ARTICLE pubs.acs.org/ac
Optical Tweezers for Synchrotron Radiation Probing of Trapped Biological and Soft Matter Objects in Aqueous Environments Silvia C. Santucci,*,†,‡ Dan Cojoc,‡ Heinz Amenitsch,§ Benedetta Marmiroli,§ Barbara Sartori,§ Manfred Burghammer,† Sebastian Schoeder,†,^ Emanuela DiCola,† Michael Reynolds,† and Christian Riekel*,† †
European Synchrotron Radiation Facility, B.P. 220, F-38043 Grenoble Cedex, France Laboratorio TASC, CNR-IOM Istituto Officina dei Materiali, Area Science Park Basovizza, I-34149, Trieste, Italy § Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, A-8042 Graz, Austria ‡
bS Supporting Information ABSTRACT: Investigations of single fragile objects manipulated by optical forces with high brilliance X-ray beams may initiate the development of new research fields such as protein crystallography in an aqueous environment. We have developed a dedicated optical tweezers setup with a compact, portable, and versatile geometry for the customary manipulation of objects for synchrotron radiation applications. Objects of a few micrometers up to a few tens of micrometers size can be trapped for extended periods of time. The selection and positioning of single objects out of a batch of many can be performed semi-automatically by software routines. The performance of the setup has been tested by wide-angle and small-angle X-ray scattering experiments on single optically trapped starch granules, using a synchrotron radiation microbeam. We demonstrate here for the first time the feasibility of microdiffraction on optically trapped protein crystals. Starch granules and insulin crystals were repeatedly raster-scanned at about 50 ms exposure/ raster-point up to the complete loss of the structural order. Radiation damage in starch granules results in the appearance of low-angle scattering due to the breakdown of the polysaccharide matrix. For insulin crystals, order along the densely packed [110] direction is preferentially maintained until complete loss of long-range order.
ptical tweezers (OT),13 based on the trapping capabilities of focused laser beams, have found numerous applications in different fields of science from physics to life sciences.46 Recently, we reported examples of synchrotron radiation (SR) investigations of optically manipulated liposome clusters7,8 and starch granules.9 The results suggest that the combination of noncontact optical forces with SR scattering and imaging techniques10 could provide powerful in situ probes, in particular for fragile, biological, and soft matter objects. Indeed, choosing a desired object in a reservoir of many, sorting and arranging an array of particles in a water suspension, and controlling their position, orientation, distance, and shape as well as the all-around visibility of the trapped objects is possible by optical forces.46 About 3 orders of magnitude weaker than the mechanical manipulators, optical forces in the femto- to nano-Newton range11 are suitable for handling fragile biological and soft matter objects such as colloidals or virus crystals. Rotations and translations of nano- to micrometric objects are possible by OTs through opportune optical components. Birefringent objects can be rotated by rotating the polarization of the trapping laser beam,12 enabling tomography or single crystal diffraction applications. Multiple traps generated via acousto-optic devices or spatial light modulators (SLM)13 provide additional
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possibilities for manipulating single and multiple objects. Serial crystallography applications14 and interface studies of merging objects (e.g., multiwall liposomes7) accompanied by conformational changes or chemical reactions are examples of the possibilities offered by the method. We also note that X-ray free electron laser (XFEL) diffraction and imaging applications rely currently on particle-loaded liquid jets or aerosols15,16 which imply random scattering events. Sequential positioning of single objects in the beam would allow correspondingly lower sample consumption. Despite the exciting possibilities offered by optical sample manipulation in high brilliance SR beams and the upcoming generation of XFEL sources, the use of OTs has, until now, been limited by the lack of OT setups adapted to the environment of a SR beamline. We have therefore realized a modular and dedicated OT setup adapted to the constrained environment of a SR microfocus beamline. Its portability also allows laboratory use. The OT setup provides a facile tool for studying fragile objects up to several tens of micrometers in diameter at room temperature Received: February 28, 2011 Accepted: May 4, 2011 Published: May 04, 2011 4863
dx.doi.org/10.1021/ac200515x | Anal. Chem. 2011, 83, 4863–4870
Analytical Chemistry
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(r.t.) in a high brilliance SR microbeam by small-angle and wideangle X-ray scattering (SAXS/WAXS) techniques. We have performed micro-SAXS/WAXS experiments on optically trapped single starch granules as a reference case to assess the performance of the setup in a beamline environment. B-type potato starch granules (see Supporting Information) have been used because of the well-known onion-shell structure of this biopolymer17 and the ease to trap single granules.9 The stability of the OT setup has allowed us to perform, for the first time, room temperature (r.t.) microcrystallography on single, optically trapped insulin crystals. Insulin has been chosen for its biological relevance at ambient temperature, as it is the only protein which is stored in a crystalline form in the human body.18 Protein crystallography is usually performed under cryogenic conditions. However, r.t. crystallography with OTs would allow the study of protein samples under conditions close to their biological activity. In combination with microfluidics (MF), optical tweezers allow in situ investigations of single particles, such as cells,19 and the study of samples sorted in their native growth solution.20 In order to obtain a better understanding of radiation damage at r.t. the loss of structural order under the X-ray exposure of these samples has been studied by consecutive raster-diffraction scans of the trapped objects.
’ MATERIALS AND METHODS Materials. Dry potato starch granules were obtained from Roquette and hydrated in deionized water. Size selection of 20 to 40 μm diameter granules was performed by selective precipitation in water. Capillaries were filled with the selected suspension by capillarity. Cubic insulin crystals were grown in 35 days from a 25 mg/mL bovine insulin solution (Sigma) in 50 mM Na2HPO4, pH 10.4, and 1 mM EDTA, according to the sitting drop method. The crystals were sucked in by capillaries directly from the sitting drop. Optical Tweezers Setup. A laser, a microscope objective with high numerical aperture (NA g 1), and an imaging system are the essential elements required for optical trapping. Diffractive optical elements or acousto-optic deflectors can be added to generate multiple traps. Our main concern has been the realization of an ultracompact and portable geometry which can be switched between several SR beamlines. Constraints are also imposed on the sample environment, mainly concerning the absorption and scattering of the materials utilized, as transparency is needed over a broad range of frequencies. In addition, its dimensions are limited by the working distance of the microscope objective, by the scattering cone, and by other elements of the beamline. The OT setup has been realized and adapted to the environment of the ESRF-ID13 microfocus beamline.21 A block diagram of the optical setup and the sample environment are shown in Figure 1A. The installation of the setup at the ID13 beamline is shown schematically in the CAD design in Figure 1B. A zoomedin view of the sample environment is shown in Figure 2A. As sample holders we use glass capillaries from VitroCom with a squared section of 200 μm external side and 50 μm thick walls. This size combines a good optical transmission for all impinging wavelengths (NIR, visible, and X-rays) with a low X-ray scattering background. The flat shape avoids optical lens effects. The capillary is coupled to a 100 μm thick glass or Mylar coverslip through a 50 μm optically clear biadhesive tape from 3M (see Figure S3 of Supporting Information). Fiber lasers offer the flexibility and the compactness desired in a beamline environment. We use an YLM-5-LP-SC ytterbium
Figure 1. A: Block diagram of the optical path in the OT setup (see also Figure 1B for the corresponding symbols). The collimated laser beam impinges on the spatial light modulator (SLM). The modulated beam is directed onto the dichroic mirror (DM) by the mirrors M1 and M2, which are used to refine the laser alignment. A telescopic arrangement of lenses (L1, L2) readapts the beam size to the microscope objective (MO) entrance pupil. This path is planar, parallel to the base of the setup. The dichroic mirror (DM) reflects the laser vertically (Z direction) into the MO, which is mounted upright. A light source illuminates the sample from above. The image is reflected by mirror M through the tube lens (TL) and focused into the CCD. The last path of the illumination runs again parallel to the base of the setup. B: CAD design in SolidWorks (Dassault Systemes SolidWorks Corp.) of the OT setup installed at the ID13 beamline. The setup fits within an idealized parallelepiped volume of 400 200 mm2 base and 230 mm height and is mounted on its own breadboard which is fixed to the motorized x/y/z sample stage of the beamline in a plug-and-play concept. The x/y/z stages are used to perform raster scans and moves the complete setup, hence the trapped sample, with respect to the SR focus. The SR beam is focused by KirkpatrickBaez (KB) mirrors which are housed in a He-filled box. The beam direction corresponds to the x-direction in Figure 1A. To position the sample holder with respect to the microscope objective (MO), a second set of motorized stages (OT x/y/z) is fixed to the breadboard. The individual components are indicated in Figure 1A.
fiber laser source from IPG Photonics which delivers a collimated, linearly polarized cw TEM00 beam at the single mode 1070 ( 1 nm wavelength. The infrared wavelength of the laser induces negligible biological damage.22 A complementary 10 mw red laser guide is used for the alignment. A 100 LUMPlanFl water immersion microscope objective from Olympus is used in upright geometry, which provides a good compromise between a high numerical aperture (NA = 1) 4864
dx.doi.org/10.1021/ac200515x |Anal. Chem. 2011, 83, 4863–4870
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Figure 2. A: Schematic design of sample environment. B: Optical microscopy image of a trapped starch granule (30 μm 40 μm linear dimensions) in a water-filled glass capillary, viewed along the beam direction by the beamline microscope. The white arrows indicate the inner edges of the glass capillary. The trace of the laser beam modulated into three traps is visible through the capillary. C: Optical microscopy image of a trapped insulin crystal (35 μm diameter). The orientation of the cubic unit cell is indicated. The X-ray beam direction is approximately along the crystallographic [001] direction. D14: Operator-assisted selection, automated trapping, displacement, and deposition of the granule indicated by an arrow (see text).
and a long working distance (WD = 1 mm) (Figures 1A,2A). The conic shape of the tip allows a large X-ray scattering cone of (55. Oil immersion could in principle also be used but would imply a shorter working distance (
