Raman microspectroscopy of optically trapped micro- and nanoobjects

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1 Raman microspectroscopy of optically trapped micro- and nanoobjects Alexandr Jonáš, Jan Ježek, Mojmír Šerý, and Pavel Zemánek Institute of Scientific Instruments of the ASCR, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 147, Brno, Czech Republic ABSTRACT We describe and characterize an experimental system for Raman microspectroscopy of micro- and nanoobjects optically trapped in aqueous suspensions with the use of a single-beam gradient optical trap (Raman tweezers). This system features two separate lasers providing light for the optical trapping and excitation of the Raman scattering spectra from the trapped specimen, respectively. Using independent laser beams for trapping and spectroscopy enables optimizing the parameters of both beams for their respective purposes. Moreover, it is possible to modulate the position of the trapped object relative to the Raman beam focus for maximizing the detected Raman signal and obtaining spatially resolved images of the trapped specimen. Using this experimental system, we have obtained Raman scattering spectra of individual optically confined micron and sub-micron sized polystyrene beads and baker s yeast cells. Sufficiently high signal-to-noise ratio of the spectra could be achieved using a few tens of milliwatts of the Raman beam power and detector integration times on the order of seconds. Keywords: optical trapping, Raman microspectroscopy, Raman tweezers, microparticles and nanoparticles 1. INTRODUCTION Raman spectroscopy is an extremely powerful analytical technique that allows selective, external label-free analysis of the molecular composition of studied samples based on the identification of characteristic peaks in the spectrum of inelastic scattering of the incident monochromatic radiation from vibrating molecular bonds. When carried out in the so-called fingerprint spectral region, Raman spectroscopy is capable of distinguishing between molecules with very small structural differences (e.g. the presence of a single unsaturated bond in a lipid molecule). Raman microspectroscopy the combination of spectroscopy with optical microscopy in either far-field or near-field imaging modes then allows analyzing the composition of sub-micron-sized specimens or obtaining composition maps of larger samples with micron- or even sub-micron resolution. 1, 2 In addition to providing spatially-resolved compositional information, Raman microspectroscopy can be performed in the timelapse mode thus permitting to follow the temporal development of the processes that take place in the studied sample. 3 In order to conduct Raman microspectroscopic measurements one must be able to control the position of the analyzed specimen with respect to the Raman probe beam. Sample position control can be easily achieved when the sample is fixed or when it can be attached to a solid support that is subsequently scanned through the probe beam focus. There are, however, a number of important specimens with sizes in the micron and sub-micron range (e.g. colloidal particles, microdroplets, vesicles, or living cells) that exist normally in suspensions or aerosols and whose function or structure require the presence of the fluid environment. Such specimens are subject to the Brownian motion and streaming of the surrounding fluid and, consequently, they can travel a significant distance within the time required for acquiring the spectrum. Attempts to apply standard immobilization procedures (e.g. chemical bonding or adsorption to a substrate, micropipette suction) to these specimens often lead to an undesired alternation of their properties. Moreover, the presence of a support substrate can cause a significant spectral background obscuring the observed Raman scattering spectra of the target specimen. Optical trapping represents an elegant approach for addressing the above mentioned experimental challenges. In its most widely used experimental configuration - optical tweezers - particles with sizes from tens of nanometers Further author information: Send correspondence to A.J. ( sasa@isibrno.cz, Phone: ) 16th Polish-Slovak-Czech Optical Conference on Wave and Quantum Aspects of Contemporary Optics, edited by Agnieszka Popiolek-Masajada, Elzbieta Jankowska, Waclaw Urbanczyk, Proc. of SPIE Vol. 7141, SPIE CCC code: X/08/$18 doi: / Proc. of SPIE Vol

2 to tens of micrometers and refractive index higher than that of the surrounding medium are confined in the vicinity of the focus of a tightly focused laser beam. 4 Since both optical tweezers and Raman microspectroscopy rely on the use of a strongly-focused laser beam, it is straightforward to combine both techniques in a single experimental setup. The combination of Raman microspectroscopy with optical trapping usually termed Raman tweezers was first proposed almost 25 years ago 5 and, since its introduction, it has found numerous applications especially in the fields of analytical and physical chemistry and cell and molecular biology (see review 6 ). Typical target specimens for Raman tweezers are aerosols, suspended microdroplets or colloidal particles, and living cells that can be studied in situ under relevant environmental conditions. Moreover, the possibility of analyzing individual target microobjects enables to detect directly the variations of their properties and composition which would be otherwise lost in an ensemble averaged measurement. In this article, we present a dual-beam Raman tweezers system for Raman microspectroscopy of optically trapped micro- and nanoobjects featuring two independent beams for the spectrum recording and optical trapping. We demonstrate the potential of this setup by recording the Raman scattering spectra of individual optically confined micron and sub-micron sized polystyrene beads and baker s yeast cells. Finally, we discuss possible directions for further development and expansion of the capacities of the described setup. 2. EXPERIMENTAL SETUP In order to carry out Raman microspectroscopy of optically trapped micro- and nanoobjects, we have designed and built an experimental system based on a custom-made inverted microscope frame with a single microscope objective lens serving to generate the optical trap and excite and collect the Raman scattering from the specimen. The layout of this system is depicted in Fig. 1. We used two independent lasers to implement the optical tweezers and the Raman spectroscopic measurements, respectively. This approach allows maximal flexibility in optimizing the parameters of both beams (total power, beam waist size) to their respective tasks. Moreover, it enables modulating the position of the trapped object relative to the Raman beam focus for maximizing the detected Raman signal and obtaining spatially resolved images of the trapped specimen. Since our ultimate samples are living cells and other biological material, both used lasers operate in the near-infrared spectral region in order to avoid unwanted background fluorescence from the samples that obscures the Raman spectra and to reduce photodamage of the specimen by light absorption. The trapping laser beam (Nd:YAG, λ = 1064 nm, beam diameter 1 mm; DPY 321 II, Adlas) passed first through a λ/2 wave plate to adjust its polarization for maximal reflection from dichroic mirror D2 (custommade; T , T > 0.9) that coupled the trapping beam into the objective lens. Before entering the objective lens, beam diameter was enlarged by 9x beam expander Exp2 in order to overfill the back aperture of the objective and achieve stable 3-D trapping. 7 The Raman laser beam (Ti:Sapphire, λ = 785 nm, beam diameter 0.6 mm; , Coherent) was delivered to the setup by an optical fiber that also expanded the beam diameter by a factor of 3 (not shown in the picture) and further enlarged by 3x beam expander Exp1 before coupling to the objective lens via dichroic mirror D1 (LPD01-785RS, Semrock). The power of the Raman laser beam could be adjusted by neutral density filter NDF1 with continuously variable optical density (Thorlabs). Objective lens is a crucial component of the setup as its quality determines the stability of the optical trap and also the intensity of the Raman scattering signal and resolution of the spectroscopic measurements. Optical trapping and spectroscopy of living objects (cells) require the specimen to be suspended in aqueous solution. Furthermore, since both trapping and Raman lasers operate in the near-infrared part of the spectrum, the lens must also have a sufficiently high transmission and very good corrections of optical aberrations in this spectral region. For these reasons, we used an IR-optimized water-immersion objective lens (Olympus UPLSAPO 60x, NA 1.20) that allowed us to work up to 200 µm deep into the specimen without compromising significantly the quality of the optical trap and the spectroscopic measurements. The lens was mounted on a custom-made aluminum frame that also provided a stable support for condenser and illumination light source and for 3- axis piezo-driven stage (P-517.3CL, Physik Instrumente) that served for nanometer-precise positioning of the sample relative to the objective lens. The sample holder attached to the piezo-stage consisted of two microscope coverslips sandwiched together with double-sided adhesive tape forming a gap of 100 µm thickness between the coverslips (see also inset in Fig. 1) into which the sample (suspension of microobjects) could be conveniently loaded. Proc. of SPIE Vol

3 Microscope frame illumination condenser 3-axis piezostage Raman laser NDF1 Exp1 objective lens D1 Exp2 WP Sample detail cover slip (condenser side) D2 trapping laser trapped object Raman scattering cover slip (objective side) overlapping trapping and Raman beams FM NF1 NF2 L2 NDF2 L1 CCD to control PC control PC spectroscopic CCD camera imaging spectrograf Figure 1. Experimental setup for Raman tweezers. D1, D2 - dichroic mirrors, Exp1, Exp2 - beam expanders, FM - flipping mirror, L1, L2 - lenses, NDF1, NDF2 - neutral density filters, NF1, NF2 - notch filters, WP - λ/2 wave plate. Dashed rectangle indicates parts of the setup attached to the microscope frame. The inset shows the overlapping focused trapping and Raman laser beams at the specimen. Raman scattering spectra from the optically trapped specimen were collected by the objective lens and subsequently focused by lens L2 into the entrance slit of an imaging spectrograph (focal length 480 mm, f/7.8; Digikröm DKSP480-I, CVI). Two notch filters NF1 (HNF , Kaiser Optical Systems) and NF2 (LP02-785RS, Semrock) were placed in the detection light path that filtered out the trapping and Raman excitation light, respectively. The Raman scattered light was dispersed with a 1200 gr/mm diffraction grating, imaged on the chip of a high-sensitivity liquid-nitrogen-cooled spectroscopic CCD camera (Spec-10:100BR/LN, Princeton Instruments), and recorded using the camera control software (WinSpec). Recorded spectra were processed off-line using custom-written routines implemented in IgorPro software (WaveMetrics). In order to facilitate the observation of the specimen, adjust coarse overlap of the Raman and trapping beams, and aid the trapping of a selected microobject in the sample chamber, light in the imaging path could be diverted via flip mirror FM to a standard CCD camera connected to a monitor and experiment control PC. 3. RESULTS Before carrying out Raman spectroscopic measurements with optically trapped microobjects, we tested the performance of our experimental setup by recording the spectra of a bulk sample. Specifically, we used polystyrene that has a set of well-defined spectral lines in the range of cm 1 with known assignment to the molecular vibration modes (see Table 1) and that is often used as a benchmark in Raman spectroscopy. 8 Fig. 2 shows the Raman scattering spectrum of polystyrene within the spectral range of 560 cm 1 to 2150 cm 1. During Proc. of SPIE Vol

4 2000 Intensity [a.u.] Raman shift [cm -1 ] Figure 2. Raman scattering spectrum recorded from a polystyrene block with 1 s integration time and 20 mw Raman beam power at the specimen. During spectrum recording, Raman beam was focused 40 µm into the polystyrene block and trapping beam with power 200 mw at the specimen was also turned on. the spectrum acquisition, the specimen was also illuminated with the focused trapping beam whose power was 10-times higher than the Raman beam power; this was done to inspect the influence of the trapping beam stray light on the quality of the spectrum. As documented in Fig. 2, all the polystyrene spectral peaks are clearly visible and there is no detectable influence of the trapping beam presence on the recorded spectrum. Thus, the wavelength separation of the Raman and trapping beams is sufficient to observe undisturbed spectra within the wavenumber range cm 1 relevant for Raman spectroscopy of organic molecules (so-called fingerprint region 9 ). Table 1. Characteristic Raman spectral peaks of polystyrene and their assignment to molecular vibrational modes. Raman shift wavenumber [cm 1 ] Vibrational mode 621 ring deformation 795 ring breathing 1000 ring breathing 1032 CH deformation 1155 C-C stretching 1181 C-C stretching 1450 CH 2 scissoring 1583 ring-skeletal stretching 1598 ring-skeletal stretching After testing performance of the spectroscopic part of the Raman tweezers setup with bulk sample, first spectra of optically trapped microobjects were acquired. We used polystyrene beads of 10 µm diameter(duke Scientific) suspended in deionized water as the test specimen. Consequently, their spectra could be readily compared to the previously acquired spectrum of bulk polystyrene. Fig. 3 shows the spectra of both polystyrene bead (green full curve) and bulk polystyrene (red dashed curve) recorded within the same wavenumber range. In the case of optically trapped bead, Raman scattering from the aqueous immersion medium contributes a broad weak non-specific background to the spectrum within the examined wavenumber range. Therefore, the bead spectrum was corrected by subtracting the low-pass filtered solvent background that was recorded with the same Proc. of SPIE Vol

5 settings of the Raman and trapping beams without the presence of optically trapped bead. Moreover, the bead spectrum was re-scaled and vertically offset in order to facilitate the comparison of both spectra. From Fig. 3, it is evident that the bead spectrum contains all the features that can be observed in the bulk material spectrum and the relative intensity of individual spectral peaks with respect to the dominant spectral peak at 1000 cm 1 is identical in both cases Intensity [a.u.] micrometer bead Polystyrene block Raman shift [cm -1 ] Figure 3. Background-corrected Raman scattering spectrum recorded from an optically trapped 10 µm diameter polystyrene bead with 5 s integration time, 25 mw Raman beam power at the specimen, and 200 mw trapping beam power at the specimen (solid, green). For comparison, Raman scattering spectrum recorded from a polystyrene block under conditions stated in Fig. 2 is also shown (dashed, red). For the sake of clarity, the bead spectrum was re-scaled and vertically offset. In the next step, we moved on to yet smaller sample objects and acquired the spectra of optically trapped polystyrene beads of 930 nm diameter (Duke Scientific). Fig. 4 displays the background-corrected spectrum of such a bead along with the reference spectrum of bulk polystyrene. Comparison of both spectra reveals that the dominant polystyrene peaks at 795 cm 1, 1000 cm 1, 1032 cm 1, and 1155 cm 1 can still be well identified in the trapped bead spectrum; however, more subtle spectral details (such as the structure of the peak at 1181 cm 1 ) are somewhat obscured by noise. This stems from the fact that the amount of specimen material contributing to the detected signal is much smaller for 930 nm beads (whose size is smaller than the Raman beam focal volume size) in comparison to bulk polystyrene and 10 µm beads and the signal-to-noise ratio (SNR) of the spectrum is then correspondingly lower. Table 2 summarizes the SNRs of individual spectra (bulk polystyrene, 10 µm beads, 930 nm beads) determined from the amplitude I S of the dominant spectral peak at 1000 cm 1 and the standard deviation σ S of the spectrum noise within the wavenumber range cm 1 that does not contain any characteristic peaks: SNR = I S /σ S. Since the spectra of various specimens were acquired with different Raman beam powers and detector integration times, normalized SNRs were also calculated assuming that I S is directly proportional to the product of the Raman beam power P and integration time t I while σ S remains constant. From Fig. 4 and Table 2, one can estimate that the minimal SNR necessary for reliable identification of polystyrene, when both peaks at 1000 cm 1 and 1032 cm 1 are still distinguishable by eye, is 20. Thus, if the power of the Raman beam is kept at 10 mw, usable spectrum of a 930 nm optically trapped bead can be obtained in 3 s. Alternatively, with Raman beam power 30 mw, spectrum acquisition time drops to 1s. Spatially-resolved Raman microspectroscopy requires the detection of the Raman scattering signal to be confined to a close proximity of the Raman beam focus. Consequently, localized information about the specimen chemical composition can be obtained by its scanning relative to the beam focus. Table 2 illustrates that the condition of the detection confinement is not perfectly fulfilled in our setup as the SNR of the spectrum depends significantly on the specimen size. This phenomenon is especially pronounced along the axial direction of the focusing / collection optical system where the confinement of both excitation and detection point spread function Proc. of SPIE Vol

6 400 Intensity [a.u.] nanometer bead 0 Polystyrene block Raman shift [cm -1 ] Figure 4. Background-corrected Raman scattering spectrum recorded from an optically trapped 930 nm diameter polystyrene bead with 10 s integration time, 8 mw Raman beam power at the specimen, and 100 mw trapping beam power at the specimen (solid, green). For comparison, Raman scattering spectrum recorded from a polystyrene block under conditions stated in Fig. 2 is also shown (dashed, red). For the sake of clarity, the bead spectrum was re-scaled and vertically offset. Table 2. Signal-to-noise ratios of Raman scattering spectra recorded from various specimens. Spectra acquisition conditions: P =20mW,t I = 1 s (bulk polystyrene), P =25mW,t I =5s(10µm polystyrene bead), P =8mW,t I =10s (930 nm polystyrene bead). Normalized SNR of individual spectra were calculated for 10 mw Raman beam power and 1 s integration time. Sample Measured SNR Normalized SNR Bulk polystyrene µm polystyrene bead nm polystyrene bead (PSF) is the weakest. In order to characterize the axial response of our Raman tweezers system, we scanned a 930 nm bead attached firmly to a glass substrate through the Raman beam focus with the use of a piezo-driven positioning stage (see inset of Fig. 5). At each axial position, we verified that the adhering bead was positioned on the beam axis and then recorded its spectrum. Subsequently, we analyzed the amplitude of the spectral peak at 1000 cm 1 as a function of the axial bead distance from the Raman beam focus. Fig. 5 shows the experimentally determined axial response function (crosses) that was fitted with a Gaussian (solid line) to determine its width of 2.1 µm. The effective axial PSF representing the axial resolution of the system could then be determined by de-convolving the measured data with the bead size and its width was estimated to be 2 µm. This value can be improved by employing confocal detection scheme for effective rejection of the out-of-focus light without reducing significantly the intensity of the detected signal. 10 Ultimate specimens for the Raman tweezers are suspended micro- and nanoobjects with complex chemical composition that generally changes in time. A prime example of such objects are individual living cells and artificial cell model systems (membrane vesicles). Biological cells consist of complex mixtures of various molecular types (proteins, nucleic acids, lipids, and polysaccharides) whose spatial and temporal concentration distributions are intimately connected to the cell physiological state. Raman tweezers enable non-contact confinement of the freely floating suspended cells and subsequent identification of individual cell constituents without the need for their exogenous labeling. This is vital as both cell immobilization on a support surface and labeling with exogenous markers (e.g. fluorescent dyes) can alter significantly their physiological state. We tested the performance of our Raman tweezers setup in live cell imaging by acquiring the spectra of optically trapped Proc. of SPIE Vol

7 Intensity of spectral peak at 1000 cm -1 [a.u.] immobilized 930 nm bead Raman beam cover slip scan direction Bead axial position [µm] Figure 5. Axial resolution of the Raman tweezers setup. Measured height of the dominant polystyrene peak at 1000 cm 1 recorded from a 930 nm diameter polystyrene bead immobilized on a glass surface and scanned axially through the Raman beam focus is displayed as a function of the bead position relative to the beam focus (crosses). Solid line shows the fit of the experimental data with a Gaussian of width (2.14 ± 0.10) µm. Spectrum acquisition parameters: integration time 5 s, Raman beam power at the specimen 8 mw. Intensity [a.u.] DNA O-P-O - Amide III Lipids Aromatic AA Amide I 5 µm Raman shift [cm -1 ] Figure 6. Background-corrected Raman scattering spectrum recorded from an optically trapped baker s yeast cell with 30 s integration time, 65 mw Raman beam power at the specimen, and 100 mw trapping beam power at the specimen. 11, 12 Most prominent spectral features have been assigned according to references. Amide I, III - protein amide bands, Aromatic AA - aromatic amino acids, DNA O P O -DNAPO 2 group stretching. Inset shows a bright field image of a typical yeast cell. baker s yeast cells Saccharomyces cerevisiae. These cells represent a convenient and robust model system as they are easy to handle (they do not require special conditions for observation, e.g. maintaining constant temperature and ph of the sample chamber), their structure and composition are relatively simple and well characterized, and their life cycle can be easily controlled. Fig. 6 shows a typical background-corrected Raman scattering spectrum recorded from an optically trapped yeast cell suspended in water that did not contain any additional nutrients or ph- and osmotic pressure-buffering substances. In contrast to polystyrene beads, cells contain a number of different molecules; therefore, their spectra are fairly complex and feature a multitude of peaks (see assignment Proc. of SPIE Vol

8 of the most prominent ones in the figure) whose relative amplitudes change with time as the cell goes through its life cycle. Moreover, because of the compositional complexity, the overall amplitude of the spectral peaks is lower which requires longer exposure time and/or Raman beam power in order to acquire spectra with sufficient SNR. Nevertheless, the Raman spectral patterns of the cells of particular kind are fairly stable and reproducible as has been verified by repeated measurements (data not shown). 4. CONCLUSION In this article, we have presented an experimental setup for Raman microspectroscopy of optically trapped micro- and nanoobjects (Raman tweezers) and characterized its performance by recording the Raman scattering spectra of polystyrene beads and living yeast cells. We have demonstrated that this system is capable of acquiring spatially and temporally-resolved spectra of the trapped specimen and, therefore, it represents a powerful and versatile tool for applications in colloidal and surface physics, analytical chemistry, and cell and molecular biology. Potential for further development of the apparatus lies in increasing the axial resolution of the setup by employing confocal detection scheme and improving the light collection efficiency and throughput by utilizing a lower f/ spectrograph. Acknowledgements Authors acknowledge support from the MEYS CR (LC06007, OC08034) and ISI IRP (AV0Z ). REFERENCES 1. Navratil, M., Mabbott, G., and Arriaga, E., Chemical microscopy applied to biological systems, Anal. Chem. 78, (2006). 2. Novotny, L. and Stranick, S., Near-field optical microscopy and spectroscopy with pointed probes, Annu. Rev. Phys. Chem. 57, (2006). 3. Urlaub, E., Popp, J., Roman, V., Kiefer, W., Lankers, M., and Rossling, G., Raman spectroscopic monitoring of the polymerization of cyanacrylate, Chem. Phys. Lett. 298, (1998). 4. Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., and Chu, S., Observation of a single-beam gradient force optical trap for dielectric particles, Opt. Lett. 11, (1986). 5. Thurn, R. and Kiefer, W., Raman-Microsampling Technique Applying Optical Levitation by Radiation Pressure, Applied Spectroscopy 38(1), (1984). 6. Petrov, D. V., Raman spectroscopy of optically trapped particles, J. Opt. A: Pure Appl. Opt. 9, S139 S156 (2007). 7. Rohrbach, A., Tischer, C., Neumayer, D., Florin, E.-L., and Stelzer, E. H. K., Trapping and tracking a local probe with a photonic force microscope, Rev. Sci. Instrum. 75, (2004). 8. Wesley, I. and Hendra, P., The low-temperature Fourier-transform Raman-spectrum of polystyrenes, Spectrochimica Acta Part A-Molecular and Biomolecular Spectroscopy 50, (1994). 9. Caspers, P., Lucassen, G., and Puppels, G., Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin, Biophysical Journal 85, (2003). 10. Houlne, M., Sjostrom, C., Uibel, R., Kleimeyer, J., and Harris, J., Confocal Raman microscopy for monitoring chemical reactions on single optically trapped, solid-phase support particles, Anal. Chem. 74, (2002). 11. Xie, C., Li, Y., Tang, W., and Newton, R., Study of dynamical process of heat denaturation in optically trapped single microorganisms by near-infrared Raman spectroscopy, J. Appl. Phys. 94, (2003). 12. Notingher, I., Bisson, I., Bishop, A., Randle, W., Polak, J., and Hench, L., In situ spectral monitoring of mrna translation in embryonic stem cells during differentiation in vitro, Analytical Chemistry 76, (2004). Proc. of SPIE Vol