Acoustophoretic 2D-Prefocusing in a Glass Microfluidic Chip for Optical-Stretching

The optical stretcher is a very promising tool for the analysis of single-cell mechanical properties, and several methods to realize a compact and reliable integrated device have been proposed and discussed in the literature, exploiting different layouts and offering different advantages. However, a constant feature of integrated optical stretchers is that the height of the region interested by the laser beams cannot be dynamically adjusted so as to trap and stretch objects flowing at different levels, because of the fixed path of laser radiation (carried by either optical fibers or waveguides). The consequence of this limitation is thus that only cells flowing with the height of the laser-path can be properly manipulated, leading to a low measurement throughput, and even causing a pre-selection of analyzed cells based on their “flow-height”. A commonly proposed solution to this problem is given by the hydrodynamic focusing technique [1], but an alternative suggestion is to use acoustophoretic focusing, as in many cases it can be applied to already existing chips, without requiring the design of complex circuits or the setting of specific flow speeds [2]. Acoustophoretic focusing in fact just requires that the right frequency of the acoustic wave is applied to the chip [3]. In this abstract, we report the experimental results we obtained by applying acoustophoretic prefocusing in a square-channel integrated optical stretcher realized into a glass substrate by 3D fs-laser micromachining technique [4]. The setup, schematically illustrated in Fig. 1a) includes, in addition to the standard components of integrated optical stretchers, a piezoelectric crystal attached underneath the microchip with a resonance frequency matching that of the glass chip [3]. This combination allows for the use of acoustophoretic forces to line-up cells in the center of the microfluidic channel, while optical forces can be used for trapping and stretching. Thanks to the chip being fabricated in glass, acoustophoretic prefocusing requires a lower power with respect to what was previously reported with a hybrid glass-PDMS-glass chip [2], and even if the piezoelectric crystal is positioned at one end of the chip, the focusing effect is observed along the whole channel length. Additionally, thanks to the square channel, focusing is simultaneously obtained both in the horizontal and vertical dimension, as it is evident by comparing Fig. 1c) (where beads are spread in the channel and not all of them are properly in-focus) and Fig. 1d), where all the beads lye in the focal plane of the objective and form a single line in the center of the channel. This solution thus enables the analysis of all the cells flowing in the microchannel, improving measurement throughput and reliability of the results.