Microfluidic integration of high power dual-beam laser ...

Microfluidic integration of high power dual-beam laser traps for cell mechanical measurements F Lautenschlaeger, J R Guck Department of Physics University of Cambridge Cambridge, UK [email protected]

Abstract—The combination of microfluidic systems with laser optical manipulation of suspended objects extends the range of possible investigations in lab-on-chip environments. As an example, mechanical properties of cells can be measured with a specific dual-beam laser trap called the optical stretcher on a single cell basis. The combination of high power laser beams in excess of 1W into a microfluidic environment with high spatial accuracy presents considerable challenges. Here we discuss three alternatives to achieve this goal: a simple glass-capillary setup with only one flow channel, a more elaborate optofluidic chip made of Polydimethylsiloxane (PDMS) for rapid prototyping, and a monolithic glass chip for high durability, damage threshold and optical clarity. Advantages and disadvantage are being discussed. Such microfluidic optical stretcher setups open new possibilities for label-free characterization of cells with biotechnological applications. Keywords-microfluidics; optical trap; optical stretcher; cell mechanics; PDMS; glass chip

I.

INTRODUCTION

Microfabrication and microfluidics are fast growing research areas. Especially in the life sciences, lab-on-chip solutions seem appropriate since often only limited amounts of sample volume are available and the objects to be investigated are on the micrometer or nanometer scale, ideally suited for microfluidic delivery. Possible investigations of cells or molecules include optical inspection or manipulation, biochemical reactions, or spectroscopic interrogation with the ultimate goal of sorting based on the outcome of such investigation. One interesting aspect is the temporary immobilization of the objects under study to permit better data acquisition. This can be achieved by dielectrophoretic [1] or by thermophoretic trapping [2]. A third alternative, optical trapping, is described in the present manuscript. As shown by Ashkin in 1970, light can create forces by radiation pressure [3], an effect that can be exploited to build laser traps – the most common one being so called optical tweezers [4]. Optical tweezers trap beads in a highly focussed laser beam which is often exploited in biology to manipulate biological material such as molecules or cells [5-9]. This can provide very precise and local measurements of e.g. mechanical properties of cells. One disadvantage of optical tweezers is the very low throughput and the need of physical

contact of the trapped bead to the object. These problems can be avoided by another type of optical trap, called the optical stretcher. This is a two beam laser trap where single suspended cells can not only be held stationary against a flow, but also stretched along the laser axis without any mechanical contact, using optically induced force on surface due to momentum transfer [10,11] (Fig 1). Cell deformability measurements with an optical stretcher have been shown to be sensitive enough to distinguish between healthy cells and cancerous cells in different metastatic states [12-14]. Exemplified by such results, mechanical measurements of living material such as tissue or single cells have gained enormous importance during the last decade [13,15-19]. Thus, it is worth considering other possible means of measurements. Local measurements can be done by poking the cell via atomic force microscopy (AFM) [15,20-22] or pulling it partially into a micropipette [23,24]. In addition to optical stretcher measurements, whole cells can also be deformed by letting them adhere between two movable plates whose distance from each other can be varied [25] or by shear flow [26]. The big advantage of the optical stretcher compared to all these other methods is the possibility to include microfluidics into the trapping system and to obtain high-throughput measurements. In addition, optical measurement within a flow system opens the possibility to sort single cells after the measurement and to investigate them further. Two key points are crucial for such a lab-on-chip highthroughput optical stretcher: First, the optical fibers have to be perfectly aligned opposite of each other to guarantee stable optical trapping. Second, the material, of which such a chip is made, needs to be durable and very low absorbent in the range of the wavelength used for the trapping as well as optically clear enough to transmit small amounts of light in case cellular features stained with fluorescent markers are investigated. In the following text we present three different approaches to achieve such requirements.

Financial support was provided by an R&D grant (SAB, Project 9889/1519) from the European Fund for Regional Development (EFRE) 2000-2006.

Figure 1: Principle of an optical stretcher. Two-counter propagating NIR laser beams (
= 1064nm) emanating from the cores of single-mode optical fibers are used to trap and deform single cells.

II.

EXPERIMENTAL REALIZATIONS OF MICROFLUIDIC DUALBEAM LASER TRAPS

A.

Glass capillary setup The fact that the optical stretcher can be combined with microfluidic delivery and therefore increases the throughput to the point where screening cell populations for non-normal cells becomes possible is one of the biggest advantages compared to others method used to measure single cell mechanics. An easy, yet very robust way of implementing a controlled flow of cell suspension into the optical stretcher was demonstrated by Lincoln [27] in 2007. In this setup, two optical fibers are aligned orthogonally to a glass capillary with help of a two dimensional channel structure produced directly in a thick layer of SU-8 photoresist by standard photolithography (Fig 2). Index matching gel is applied in the gaps between the fiber ends and the glass capillary to avoid Fresnel reflection. The two ends of the capillary are connected to two reservoirs which can be adjusted in height. This creates a differential pressure which is used to control the flow inside the capillary. The technical details can be found in a previous publications [27,28]. A big advantage of this capillary setup is the high damage threshold of glass. Unfortunately, glass capillaries with appropriately sized dimensions and a square cross section are not available with a Y-junction and therefore only obtainable with one output. As a result, all cells will be collected together regardless their position in the flow channel. This is not suitable to sort single cells for further analysis and another approach has to be used when this is the intention.

B. Optofluidic PDMS chip Polydimethylsiloxane (PDMS), a two component silicone based elastomeric material, seems to be the ideal candidate for creating arbitrary flow geometries. It is liquid and gets hardened by baking for about 1h at 50-100 ºC. The result is an elastic, optically clear material which is often used to build microfluidic devices [29]. The liquid PDMS is poured on top of a negative mask of flow and fiber channels, which can be produced e.g. by photolithography. After heating, the PDMS can be separated from the mask and has now the desired channel structure. Pressing a microscope cover slip on top of it

Figure 2: Glass capillary setup for optical stretcher. a) Microfluidic chamber mounted on a microscope stage with the optical fibers aligned opposite of each other and a glass capillary placed orthogonally to them. The ends of the glass capillary are connected with graphite ferrules to standard tubing leading to corresponding reservoirs. b) Phase contrast image of the microfluidic chamber with a cell trapped between the two optical fibers.

seals the channel structure via adhesion. PDMS chips have the advantage that the design can be easily changed and produced at a low cost. Combining the microfluidic PDMS part with a holder made of Poly(methyl methacrylate) (PMMA) is possible and allows the use of standard fluidic connectors (Fig 3). The challenge of accurate fiber insertion necessary to complete a working optofluidic PDMS chip has successfully been addressed by using a fiber insertion wheel [29]. Since PDMS and the required production steps are very economical, this optofluidic PDMS chip can be produced for single use. This aspect is particularly important in cancer diagnosis applications to avoid cross contamination between patients. For extended use in a research laboratory, however, PDMS has some disadvantages such as thermally activated depolymerisation mechanisms at increased temperatures [30]. These might occur when laser light is absorbed by dirt particles in the system. Therefore, it is desirable to consider a third option, which provides both microfluidic flexibility as well as durability. C. Monolithic optofluidic glass chip Arbitrary microfluidic channels can also be produced in glass by photolithographic etching using chrome masks and etching with hydrofluoric acid. In this example, the channels

Figure 3 : Optofluidic PDMS chip for optical stretcher. A thin PDMS layer containing the optical fiber and fluidic channels, sealed by a thin cover slip, is created directly on top of a PMMA carrier, which provides convenient fluidic connectorization. Fibers can be inserted after mounting on the microscope by a custom device. The size of the PDMS/PMMA hybrid chup is approximately that of a LEGO piece.

Figure 4 : Monolithic glass chip of the optical stretcher including fluidic connector.

are isotropically etched. The two halves of a final chip can both be processed and then chemically bonded which results in one piece of glass with channel structures inside. The glass chip, including a fitted microfluidic adaptor to external tubing also acting as a strain relieve for the optical fibers, was professionally produced (Dolomite Centre Ltd., Royston, UK) (Fig 4). The design of the fiber and flow channels builds on previous experience with the other microfluidic manifestations of the optical stretcher to ensure efficient trapping of cells. Obviously, one concern with this solution is the curved interfaces of the various channels, which the laser beam has to pass. A first proof of promise of trapping and stretching of single cells inside such a glass chip has been obtained (Fig 5). This monolithic optofluidic glass chip combines the advantages of the capillary setup such as a high damage threshold and good transparency of glass with the arbitrary channel design of the optofluidic PDMS chip. Unfortunately, this chip is considerably more difficult to produce, which results in higher manufacturing costs compared to the PDMS alternative.

The combination of dual-beam laser traps into lab-on-chip environments provides added capabilities for the extended investigation of biological samples. One such capability is the measurement of cell mechanical properties with highthroughput using an optical stretcher. We have shown three different ways of combining this tool with microfluidic delivery, addressing the various requirements of such a system.

III.

CONCLUSION

The simplest yet very reliable and robust solution consists in the use of a straight glass capillary for cell delivery between the optical fiber ends. If the possibility to sort single cells after measurements is of interest, more than one outlet is necessary, which can be achieved in two ways. One is a low-cost PDMS chip which can be produced for single use due to is low manufacturing cost, but where the potential damage of PDMS when irradiated with high-power laser beams needs to be considered. This problem is better balanced in a monolithic glass chip, which has a much higher damage threshold but quite high manufacturing costs. Either one has applications in the characterization of cells by assessing their mechanical properties. Beyond mechanical characterization, optically trapping cells inside microfluidic systems with such dual-beam traps will also be useful for general high-content analysis.

Figure 5: Stretching in the monolithic glass chip. a) Light microscopy image of a cell trapped inside the monolithic glass chip. b) Relative deformation along the laser axis of a single cell deformed in the monolithic glass chip. Stress application indicated by continuous line.

ACKNOWLEDGMENT The authors would like to thank B. Lincoln, F. Wottawah, S. Schinkinger, J. Käs, Ch. Dietrich, G. Whyte, E. MartinBadosa, K. Chalut, GeSiM mbH (Dresden, Germany), and Dolomite Centre Ltd (Royston, UK) for technical assistance, advice, and helpful discussions.

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