Fast Optical Scanning for - PhysicsOklahoma State University快速掃描光學(xué)-美國俄克拉荷馬州立大學(xué)物理學(xué)

1、.Confocal Raman Tweezers for a Nanotoxicology ApplicationEmanuela EneAbstractThe Confocal Raman Tweezers 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 real time monit

2、oring the chemical and functional changes in nanoparticles-embedded cells. A two-axis acousto-optic deflector (AOD) and a piezo-positioner are designed to be included in our existing Confocal Raman Tweezing Spectrometer in order to achieve fast and precise laser trap displacements. The life time of

3、trapped cells will be measured based on the fluorescence signal excited with a tunable Coherent laser Resonance Raman spectra for individual nanoparticles will be mapped spatially, near resonance, using a tunable Coherent laser. A living cell embedded with nanoparticles will be monitored via CRTS ov

4、er a series of different time points and distinguish the death or chemical changes in the cell.From http :/www.uni-Mainz.de/FB/Medizin/Anatomie/Leube/images/ogolivingcell/jpga) A single beam traps that part of a living cell with the highest refractive index.The trapped cell can have different orient

5、ations inside the trap.Images of a trapped budding yeast cell immediately after the trapping event (b), after 2 s (c) and 5 s (d). Our CTRS setting The Confocal Raman Tweezers Spectroscopy has the ability to provide precise characterization of a living cell without physical or chemical contact. The

6、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. *;Figure 1 is a picture of our actual CRT syst

7、em working with a green 514.5nm Ar+ ion laser. Figure2 is a detail for the Plan Apochromat focusing objective schematics. Experimental setup Figure 1Figure 2Slide with 1.5mm depression, filled with 5m polystyrene (PS) spheres in water. Focus may move 440 m from the cover glass. Cover glass (n=1.525,

8、 t=150m)Aqueous solution of PS spheres (m=1.19)SlideOil layer (n=1.515)Oil immersion objective (NA=1.25)Backward scattered Raman light Incident laser beamz440m Figure 2 Focusing objective and sample for calibration the CRTS We have calibrated our CRTS system using polystyrene spheres and taking sepa

9、rate confocal scans from the cover glass and the PS spheres, as shown in Figure 3. Here we show the CRT spectrum collected from a single 5.0m, polystyrene sphere ( BangsLaboratoratories) continuously trapped for more than eight hours with a Meredith 632.8nm HeNe laser, 5mW in front of the objective.

10、 The total collection time was 1500s, with 2.0s per each 0.2cm-1 step. Figure 3Calibration spectrum The actual trapping-excitation laser has a Gaussian profile with the exit waist of 1.25mm, 632.8nm wavelength, and it is linearly polarized. The 4X beam expander keeps the beam collimated with a 6.0mm

11、 waist size. The expanded beam “fills” the 6.0mm-radius of the microscope aperture; the beam is truncated by this aperture to its 1/e2 diameter. For a Gaussian beam, incident on the objective lens, the magnification is written: (1)For the focused beam, in the paraxial image plane, the radius of the

12、wave-front is (2) where R is the radius of curvature of the “object” beam. Setting 1/R=0, we get for the f=2mm perfect lens, R= -200mm at the lens surface. From (3)we get for the distance AOD objective the value z=202mm. An OSLO simulation gives the trap profile for a truncated Gaussian beam “fillin

13、g” the objective aperture, passing the0.17mm thickness cover glass (n=1.52216632.8nm), respectively the aqueous sample. The simulation gives the ranges for the angular lateral deflection, via AODs, and the z-axial deflection. The beam steering must be faster than the cell motility in an aqueous solu

14、tion at 37°C, previously measured as less than 50m/s. The pinhole will be initially aligned in the conjugate plane of the objective focal plane7. This alignment will be stable while both scanning with the trap the x-y plane in the range of 0-100m for a pinhole size in the range 200-400m and whe

15、n moving the infinity corrected objective on the z-optical axis in the range of 0-440 m. 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 success

16、ive spectra collection, and repeatability are required.1,2,3,4,5,6 The improved CRTS setup is shown in Figure 4. 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

17、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 Rama

18、n 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.Imaging systemLaser4X be

19、amexpander Confocal pinholeMicroscope objective piezo controlledDual axis AODEntranceslitRamansystemFigure 4 The CRTS systemReferences1. Carls, J.C. et al, Time- resolved Raman spectroscopy from reacting optically levitated microdroplets, Appl. Optics, 29, 1990, pp. 2913-182. Cao, Y.C. et al, Raman Dye-Labeled Nanoparticle Probes for Proteins , J. Am. Chem. Soc., 125 (48), 14676 -14677, 20033. C. Xie, Y-qing Li, Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shift