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Home , Optical tweezers, Raman spectroscopy

Communications 245 (2005) 465–470 www.elsevier.com/locate/optcom

Dual for confocal Raman

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 . 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 could be trapped and monitored for over 2 h without incurring damage. 2004 Elsevier B.V. All rights reserved.

PACS: 87.80.Cc; 87.64.Je; 87.64.Tt Keywords: Optical tweezers; ; 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 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 . 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 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 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 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 . 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 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 onto the 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 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 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 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. This spectrum also served as a Raman shift frequency standard (a) for our experiments using literature values [18]. The monitoring of the fission yeast Saccharomyces pombe using Raman microspectroscopy was re- ported in [19]. In this study the cell was adhered on a glass slide and the Raman spectra recorded the spectral changes due to cell mitosis. However, refer- ence [9] cites some problems associated with acquir- ing Raman spectra from cells in this manner including larger background and fluorescence from (b) the glass slide, and showed how optically trapping the cell at some distance from the slide could avoid such problems. Another experimental consideration is that trapping over long periods of time must not damage the cell. Here, we report living yeast cells

trapped by near-IR light for over 2 h. The spectra Intensity (a.u.) are shown in Fig. 3 with tentative peak assignments as indicated in [9,20,21]. Changes can be easily seen in the spectra after (c) different times, for example the ratio of peak inten- sities of adenine to phenylalanine changes drasti- cally between 3 and 126 min. Xie and Lie [9] report a large change in the ratio of I1004/I946 from 1.33 to 12.5 after heat treating cells which they suggest is a measure of the increase of the amount of in the a helical state. By comparison the peak intensities from our background sub- tracted data show a ratio of I1002/I943 that increases Raman shift (cm-1) only slightly from 1.12 to 1.40 after 126 min of trapping indicating that there is little change in Fig. 3. Raman spectra of yeast cells trapped for a total of three: conformation during the experiment. The (a) 3, (b) 33 and 126 (c) min, 180 s accumulation time, laser À1 power >10 mW. Spectra are background subtracted using the comparatively large increase in the 1004 cm tweezers method and subsequently baseline subtracted. peak seen after heating yeast cells as reported in Xie. indicated that the phenylalanine side chain was more exposed after protein denaturation. cells. In each case the environment of the cell will Although the intensity of the phenylalanine peak be slightly different, for example in the distance in our experiment increases slightly as time pro- from the substrate. To accurately perform com- gresses it does not do so to the same extent. This parative kinetic studies on different cell samples is some indication that for our set up localised background subtraction must account for these heating of the cell due to trapping for long periods sample to sample differences. Fig. 4 illustrates the of time is not taking place on an appreciable scale. sizeable effect background subtraction has on the Another example of information inferred from Raman peak intensities. Unlike the background changes in the Raman peak intensities is character- spectrum of the buffer seen in Fig. 2 the back- ising the level of RNA translation in a cell by com- ground spectrum of the SDC shows peaks in this puting the ratio of a peak for DNA to a protein region due to itÕs composition of amino acids peak [22]. The relative intensity of a Raman line and other nutritional supplements. Thus, back- is crucial when comparing processes from different ground subtraction will have an effect on the ratio C.M. Creely et al. / Optics Communications 245 (2005) 465–470 469

background spectrum for each sample. Further- more a relatively short accumulation time and low power was needed to achieve well resolved spectra. An investigation is currently under way to monitor the effect of chemically induced stress on live yeast cells. (a)

Acknowledgements

This research was carried out in the framework of ESF/PESC (Eurocores on Sons) through Grant 02-PE-SONS-063-NOMSAN, and with the finan- intensity (a.u.) cial support of the Spanish Ministry of Science (b) and Technology. The yeast cells were kindly pro- vided by the group of Dr. M.I. Geli of the IBMB, CSIC, Barcelona, and grown by Ms. H. Groetsch. We acknowledge the ongoing collaboration with Dr. T.M. Thomson and M. Soler of the IBMB, Barcelona.

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