Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay.

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.

Effect of long- and short-term exposure to laser light at 1070 nm on growth of Saccharomyces cerevisiae.

The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.

Three-dimensional imaging in three-dimensional optical multi-beam micromanipulation.

In this work, we present a method providing real-time, low cost, three-dimensional imaging in a three-dimensional optical micromanipulation system. The three-dimensional imaging system is based on a small form factor LED based projector. The projector is used to dynamically shape the rear illumination light in a counter-propagating beam-trapping setup. This allows us to produce stereoscopic images, from which the human brain can construct a three-dimensional image, or alternatively image analysis can be applied by a computer, thereby obtaining true three-dimensional coordinates in real-time for the trapped objects.

Computerized "drag-and-drop" alignment of GPC-based optical micromanipulation system.

In the past, aligning the counterpropagating beams in our 3D real-time generalized phase contrast (GPC) trapping system has been a task requiring moderate skills and prior experience with optical instrumentation. A ray transfer matrix analysis and computer-controlled actuation of mirrors, objective, and sample stage has made this process user friendly. The alignment procedure can now be done in a very short time with just a few drag-and-drop tasks in the user-interface. The future inclusion of an image recognition algorithm will allow the alignment process to be executed completely without any user interaction. An automated sample loading tray with a loading precision of a few microns has also been added to simplify the switching of samples under study. These enhancements have significantly reduced the level of skill and experience required to operate the system, thus making the GPC-based micromanipulation system more accessible to people with little or no technical expertise in optics.

Fully automated beam-alignment and single stroke guided manual alignment of counter-propagating multi-beam based optical micromanipulation systems.

In a previous paper [J. S. Dam et al, Opt. Express 15, 1923 (2007)] we demonstrated computerized "drag-and-drop" optical alignment of a counter-propagating multi-beam based micromanipulation system. By inclusion of image analysis, we report here on the extension of this work to accommodate a completely automated beam-alignment process. Additionally, to maintain a cost-effective and technically less demanding system architecture, we also report on a computer-guided manual alignment procedure. In the manual version, the computer analyzes the initial misalignment and the required compensations for each mirror in the system are calculated. Subsequently, the user is guided in adjusting the mirrors exactly by the requisite amount. This way, all mirrors only need to be moved once. The image analysis utilized in both calibration schemes employs a fitting algorithm to determine the position of beam-center with sub-pixel accuracy, thereby providing "better than human" alignment.

2D optical manipulation and assembly of shape-complementary planar microstructures.

Optical trapping and manipulation offer great flexibility as a non-contact microassembly tool. Its application to the assembly of microscale building blocks may open new doors for micromachine technology. In this work, we demonstrate all-optical assembly of microscopic puzzle pieces in a fluidic environment using programmable arrays of trapping beams. Identical shape-complimentary pieces are optically fabricated with submicron resolution using two-photon polymerization (2PP) technique. These are efficiently assembled into space-filling tessellations by a multiple-beam optical micromanipulation system. The flexibility of the system allows us to demonstrate both user-interactive and computer-automated modes of serial and parallel assembly of microscale objects with high spatial and angular positioning precision.

High-speed phase modulation using the RPC method with a digital micromirror-array device.

An improved implementation of the reverse phase contrast (RPC) method for rapid optical transformation of amplitude patterns into spatially similar phase patterns using a high-speed digital micromirror-array device (DMD) is presented. Aside from its fast response, the DMD also provides an electronically adjustable and inherently aligned input iris that simplifies the optimization of the RPC system. In the RPC optimization, we illustrate good agreement between experimentally obtained and theoretically predicted optimal iris size. Finally, we demonstrate the conversion of a binary amplitude grating encoded on the DMD into a binary (0-pi) phase grating.

Three-dimensional forces in GPC-based counterpropagating-beam traps.

We theoretically investigate the three-dimensional (3D) trapping force acting on a microsphere held in a pair of counterpropagating beams produced by the generalized phase contrast (GPC) method. In the case of opposing beams of equal power, we identify the range of beam waist separation s that results in a stable 3D optical potential-well by assessing the dependence of the axial and transverse force curves on s. We also examine how the force curves are influenced by other parameters such as size and refractive index of the microsphere. Aside from force curves of beam tandems with equal powers, we also numerically calculate force curves for cases of beam pairs having disparate relative strengths. These calculations enable us to elucidate the large dynamic range for axial position control of microparticles in GPC-based counterpropagting-beam traps.

Autonomous and 3D real-time multi-beam manipulation in a microfluidic environment.

The Generalized Phase Contrast (GPC) method of optical 3D manipulation has previously been used for controlled spatial manipulation of live biological specimen in real-time. These biological experiments were carried out over a time-span of several hours while an operator intermittently optimized the optical system. Here we present GPC-based optical micromanipulation in a microfluidic system where trapping experiments are computer-automated and thereby capable of running with only limited supervision. The system is able to dynamically detect living yeast cells using a computer-interfaced CCD camera, and respond to this by instantly creating traps at positions of the spotted cells streaming at flow velocities that would be difficult for a human operator to handle. With the added ability to control flow rates, experiments were also carried out to confirm the theoretically predicted axially dependent lateral stiffness of GPC-based optical traps.

GPC-based optical micromanipulation in 3D real-time using a single spatial light modulator.

Using a novel dual-beam readout with the generalized phase contrast (GPC) method, a multiple-beam 3D real-time micromanipulation system requiring only one spatial light modulator (SLM) has been realized. A theoretical framework for the new GPC scheme with two parallel illumination beams is presented and corroborated with an experimental demonstration. Three-dimensional arrays of polystyrene microbeads were assembled in the newly described system. The use of air immersion objective lenses with GPC-based optical trapping allowed the simultaneous viewing of the assemblies in two orthogonal bright-field imaging perspectives.

Real-time interactive 3D manipulation of particles viewed in two orthogonal observation planes.

The generalized phase contrast (GPC) method has been applied to transform a single TEM00 beam into a manifold of counterpropagating-beam traps capable of real-time interactive manipulation of multiple microparticles in three dimensions (3D). This paper reports on the use of low numerical aperture (NA), non-immersion, objective lenses in an implementation of the GPC-based 3D trapping system. Contrary to high-NA based optical tweezers, the GPC trapping system demonstrated here operates with long working distance (>10 mm), and offers a wider manipulation region and a larger field of view for imaging through each of the two opposing objective lenses. As a consequence of the large working distance, simultaneous monitoring of the trapped particles in a second orthogonal observation plane is demonstrated.

Actuation of microfabricated tools using multiple GPC-based counterpropagating-beam traps.

We explore the functionalities of a generalized phase contrast (GPC) -based multiple-beam trapping system for the actuation of various microfabricated SiO2 structures in liquid suspension. The arrays of optical traps are formed using two counterpropagating light fields, each of which is spatially reconfigurable in both cross-sectional geometry and intensity distribution, either in a user-interactive manner or under computer supervision. Design of microtools includes multiple appendages with rounded endings by which optical traps hold and three-dimensionally actuate individual tools. Proof-of-principle demonstrations show the collective and user-coordinated utility of multiple beams for driving microstructured objects. The potential to integrate these optically powered microtools may lead to more complex miniaturized machineries - a closely achievable goal with the real-time reconfigurable optical traps employed in this work.