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Dynamic micro-bead arrays offer great flexibility and potential as sensing tools in various scientific fields. Two optical trapping techniques, the GPC method using a spatial light modulator and a mechanical scanning method using galvano mirrors, are combined in a hybrid optical tweezers system to handle dynamic micro-bead arrays. This system provides greater versatility while the GPC method creates massive micro-bead arrays in a 2D space, where the trapped beads can be manipulated smoothly and very quickly in a 3D space using the mechanical scanning method. Four typical examples are demonstrated in real time.
©2011 Optical Society of America
1. Introduction
Micro/Nanosystems for biomedical fields, such as microarrays [1] and microfluidic systems [2], are currently an area of intensive research. DNA-chips, using micro-spots of bio-molecules on a static solid support, represent a widely used group of static microarrays for basic studies in biomedical fields, diagnostics, drug discovery, etc. Compared with such static microarrays as DNA-chips, dynamic microarrays using mobile substrata, usually micro-beads coated with bio-molecules or chemicals, offer greater flexibility and have the potential to be used as sensing tools for advanced research in biomedical fields [1]. To realize the potential of dynamic microarrays, micro-bead handling techniques allowing us to transport the selected beads and to immobilize them for signal detection are essential. In several demonstrated approaches [3–5], multi-beam optical tweezers [6–10] are the most suitable techniques for the simultaneous manipulation of numerous micro-beads, and the dynamic micro-bead arrays assembled by these optical tweezers have several advantages: superior cost performance due to the software-oriented configuration, easy applications based on computer vision techniques, and so forth [11]. In our previous paper [11], we chose the Time-Sharing Synchronized Scanning (T3S) technique for the physical method of multi-beam optical tweezers to demonstrate the proposed software-oriented approach. However, the individual use of these multi-beam techniques including the T3S optical tweezers allows for motion of the traps with their specific strengths and limitations [12].
For the dynamic handling of massive micro-bead arrays, we present in this paper a hybrid system consisting of two multi-beam optical tweezers techniques: a generalized phase contrast (GPC) method using a spatial light modulator (SLM) [8,13], and a mechanical scanning method using galvano mirrors (GMs) [11]. This system provides greater versatility while the GPC method creates the trap fields for immobilizing massive arrays, where the beads can be manipulated smoothly and very quickly by the mechanical scanning method. We demonstrate four typical examples: the interactive handling of a massive 12 × 12 array and its elements, the dynamic manipulation of two arrays in two-and-half-dimensional (2.5D) space, the high-speed manipulation of the elements in four sets of a 4 × 4 array, and the parallel manipulation of two sets of a 4 × 4 array using both the GPC and GM scanning methods. We also describe the system features and the configuration of the hybrid system.
2. System features and experimental setup
In our previous paper [11], we applied the T3S optical tweezers with GMs to the fully automated assembly of dynamic micro-bead arrays in a 2.5D working space. This GM scanning method was extremely useful for testing new control algorithms of the dynamic arrays, since it was simple to rapidly change multiple trapping positions. However, the number of beads which could be trapped was limited to ten-odd because of the scanning speed and the dwell time necessary for stable trapping; for example, the dwell time required was 10 ms for each bead. On the other hand, the GPC method using a SLM is a more suitable technique for manipulating a large number of beads under real-time control, since it is a straightforward procedure which can generate numerous and complex intensity patterns in the observation plane of a microscope. Another approach using a SLM is the holographic optical tweezers (HOT) [6]. However, real-time HOT-based manipulation of individual beads in large arrays is limited because of the complex computation process, the high requirements to the space-bandwidth product of the SLM, and the inherent diffraction losses that could affect the trapping efficiency of each beam [8,13,14]. For the real-time handling of massive arrays, low computational cost of generating traps and high light efficiency are compulsory. Therefore, we chose the GPC method to manipulate/immobilize many beads in massive arrays. However, the conventional GPC tweezers linked with a standard inverted microscope do not provide 3D traps but only 2D traps, where beads are normally held against the microscope cover glass [8]; consequently, the potential for the undesired stacking of beads along the beam axis increases with the array size. Therefore, for the practical utilization of GPC tweezers except for the counterpropagating-beam GPC using the special microscope with two objective lenses [13,15], a user interface which can deal with this problem efficiently is required [16].
Thus, under the practical and reasonable restrictions of using a commercially available standard microscope and a single laser source, we developed a hybrid system combining two optical trapping techniques (the GPC method using a SLM and a mechanical scanning method using GMs) for the dynamic assembling/handling of massive micro-bead arrays. This system provides greater versatility, because the GPC method creates massive micro-bead arrays in a 2D space where the beads thus trapped can be manipulated very quickly and smoothly in a 3D space using the GM scanning method. Figure 1 shows the schematic diagram for the implementation of the hybrid system. This optical structure is linked to the inverted microscope (Olympus, IX70) via its epi-fluorescence port. The single laser source is a continuous wave (cw) Nd:YAG laser (Laser Quantum, forte, 1064 nm, TEM00, 700 mW), and its laser beam passing through a half-wave plate (λ/2) is split into two beams (p- and s-polarized beams) by a polarized beam splitter (PBS). One set of optical tweezers, based on the GPC method, is composed of a SLM (Hamamatsu Photonics, LCOS-SLM), a phase contrast filter (PCF) and lenses (L1(f 1 = 300 mm), L2(f 2 = 200 mm), L3(f 3 = 400 mm)), and uses the p-polarized beam. The custom PCF for the GPC tweezers was fabricated by the dry etch of a synthesized quartz glass plate, etching a 40-μm diameter circular area on its surface. Note that the optimal etching depth, d PCF, is
where λ is the wavelength of laser beam, and nλ is the refraction index of the glass plate. The other set of optical tweezers, based on the GM scanning method, is composed of GMs and a lens (Lz) mounted on a PC-controlled linear stage, and uses the s-polarized beam.
Fig. 1 Schematic diagram of a hybrid system combined GPC optical tweezers (orange beam) and GM scanning tweezers (red beam) for interactive/automatic handling of dynamic arrays.
The trapping beams of these tweezers are introduced coaxially into the microscope via a dichroic mirror and an oil-immersion objective lens. The laser power can be distributed between the two methods in varying proportions with the half-wave plate. The geometric shape of the trap fields formed by these tweezers can also be controlled independently, since the p- and the s-polarized beams do not interfere with each other. In the hybrid system, micro-beads trapped by the GPC tweezers are normally trapped at a microscope’s imaging plane, fo, against the upper surface of a closed space. On the other hand, a micro-bead trapped by the GM scanning tweezers at a beam’s focal point, fs, can be manipulated in a 3D space. Note that in our system arrays formed by the GM scanning tweezers based on the T3S technique can be handled only in a 2.5D space, where the arrays can be translated/rotated in the XY-plane at an arbitrary Z-coordinate. This limitation arises from the lower bandwidth (several Hz in our system) of Z-axis manipulation due to the lens Lz translation using the linear stage, since the lens translation has large inertia and requires mm order motion for Z-axis manipulation. For true 3D T3S array manipulation, therefore, an alternative Z-axis manipulation method with higher bandwidth (for example, using a deformable mirror [17]) is required.
3. Demonstrations
3.1 Interactive handling of arrays
Here we demonstrate the interactive assembly/handling of micro-bead arrays. The sample is polystyrene micro-beads (Polysciences, 2 μm) dispersed in water, and the objective lens employed is an oil-immersion lens (Olympus, UPlanFLN × 60, NA = 1.25, IR).
Figure 2 (Media 1) is a sequence of images recorded with the CCD camera showing the results of the interactive assembling of a 12 × 12 array and the subsequent handling of its elements. The laser power for the GPC tweezers (p-polarized beam) at the entrance pupil of the objective lens was 240 mW, and that for the GM scanning tweezers (s-polarized beam) was 28 mW. First, a 14 × 14 matrix pattern of disk-shaped beams illustrated in the inset of Fig. 2(a) was irradiated with the GPC tweezers, where the irradiation area was 36 μm in diameter, and each disk-shaped beam with a diameter 2 μm was able to trap a single bead at the center. Secondly, we interactively transported 144 beads into the array, one by one, to form a massive 12 × 12 array of micro-beads using a drag-and-drop user interface with the PC-mouse controlled GM scanning tweezers (Fig. 2(a)). This drag-and-drop user interface using a PC-mouse (where the right button and the wheel button were assigned the shutter on/off command and the Z-coordinate movements of the GM scanning tweezers, respectively) allowed us to collect and arrange beads smoothly into the lattice points generated by the GPC tweezers; for example, it took less than 10 minutes to assemble the 12 × 12 array. Thirdly, three beads (indicated by the white arrows and the ellipse in Fig. 2) in the sixth column of the 12 × 12 array were taken out one by one using the user interface (Figs. 2(b) and 2(c)). Fourthly, these beads were returned into the array to re-form the complete 12 × 12 matrix (Media 1). Finally, the 12 × 12 array was broken after shutting out the irradiation of the disk-shaped beams. To our knowledge, this is the first demonstration of the optical assembly of significantly large micro-bead arrays composed of over a hundred beads, without lattice defects and without undesired stacking of beads along the beam axis.
Fig. 2 (Media 1) Video frame sequence of interactive manipulation of micro-beads to assemble and handle a massive array. Inset in (a) shows the 14 × 14 matrix pattern of disk-shaped beams and its irradiation area (dotted circle) in imaging plane, with GPC tweezers. (b-c): The elements of the array are taken out one by one from the 12 × 12 array using the PC-mouse controlled GM scanning tweezers.
In another demonstration shown in Fig. 3 (Media 2), a 2 × 2 array trapped by the T3S optical tweezers was interactively manipulated in a 2.5D space while the 24 beads trapped by the GPC tweezers formed a square. The laser power for the GPC tweezers was 172 mW, and that for the GM scanning tweezers was 72 mW. First, in order to assemble a 2 × 2 array and a square of micro-beads, the beads were arranged in a 2D pattern of disk-shaped beams with the GPC tweezers using the drag-and-drop user interface with the GM scanning tweezers in the same manner as in the first demonstration, where the beads in the 2 × 2 array and the square were trapped against the lower surface of an upper cover glass in the same XY-plane, namely the microscope’s imaging plane (Fig. 3(a)). Secondly, four beads forming the 2 × 2 array were firmly and simultaneously trapped, using the T3S optical tweezers instead of the GPC tweezers, while the 24 beads forming the square were still trapped by the GPC tweezers. Finally, subsequent 2.5D movements of the 2 × 2 array, namely descent (Fig. 3(b)), translation in another XY-plane (Figs. 3(c) and 3(d)) and ascent (Fig. 3(e)), were able to traverse the square in the 3D space. Consequently, the 2 × 2 array which was outside the square was able to reach its destination within the square, whilst maintaining its geometrical shape. Figure 3(f) illustrates these 2.5D movements of the 2 × 2 array in cross-sectional view, where yellow circles indicate the 2 × 2 array and red circles indicate the square.
Fig. 3 (Media 2) Video frame sequence of the interactive manipulation of a 2 × 2 array in a 2.5D space. The 2 × 2 array is controlled by the time-sharing synchronized scanning technique while the 24 beads form a square with GPC tweezers. (f): Movements of the 2 × 2 array in cross-sectional view, where yellow circles denote the 2 × 2 array.
3.2 High-speed and parallel handling of arrays
To verify the performance of the hybrid system for the automatic handling of both the arrays and their components, here we demonstrate the high-speed manipulation of the array’s elements and the parallel handling of multiple arrays. The sample and the objective lens are the same as mentioned in Section 3.1.
Figure 4 (Media 3) is a sequence of images recorded with the CCD camera showing the results of the high-speed manipulation of the elements in the four sets of a 4 × 4 array. The laser power for the GPC tweezers was 198 mW, and that for the GM scanning tweezers was 47 mW. First, in the same manner as in the first demonstration in Section 3.1, four sets of a 4 × 4 array were assembled in a 12 × 12 matrix pattern of disk-shaped beams with the GPC tweezers, where each disk-shaped beam was 2 μm in diameter (Fig. 4(a)). Next, using the GM scanning tweezers, after a bead at a corner of single 4 × 4 array was taken out from the array, the bead was driven twice along an 8-shaped path at super-high speeds (for example, the path for bead numbered ‘one’ is indicated by black arrows in Figs. 4(b) and 4(c)), and returned again to its original position. These automated procedures were executed for the four beads at a corner of the 4 × 4 arrays in the numbered order in Fig. 4(a). To detect the trajectories by visual observation and by color CCD camera images recorded at a sampling rate of 1/30 second, we introduced a He-Ne laser beam as a marker for the GM scanning tweezers, and the velocity along the 8-shaped paths was specified at 85 μm/s, although we could have manipulated the bead more quickly. Note that in the accompanying movie, the 8-shaped trajectories during the high-speed manipulation are observed by the dim red line (i.e. as an afterimage of the bead).
Fig. 4 (Media 3) Video frame sequence of the high-speed manipulation of the elements of micro-bead arrays forming the four sets of a 4 × 4 array with GPC tweezers. Four beads indicated by numbers in (a) are sequentially manipulated at super-high speeds along the paths indicated by black arrows in (b) and (c). The accompanying movie is in real time, not accelerated.
In another demonstration shown in Fig. 5 (Media 4), all beads in the image were manipulated fully automatically by the GPC tweezers to form two sets of a 4 × 4 array under the intelligent control techniques as described in our previous paper [11], and subsequent parallel manipulation using both the GPC and the GM scanning tweezers transferred these two arrays in opposite directions. The laser power for the GPC tweezers was 220 mW, and that for the GM scanning tweezers was 41 mW. First, in order to prevent diffusion of the beads by Brownian motion during the bead recognition process based on the circular Hough transform [18], the beads were trapped at the grids of the disk-shaped beams generated by the GPC tweezers, where each disk-shaped beam was 2 μm in diameter and the distance between the grids was 3.5 μm (Fig. 5(a)). The segments of the grids at which the beads are trapped are indicated by the red circles in the inset of Fig. 5(a). Secondly, after detection of the bead positions by the Hough transform, all beads were automatically re-trapped by the disk-shaped beams which only irradiated at the detected positions, instead of at the grid pattern (Fig. 5(b)). Thirdly, these trapped beads were simultaneously transported by the GPC tweezers along the collision-less trajectories to form two sets of a 4 × 4 array (Figs. 5(b)–5(d)). Fourthly, the sixteen beads forming the upper-left 4 × 4 array were transported one by one with the GM scanning tweezers at super-high speeds while another sixteen beads forming the lower-right 4 × 4 array were simultaneously transported with the GPC tweezers (Figs. 5(d) and 5(e)). Note that the upper 4 × 8 matrix of the disk-shaped beams illustrated in each inset of Figs. 5(d)–5(f) was used to immobilize the beads in the 4 × 4 arrays before and after the one-by-one high-speed transportation with the GM scanning tweezers. Finally, parallel manipulation using both the GPC and the GM scanning tweezers was completed to transfer the two arrays in opposite directions (Fig. 5(f)).
Fig. 5 (Media 4) Video frame sequence of the parallel manipulation of the elements of micro-bead arrays forming the two sets of a 4 × 4 array. (a)–(d): Full-automated assembling of two sets of a 4 × 4 array using the GPC tweezers. (d)–(f): Sixteen beads forming an upper 4 × 4 array are transported one by one using the GM scanning tweezers at super-high speeds while another sixteen beads forming a lower 4 × 4 array are simultaneously transported using the GPC tweezers. Inset in each figure shows the 2D pattern of disk-shaped beams generated by the GPC method. The accompanying movie is in real time, not accelerated.
4. Conclusion
We have developed hybrid optical tweezers consisting of GPC tweezers and GM scanning tweezers, and demonstrated four typical examples of interactive or automatic handling of the massive arrays. Although we have dealt with arrays composed of micro-spheres alone, the hybrid system combined with image processing techniques would enable us to apply the multiple-force optical clamp techniques to massive arrays composed of non-spherical micro-objects (for example, rod-shaped, ellipse-shaped, etc.) such as diatoms and whiskers [19]. Additionally, the hybrid system has great versatility as a non-contact micromanipulation tool for various biomedical applications as well as for the demonstrated applications of massive micro-bead arrays; therefore, the system will enable exciting applications not only in microfluidic systems [20] but also in cell biology, such as non-contact mechanotransduction in live cells [21]. Furthermore, under the visual feedback control schemes, the use of the force feedback device instead of the PC-mouse for the user interface of the interactive control of the GM scanning tweezers, may enable higher dexterous micromanipulations [22], since even conventional GM scanning tweezers can respond within 1 ms.
Acknowledgments
This work was partly supported by the Japan Society for the Promotion of Science (Grants-in-Aid for Scientific Research (C, #20560252)).
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