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Dual Wavelength Optical Tweezers for Confocal Raman Spectroscopy

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Dual Wavelength Optical Tweezers for Confocal Raman Spectroscopy Optics Communications 245 (2005) 465–470 www.elsevier.com/locate/optcom Dual wavelength optical tweezers for confocal Raman spectroscopy C.M. Creely a, G.P. Singh a, D. Petrov a,b,* a ICFO – Institut de Cie`ncies Foto`niques, c/Jordi Girona 29, 08034, Barcelona, Spain b ICREA – Institucio´ Catalana de Recerca i Estudis Avanc¸at, Barcelona, Spain Received 16 September 2004; received in revised form 5 October 2004; accepted 6 October 2004 Abstract We describe the use of dual optical tweezers to manipulate micron-size particles in and out of the focus of a confocal Raman microscope. One of the beams excites the Raman spectrum while the second tweezers improves the sensitivity of the technique and also allows for the manipulation of the environment of the trapped objects. We concentrated on optimising the alignment of both trapping and Raman excitation beams and on the background subtraction method. Even at the low trapping/excitation powers used a single living cell could be trapped and monitored for over 2 h without incurring damage. Ó 2004 Elsevier B.V. All rights reserved. PACS: 87.80.Cc; 87.64.Je; 87.64.Tt Keywords: Optical tweezers; Raman spectroscopy; Microspectroscopy Optical tweezers utilise the force of radiation perform spectroscopy on a single object of micron pressure to trap and manipulate micron and sub- size. A greater signal to noise ratio from the micron sized particles whose refractive index dif- trapped object can be achieved when background fers from that of the environment [1,2]. Since signals from the cover slip and immersion oil are 1984 radiation forces have been used to perform minimised by manipulating the particle inside a spectroscopy on trapped particles, aerosols and sample holder. In most previous studies the same living cells (for example [3–13]). The important beam has been used to trap an object and excite advantage of such techniques is the ability to either fluorescence or Raman scattering. Such a technique does not allow one to perform spatially * Corresponding author. Tel.: +34 93 413 7942; fax: +34 93 resolved spectroscopy of the trapped objects or to 413 7943. take the background for a given position of the E-mail address: [email protected] (D. Petrov). trapping beam. A second trapping beam can both 0030-4018/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.optcom.2004.10.011 466 C.M. Creely et al. / Optics Communications 245 (2005) 465–470 improve the sensitivity of the optical tweezers Ra- man spectroscopy system and allow one to manip- ulate the micro-environment of the object. Recently novel setups consisting of two separate laser beams for trapping and Raman spectra exci- tation were proposed in [14,15]. The system inves- tigated in [14] consisted of two independent microscopes: one to trap an object and another to excite the Raman spectra and collect scattering light. The functionality of the system was demon- strated by measurements of resonance Raman spectra of red blood cells. In [15] two optical beams of different frequencies were also used for trapping and spectroscopy. It was shown that the set-up which included only one microscope has a better signal to noise ratio and also obviated prob- lems arising from replacing the condenser. In this Letter, we develop a variant of dual fre- quency Raman microspectroscopy combined with optical tweezers that includes only one microscope system. In particular, we concentrated on optimis- Fig. 1. The confocal Raman spectroscopy set-up with dual tweezers. ing the alignment of both trapping and Raman excitation beam and on the background subtrac- tion method. Our system allows us to achieve a objective NA = 1.25 (Edmund Optics) and non flu- high signal to noise ratio such that at <10 mW orescent oil (Cargille, Type DF). Back scattered input power the Raman spectrum of a trapped light collected by the objective passed through a polystyrene bead can be monitored in real time holographic notch filter (NF) (Kaiser) and a con- and hence used to align our confocal system with focal system formed by lenses L1 and L2 and a greater accuracy. The power of our 1064 nm trap- 100 lm pinhole, before finally being focussed by ping beam is about 50 times less than that used in a cylindrical lens onto the spectrometer slit. The [14] and the laser excitation power required is five spectrometer was a SpectraPro 2500Ài from Acton times less than that used in [15]. These conditions containing a 600 lines/mm grating (750 nm blaze together with using IR wavelengths for trapping wavelength) and incorporating a Princeton Instru- allow us to study living cells for long periods of ments Spec-10 CCD, cooled to À100 °C. A 1064 time, thus kinetics for biochemical processes can nm Nd:YAG laser operating at less than 2.0 mW be obtained. Here, we show results for non reso- of power at the sample was used as the auxiliary nant Raman spectra where living yeast cells are laser tweezers. By adjusting mirror M and varying trapped and spectra recorded for over 2 h with the distance between lenses L3 and L4 we manipu- no detrimental effects to the cell. lated a trapped object along all three coordinates. The main parts of our experimental set-up are A CCD camera (JAI) attached to the microscope as follows (Fig. 1). A diode laser (CrystaLaser) provided optical images during experiments. operating at 785 nm was used for excitation of We found that the optimum alignment for Ra- Raman spectra with an average power of <10 man spectra of a trapped object differs greatly to mW at the sample. Micro-particles or cells were that of an object flat on a surface. The system trapped inside a custom-made holder using a was optimised for Raman spectra from a trapped 100-lm thick fused silica cover slip (UQG Optics). spherical object by using a 5-lm diameter polysty- The holder was placed on an inverse Olympus IX rene bead. First, the optical systems were aligned 51 microscope equipped with a 100· oil immersion in such a way that the trap could be achieved at C.M. Creely et al. / Optics Communications 245 (2005) 465–470 467 the same place inside the sample holder for both After this measurement was made the 1064 nm beams. Then the bead was trapped using the 785 tweezers were switched on and used to trap and nm laser and the Raman spectrum was recorded then move the particle out of the Raman laser in real time. The signal to noise ratio for our beam waist in order to take a background meas- system was large enough to be able to align the urement under the exact same experimental condi- system using a 0.2 s or even 0.1 s accumulation tions (see Fig. 2(c)). The final back subtracted data time (Fig. 2(a)). By adjusting the position of the is seen in Fig. 2(d). All background subtractions bead inside the sample holder and the orientation for trapped objects were made in the same way. of the pinhole we optimised the signal to noise ra- The spectra were processed by background sub- tio for the trapped bead. A sample spectrum of traction and subsequent Savitsky–Golay smooth- polystyrene taken for 10 s is shown in Fig. 2(b). ing. After the system was aligned for polystyrene beads, measurements were made on living cells using the same procedure for the background subtraction. The Saccharomyces cerevisiae yeast cell is widely used as a model for fundamental studies of cell processes including cell stress response, as many fundamental cellular processes are con- served from yeast to human cells and correspond- ing genes can often complement each other. Many disease-related genes have been identified from studies of this organism, forming the basis for understanding more complex cell-signalling events [16,17]. Cell processes such as stress response have been monitored by biochemical methods but not on a single cell basis. To be able to monitor dy- namic processes such as mitosis or metabolic changes in single cells a method must be available to immobilise and if necessary manipulate a single cell over a period of time relative to the length of the cell cycle. For our experiments yeast cells (S. cerevisiae) were grown in synthetic defined media (SDC) with complete supplements under standard conditions. For Raman measurements the cells were diluted further in SDC such that a single cell could be trapped with no other cells in the surrounding medium. This medium was used to ensure cell via- bility over the long time scale of our experiments, providing nutrients for the cell to live on. Raman spectra of single, budding live yeast cells were Fig. 2. (a) Spectrum of trapped polystyrene bead in the optical recorded every 3 min for 180 s accumulation time, trap used for alignment, 0.2 s accumulation at <10 mW power. for a total of 150 min in the same optical trap. (b) Spectrum for trapped bead for 10 s accumulation. (c) The After the experiment the buds were seen to have subsequent background spectrum after the bead had been grown appreciably, indicating the normal growth removed using the 1064 nm tweezers. (d) Background subtracted spectrum, no baseline subtraction used. Insets: of the cell in the optical trap. The resolution for À1 Images of trapped bead, X denotes the position of the 785 nm our experiments was 8.5 cm as estimated from beam. the polystyrene spectrum by measuring the 468 C.M. Creely et al. / Optics Communications 245 (2005) 465–470 FWHM of the 1001.4 cmÀ1 peak.
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