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• Abstract

Digital holography (DH) is presented as a versatile tool for specimen analysis in the field of noninvasive microscopy applied to biology and microfluidics. The main feature of DH is the possibility to perform quantitative phase measurements that is particularly requested in the case of biological samples.

Recently, at the University of California at Los Angeles (UCLA), researchers have developed a compact and efficient device able to integrate DH technique with microfluidics. The novelty stands in the ability to perform tomography on biological samples while they flow into a microchannel. The optofluidic tomographic microscope, realized by Ozcan et al., presents high resolution in 3-D (about 1 $\mu\hbox{m}$ as lateral resolution and 3 $\mu\hbox{m}$ as axial resolution) over a large field of view (FOV, 15 $\hbox{mm}^{2}$), and it is integrated as a portable on-chip imaging system [1], [2], [3], [4].

The new challenge in this research field is the capability to trap (or drive) and to measure micro objects actually floating or moving inside channels. The goal is to realize a label free and noninvasive system able to stop cells and microorganisms and, at the same time, to measure them quantitatively. The most interesting measurement in the biological sense are, for example, the volume, the shape or the forces exerted among different organisms or between the sample and its environment. Nowadays, the most promising technique is the optical tweezers one. Several research groups in the world have put a lot of effort into improving this optical method, which is able to manipulate objects without mechanical contact. The basic principle is that optical forces generated by a strongly focused laser beam can be exploited to trap and even guide microscopic objects [5], [6], [7]. One of the last advancements has been reached at the University of Innsbruck, Austria, where researchers exploited the ability to combine optical with acoustic micromanipulation in order to merge the large scale trapping of acoustic forces with the high flexibility of optical trapping [8].

Another important topic in optical micromanipulation is the capability to generate intensity light profiles tailored on the specific application. Usually, this is achieved inserting into the optical arrangement one or more reconfigurable diffractive optical elements (DOEs). DOEs are commercially available devices based on liquid crystal technology named Spatial Light Modulators (SLMs). Many efforts, in the last years, have been devoted to improving the quality of the light profile in terms of signal-to-noise ratio (SNR) and to enlarge the range of possible profiles that can be generated [9], [10], [11].

On the other side, a lot of imaging methods are widely investigated for studying trapped objects, in particular for the biological ones, such as phase contrast, differential interference microscopy, etc. [12], [13], [14], [15], [16], [17], [18], [19], [20]. This paper shows several functionalities that can be performed by DH in these fields of application. First, performing phase measurements of samples that, at same time, are manipulated by optical forces, and then computing 3-D tracking of micro-objects. The novelty of this work stands in the implementation of an optical setup able to perform phase-contrast interferometric measurements of a single cell or particle while it is flowing in a microfluidic channel. At the occurrence the apparatus can be used to manipulate and drive the particles to a desired site or to perform the 3-D tracking. One of the main issues is that very often it is not easy to image an object that is moving and/or changing its shape going out of the focus plane. The novelty of our recently developed technique consists in the fact that we are able to trap and guide an object of micrometric size in the appropriate position to be “photographed” by means of a DH microscope. In particular, here, we demonstrate that objects (polymeric particles and in vitro cells) floating in a microfluidic environment can be driven by optical forces along desired directions [14], [21]. In this way these objects can be analyzed by the DHM allowing us to get their quantitative phase-contrast images. For these aims we use two slightly off-axis laser beams coming from a single laser source; the interference between the two beams gives the possibility to record in real time a sequence of digital holograms while one of the beams creates the driving force. The configuration is very stable against the vibrations because the two beams pass through the same microscope objective. The whole setup is described in Fig. 1(a); for details, see [21]. It allows the particles to be driven into the useful FOV, or holographic pose, and to record the digital holograms. The first CCD(I) images the plane close to the beam waist, i.e., the focal plane of the first (lower) objective, in order to monitor the capturing process. The second CCD(H) detects the digital hologram, at an out-of-focus plane where the two beams interfere. Then, the hologram is recorded and numerically reconstructed by the standard algorithms [18], [19] to obtain the whole complex wavefront from which the quantitative phase map of the object can be easily retrieved. In Fig. 1(b), a sketch of the forces acting on the sample is shown. The inset shows three instants of an experiment in which a mouse cell is pushed at different depths under the action of the scattering force of the light beam [21]. The cell is driven against the upper glass wall of the chamber and, in this case, maintained in this position exhibiting adhesion to the surface. Fig. 1(c) shows the reconstructed phase map of the cell in the final stage of the “trip,” during duplication. What is important to note is that usually the observer has to look for the object to be analyzed and place it at center of the FOV area to record the interferogram. In the proposed approach, particles are driven automatically into the FOV of the holographic microscope without the need for a mechanical displacement. In other words, the specimen is forced to enter and remain in the FOV and to travel for a long path along the optical axis; it can be considered a 2-D confinement, and all the particles that fall into this “weak-trap” are pushed along the desired path.

Fig. 1. (a) Sketch of the adopted setup: One beam is used to trap and drive the object, while the other generates the hologram [13]. (b) Close-up of the sample chamber, evidencing the forces exerted on the object; in the inset, a mouse cell driven along a desired path is displayed [13]. (c) Reconstructed phase map of the duplicating cell. (d) Drawing of two interfering laser beams for 3-D tracking. (e) Interferometric recording of the flowing particles. (f) Calculated trajectories of the three particles.

Three-dimensional tracking is performed using the optical setup sketched in Fig. 1(a) by evaluating the double out-of-focus projections of the particles due to the twin-beams onto the array detector's plane, and the geometry is sketched in Fig. 1(d). Each particle forms two shadows on the CCD array, the separation between the two shadows being a function of the longitudinal position of the particle [22]. In this arrangement, three different latex particles are floating into a microfluidic chamber without a priori information. A sequence of images is recorded by the setup of Fig. 1(a). One of the interferometric recording is shown in Fig. 1(e). The presence of six points, i.e., two projection for each of the three particles, is visible here. We compute the centroids by an image-processing algorithm for each frame of the recorded sequences [22]. The 3-D paths of the particles are calculated and the corresponding 3-D plots of the paths are shown in Fig. 1(f). From the last result, it is clear that all the particles taken into account move along the same streamlines in the microfluidic flux. They experience a displacement mainly along the longitudinal axis. This is due to the driving effect on the particles described before. In this paper, we have tried to present a brief and certainly non-exhaustive overview on recent achievements in the field of optical trapping and microscopic imaging, focusing ourselves on our developments in simultaneous optical manipulation and digital holographic imaging of micrometric objects.

## Footnotes

Corresponding author: F. Merola (e-mail: francesco.merola@inoa.it).

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