1. Confocal Raman Tweezers for a Nanotoxicology Application Raman measurements from optically trapped dielectric and magnetic microparticles, under various visible laser excitation wavelengths, are being studied. Changes in the Raman spectra for trapped living cells embedded with nanoparticles will be investigated. Emanuela Ene Oklahoma State University

2. The Confocal Raman Tweezers Spectroscopy (CTRS) 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 that utilizes dried samples. In a confocal setting, the collected signal comes just from a minimum volume around the trapped-excited object. Our Confocal Raman Tweezers Setting The actual CRT system working with a green 514.5nm Ar+ ion laser Imaging system Laser 4X beam expander Confocal pinhole Microscope objective piezo controlled Dual axis AOD Entrance slit Raman spectro meter The CRT system schematics

3. Trap image (tweezing focus) in the X-Y plane The tweezing profile in the image plane The cover glass and the colloidal solution introduce aberrations Simulations for trapping with a Gaussian beam

4. Water immersed complex microobjects have been optically manipulated Cell “stuck” near a 0.8µm PMMA sphere with 6nm gold nanoparticles coating SFM image of a cluster of 0.18μm PS “spheres” coated with 110nm SWCN. Scanning range: 4.56μm Diffraction rings of trapped objects. Sub-micrometer coated clusters were optically manipulated near plant cells; both of the objects stayed in the trap for several hours. PMMA = polymethylmethacrylate

5. Calibration spectrum Slide with 1.5mm depression, filled with 5μm polystyrene (PS) spheres in water. Focus may move ≈ 440 μm from the cover glass. Cover glass (n=1.525, t=150μm) Aqueous solution of PS spheres (m=1.19) Slide Oil layer (n=1.515) Oil immersion objective (NA=1.25) Backward scattered Raman light Incident laser beam Δz≈440μm Focusing objective and sample for calibration the CRT system The CRT spectrum collected from a single 5.0μm, polystyrene sphere ( Bangs Laboratoratories) continuously trapped for more than eight hours with a Meredith 632.8nm HeNe laser, 5mW in front of the objective. The total collection time was 1500s, with 2.0s per each 0.2cm -1 step.

6. Confocal Raman Tweezers Spectra from magnetic particles 1.16μm-sized iron oxide clusters (BioMag 546, Bangs Labs) with silane (SiHx) coating The biological applications of nanoparticles, from imaging to drugs delivery, have created an increased interest in the past decades. Already in widespread use, superparamagnetic iron oxide nanoparticles associated with biological molecules are easily for manipulating and attractive for MRI contrast or targeted molecule delivery. Although used in biological and medical research, there is just little work done in investigating the effects of interactions between these magnetic particles and the living cells they are attached to.

7. Future development In our nanotoxicity study, CRTS will be used to monitor the chemical and functional changes in nanoparticle- embedded living cells. Both stability of the trap, for around eight hours of successive spectra collection, and repeatability are required. 1,2,3,4,5,6 For living cells, photodamage effects restrict the range of wavelengths to be used. We intend to employ a tunable 505 to 750nm (Coherent) beam for both tweezing and Raman excitation. The automatic fast laser beam steering will allow moving the beam focus in 3D to “ chase ” the cell that will be trapped and analyzed. For a photodamage initial evaluation, the life time of the trapped cells will be measured based on the fluorescence signal excited with the tunable laser 8. Resonance Raman spectra for individual nanoparticles will be mapped spatially, near resonance, using the same tunable laser. A living cell embedded with nanoparticles will be monitored via CRTS over a series of different time points and distinguish the death or chemical changes in the cell. References 1.Carls, J.C. et al, Time- resolved Raman spectroscopy from reacting optically levitated microdroplets, Appl. Optics, 29, 1990, pp. 2913-18 2.Cao, Y.C. et al, Raman Dye-Labeled Nanoparticle Probes for Proteins, J. Am. Chem. Soc., 125 (48), 14676 -14677, 2003 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), 2982-2986 4.Owen, C.A. et al,In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy, J. Cell. Biochem., 2006, 99, 178-186 5.Volpe, G. et al, Dynamics of a growing cell in an optical trap, Appl. Phys. Lett., 2006, 88, 231106-31108 6.Creely, S.M. et al, Raman imaging of neoplastic cells in suspension, Proc. SPIE, 2006, 6326: 63260U 7.Shaevitz, J.W., A practical Guide to Optical Trapping, web resource at www.princeton.edu/~shaevitz/links.html 8.Neumann, K.C. et al, Characterization of Photodamage to Escherichia coli in optical traps, Biophys. J., 1999, 77(5), 2856-2863