhole alignment. The pinhole will be initially aligned in the conjugate plane of the objective focal plane. This alignment will be stable while scanning the x-y plane in the range of 0-100μm (or 0-50mrad) for a pinhole size in the range 200-400μm. The alignment will be also stable when moving the infinity corrected objective on the z-optical axis of the setting in the range of 0-400 μm. If the position of the trap on the z-axis would be changed by controlling the laser beam divergence, as done in classical tweezing setups, the conjugate plane of the pinhole can not be kept fixed.

10. 9/14/2015PHYS 5123 Optical Design Project 10 Technical description The improved CRTS setup is shown in Fig. 1. Three alternatives for new parts that should be included are listed in Table 1. The effects of beam steering with the AODs and of displacing the objective with the piezo controller are shown in OSLO simulations. Potential problems which we may encounter are due to the thermal sensitivity and to the electric noise of the driving voltage for the AODs. We address these two weaknesses by designing a heat sink for the AODs and by including the highest precision voltage controllers on the market.

11. 9/14/2015PHYS 5123 Optical Design Project 11 Fig.1 – the improved CRTS system Laser 4X beam expander Imaging system Confocal pinhole Microscope objective piezo controlled Dual axis AOD Entrance slit Raman system

12. 9/14/2015PHYS 5123 Optical Design Project 12 # Company & system Total price ($) Deflection angle (mrad) Efficiency (%) Aperture (mm) Delivery time (weeks) 1 Physik Inst. 1D - piezo 6,206-100-2-4 2 IntraAction 2D -AOD 5,16542.97010 X 10Several months 3D price11,371 3 Isomet 2D -AOD 16,05150>359.3 X 9.35-8 3D price22,257 4 Physik Inst. 3D - piezo 16,15410 for 100μm linear translation 902-4 Table 1 Specifications & Prices to electronically control the tweezing position.

13. 9/14/2015PHYS 5123 Optical Design Project 13 Microscope objectives are complex systems of lenses, corrected for geometrical and chromatic aberrations; such an almost perfect system has more surfaces than we may handle in EDU version of OSLO; in the simulations we enter a PERFECT LENS with F=2mm and magnification 100X for our PLAN APOCHROMAT infinity corrected oil immersion objective the object to be “imaged” is the incident laser beam the laser beam is Gaussian, 632.8nm, is collimated, and has an expanded 6.0mm waist size the expanded beam “fills” the 6.0mm-radius of the microscope aperture; the beam is truncated by this aperture to its 1/e 2 diameter Preliminary results

14. 9/14/2015PHYS 5123 Optical Design Project 14 Microscope objectives are complex systems of lenses, corrected for geometrical and chromatic aberrations; such almost perfect systems have more surfaces than we may handle in EDU version of OSLO An 100X magnification immersion objective, under US Patent 5,978,147, comprises three groups of lenses and a total of 22 surfaces

15. 9/14/2015PHYS 5123 Optical Design Project 15 PERFECT LENS “A perfect lens is that one that forms a sharp undistorted image of an extended object on a plane surface” (from the OSLO Reference manual). OSLO uses perfect lenses obeying the exact laws of optics. The results when using these perfect lenses are different from modeling with paraxial lenses. If the lens is to obey Abbe’s sine law, rays must emerge from the surface at a different height than they enter. A real perfect lens cannot be infinitely thin. Abbe’s sine law, valid for aplanatic (coma free) lenses: with U, U’ the angles which the corresponding rays in the object and image spaces make with the axis of the system u, u’ the slopes of the corresponding rays in the object and image spaces

16. 9/14/2015PHYS 5123 Optical Design Project 16 PERFECT 100X, F=2mm, focusing LENS DATA *LENS DATA perfect 100x f=2 focusing lens SRF RADIUS THICKNESS APERTURE RADIUS GLASS OBJ -- 202.000000 6.000000 AIR AST ELEMENT GRP -- 1.732985 AS AIR * 2 PERFECT 2.020000 S 1.732985 S OIL M * PERFECT IMS -- -- 0.060000 S

17. 9/14/2015PHYS 5123 Optical Design Project 17 PERFECT 100X, F=2mm, focusing LENS No aberrations

18. 9/14/2015PHYS 5123 Optical Design Project 18 The 100X on the short conjugate side PERFECT LENS with F=2mm gives for a 632.8nm Gaussian beam a 2μm minimum waist *LENS DATA tweezing INITIAL SRF RADIUS THICKNESS APERTURE RADIUS GLASS SPE NOTE OBJ -- 1.0000e+03 6.000000 AIR AST ELEMENT GRP -- 6.000000 AS AIR * 3 PERFECT 1.400000 6.000000 S OIL M * PERFECT 4 -- 0.170000 4.804800 S COVER M 5 -- 0.356946 4.299109 S SAMPLE M IMS -- -- 0.0019921 S oil layer: n oil =1.51536@632.8nm t=(0.1- 0.6)mm cover glass: n cover = 1.52216@632.8nm t=0.17mm sample: n=1.33 t=1.75mm

19. 9/14/2015PHYS 5123 Optical Design Project 19 The beam profile in the image plane for the OSLO model The cover glass and the solution with cells change the conditions for a perfect lens The tweezing spot profile for a Gaussian beam (T>2) Truncation factor: T=D_beam(1/e 2 ) / D_apert

20. 9/14/2015PHYS 5123 Optical Design Project 20 The beam profile in the image plane, the OSLO model, for truncated Gaussian beams employed in optical tweezing Calculations based on a paraxial ray trace may be invalid for a truncated Gaussian beam T=1 OSLO computes the diffraction image of a point object (the Point Spread Function) from the information of the geometric wavefront. For a truncated Gaussian beam entering our tweezer the central normalized energy peak is 0.48. The orresponding trapping force, in the spring- like trap, is 70% of the full power force. Note: the PSF algorithm results depend on the number of points in the sampling grid Truncation factor: T=D_beam(1/e 2 ) / D_apert

21. 9/14/2015PHYS 5123 Optical Design Project 21 Trap image (tweezing focus) in the X-Y plane for a Gaussian beam

22. 9/14/2015PHYS 5123 Optical Design Project 22 Steering the tweezing focus in the X-Y plane for a Gaussian beam 2 3 1 Position #Axial displacement of the beam center (mrad) Lateral displacement of the focus (micrometers) 10.50.11 21.574.88 339.681 449.6100 AOD – objective distance: -202mm Both AOD’s are driven for equal scan angles on the X and Y directions

23. 9/14/2015PHYS 5123 Optical Design Project 23 Steering the tweezing focus on the Z-axis for a Gaussian beam t_oil=0.1mm t_oil=0.6mm t_oil=0.5mm 1 2 3 Trap position moves on the z-axis when the immersion oil layer is compressed by the piezo-controlled objective. The cover glass is 0.17mm thick for all three shown positions but the beam focusing and aperture change. The OSLO simulations show: 1) focus in the cover; 2) focus in the sample, 0.030mm from the cover; 3) focus in the sample, 0.381mm from the cover

24. 9/14/2015PHYS 5123 Optical Design Project 24 A perfect lens OSLO simulation has shown how a perfect lens focuses a Gaussian beam Beam steering OSLO computations, in both transversal and axial directions, have demonstrated the range for scan angles and linear translation For a truncated Gaussian beam, employed in optical tweezing, we expect aberrations even when focusing with a perfect lens Summary

25. 9/14/2015PHYS 5123 Optical Design Project 25 References 1.Carls, J.C. et al, Time- resolved Raman spectroscopy from reacting optically levitated microdroplets, Appl. Optics, 29, 1990, pp. 2913-18Time- resolved Raman spectroscopy from reacting optically levitated microdroplets 2. Cao, Y.C. et al, Raman Dye-Labeled Nanoparticle Probes for Proteins, J. Am. Chem. Soc., 125 (48), 14676 -14677, 2003Raman Dye-Labeled Nanoparticle Probes for Proteins, 3.C. Xie, Y-qing Li, Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shifted excitation techniques, J.Appl.Phys., 2003, 93(5), 2982-2986Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shifted excitation techniques 4.Owen, C.A. et al,In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy, J. Cell. Biochem., 2006, 99, 178-186In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy 5.Volpe, G. et al, Dynamics of a growing cell in an optical trap, Appl. Phys. Lett., 2006, 88, 231106-231108Dynamics of a growing cell in an optical trap 6.Creely, S.M. et al, Raman imaging of neoplastic cells in suspension, Proc. SPIE, 2006, 6326: 63260URaman imaging of neoplastic cells in suspension 7.Shaevitz, J.W., A practical Guide to Optical Trapping, web resource at www.princeton.edu/~shaevitz/links.html www.princeton.edu/~shaevitz/links.html