Biophysics

We investigate the application of the evanescent-wave illumination for particle velocimetry near nano-structured surfaces. Theoretical analysis shows that, when the nano-structured lower wall of a channel has to be (optically) considered as an intermediate layer, the nano-structures affect the evanescent-wave illumination in several ways: (1) the nano-structures grating period has to be set perpendicular to the optical plane of incidence, so that all diffraction orders experience total internal reflection (TIR) simultaneously; (2) the TIR has to occur at the smooth bottom of the lower wall, so that the nano-structures do not scatter the incident laser beam; (3) the nano-structures spatially modulate the illumination by occupying spaces that cannot be sampled by the tracer particles. We perform quantitative imaging measurements inside 5-µm-deep PDMS channels, where a part of the 165-µm-thick lower wall is equipped with nano-structures (2D grating, height = 200 nm, period = 2 µm). We use distilled water as the liquid, in which we dilute 40-nm-diameter fluorescent beads employed as tracer particles. The evanescent-wave, which illuminates the particles, is induced using TIR of a continuous-wave 532-nm laser beam delivered through an oil-immersion objective lens (NA=1.49). Our results show that, besides of its main aim for the 3D velocimetry, the modulated illumination is also potentially useful for: (1) estimating the actual illumination depth directly in the channel as well as (2) detecting and quantitatively measuring a thin layer of air trapped at the nano-structures, which may explain the previously reported enhanced slip length values at nano-structured surfaces. We also discuss how the fluorescence emission efficiencies of the tracer particles may depend on each particle's location, particularly relative to the nano-structured surfaces and to the neighbouring particles.

Nanoparticle image velocimetry ͑nano-PIV͒, based on total internal reflection fluorescent microscopy, is very useful to investigate fluid flows within ϳ100 nm from a surface; but so far it has only been applied to flow over smooth surfaces. Here we show that it can also be applied to flow over a topologically structured surface, provided that the surface structures can be carefully configured not to disrupt the evanescent-wave illumination. We apply nano-PIV to quantify the flow velocity distribution over a polydimethylsiloxane surface, with a periodic gratinglike structure ͑with 215 nm height and 2 m period͒ fabricated using our customized multilevel lithography method. The measured tracer displacement data are in good agreement with the computed theoretical values. These results demonstrate new possibilities to study the interactions between fluid flow and topologically structured surfaces.

Producing polymeric or hybrid microfluidic devices operating at high temperatures with reduced or no water evaporation is a challenge for many on-chip applications including polymerase chain reaction ͑PCR͒. We study sample evaporation in polymeric and hybrid devices, realized by glass microchannels for avoiding water diffusion toward the elastomer used for chip fabrication. The method dramatically reduces water evaporation in PCR devices that are found to exhibit optimal stability and effective operation under oscillating-flow. This approach maintains the flexibility, ease of fabrication, and low cost of disposable chips, and can be extended to other high-temperature microfluidic biochemical reactors.

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4 • C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16 • C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.

Artificial surfaces that exhibit unidirectional water spreading and superhydrophobicity are obtained by Strelitzia reginae leaves. Both green and dried leaves are used, thus exploiting the plant senescence. We demonstrate that the natural drying process of the leaves strongly affects the surface morphology and wettability. Polymeric stamps from the green leaf show an arrangement of periodic microridges/microgrooves that favor anisotropic wetting, with a water contact angle (WCA) variation of about 21% along the two principal directions. Instead, the shrinkage of the leaf tissue, as a consequence of the natural dehydration process, induces an enhancement of the superficial corrugation. This results in the establishment of a superhydrophobic state, which shows a WCA of up to 160°, and water rolling off. S. reginae leaves are therefore easily accessible stamps suitable for controlling wettability and realizing surfaces that exhibit various wetting behaviors.

An innovative, simple and reliable method to fabricate micro-lasers by self-assembly of rod-shaped nanocrystals is demonstrated. Dot/rod core/shell CdSe/CdS nanorods are used to form optical micro-resonators by exploiting their self-organization into well-defined coffee stain rings. The fabrication process merely consists of capillary jet deposition of a nanorod solution onto a glass substrate, and is scalable, economic, and highly reproducible. Upon optical pumping of the micro-resonators, laser emission in the red or in the blue-green spectral region is obtained, demonstrating lasing both from core and shell transitions, with low pumping thresholds. Modeling by full-wave numerical simulations according to generalized (i. e. scattering) formulation of laser theory demonstrates lasing from complex modes of the self-assembled cavity.

Multifunctional capability, flexible design,rugged lightweight construction, and selfpowered operation are desired attributes for electronicsthat directly interfacewith the human body or with advanced robotic systems.For these and related applications, piezoelectric materials, in forms that offer the ability to bend and stretch, are attractive for pressure/force sensors and mechanical energy harvesters. In this paper we introduce a large area, flexible piezoelectricmaterial thatconsists of sheets of electrospunfibers of the polymer poly[(vinylidenefluoride-co-trifluoroethylene]. The flow and mechanical conditions associated with the spinning process yield free-standing, threedimensional architectures of alignedarrangements of such fibers, in which the polymer chains adopt strongly preferential orientations. The resulting material offers exceptional piezoelectric characteristics, to enable, as an example, ultra-high sensitivity for measuring pressure, even at exceptionally small values(0.1 Pa). Quantitative analysis providesdetailed insights into the pressure sensing mechanisms, and engineering design rules. Applications range from self-powered micromechanical elements, to self-balancing robots and sensitive impact detectors.

We show how the capillary filling of microchannels is affected by posts or ridges on the sides of the channels. Ridges perpendicular to the flow direction introduce contact line pinning which slows, or sometimes prevents, filling; whereas ridges parallel to the flow provide extra surface which may enhances filling. Patterning the microchannel surface with square posts has little effect on the ability of a channel to fill for equilibrium contact angle θe 30 o . For θe 60 o , however, even a small number of posts can pin the advancing liquid front. 47.55.nb, 68.08.Bc Recent years have seen rapid progress in the technology of fabricating channels at micron length scales. Such microfluidic systems are increasingly finding applications, for example, in diagnostic testing and DNA manipulation and as microreactors. As narrower channels are used, to conserve space and reagents, surface effects will have an increasing influence on the fluid flow. In particular, it may be possible to exploit the chemical or geometrical patterning of a surface to control the flow within the channels. Indeed first steps in this direction have already been taken: for example, Joseph et al. used superhydrophobic surfaces to introduce slip lengths of the order a few µm [1], Zhao et al. used hydrophilic stripes to confine fluid flows [2], and Stroock et al. used oriented grooves to enhance mixing [3].

The significant increment of TiO 2 surface wettability upon UV irradiation makes it a promising component of materials or systems with tunable surface wetting characteristics. This remarkable property of TiO 2 is retained in the nanocomposite materials developed for this work, which consist of the elastomer PDMS enriched with organic-capped nanorods of TiO 2 . In particular, the nanocomposites demonstrate a surface transition from a hydrophobic state to a hydrophilic one under selective pulsed UV laser irradiation. This wettability change is reversible, with the hydrophobic character of the nanocomposites being fully recovered after a couple of days of samples storage in moderate vacuum. The hydrophobic-to-hydrophilic transition and recovery can be repeated tens of times on the same sample without any apparent fatigue. As verified by XPS and AFM analysis, the wettability enhancement is exclusively attributed to the TiO 2 nanorods exposed on the nanocomposite surface. The tuning of the surface wettability properties of the PDMS/TiO 2 materials, together with the easy processability of this elastomer, opens the way to the realization of microfluidic devices with controlled liquid flow. We demonstrate the potentiality of such systems by fabricating microfluidic channels with walls of PDMS and PDMS/TiO 2 nanorods composite materials. The combination of the used geometry with the hydrophobic character of both the pure and nanocomposite PDMS prohibits the penetration of water in their developed microchannels. After UV irradiation, water penetration is allowed inside the irradiated nanocomposite microfluidic channels, whereas it is still forbidden after the irradiation of the bare PDMS microchannels, revealing the essential role of the TiO 2 nanofillers.

An innovative, simple and reliable method to fabricate micro-lasers by self-assembly of rod-shaped nanocrys-tals is demonstrated. Dot/rod core/shell CdSe/CdS nanorods are used to form optical micro-resonators by exploiting their self-organization into well-defined coffee stain rings. The fabrication process merely consists of capillary jet deposition of a nanorod solution onto a glass substrate, and is scalable, eco-nomic, and highly reproducible. Upon optical pumping of the micro-resonators, laser emission in the red or in the blue-green spectral region is obtained, demonstrating lasing both from core and shell transitions, with low pumping thresholds. Modeling by full-wave numerical simulations according to generalized (i. e. scattering) formulation of laser theory demonstrates lasing from complex modes of the self-assembled cavity.

We investigate the application of the evanescent-wave illumination for particle velocimetry near nano-structured surfaces. Theoretical analysis shows that, when the nano-structured lower wall of a channel has to be (optically) considered as an intermediate layer, the nano-structures affect the evanescent-wave illumination in several ways: (1) the nano-structures grating period has to be set perpendicular to the optical plane of incidence, so that all diffraction orders experience total internal reflection (TIR) simultaneously; (2) the TIR has to occur at the smooth bottom of the lower wall, so that the nano-structures do not scatter the incident laser beam; (3) the nano-structures spatially modulate the illumination by occupying spaces that cannot be sampled by the tracer particles. We perform quantitative imaging measurements inside 5-µm-deep PDMS channels, where a part of the 165-µm-thick lower wall is equipped with nano-structures (2D grating, height = 200 nm, period = 2 µm). We use distilled water as the liquid, in which we dilute 40-nm-diameter fluorescent beads employed as tracer particles. The evanescent-wave, which illuminates the particles, is induced using TIR of a continuous-wave 532-nm laser beam delivered through an oil-immersion objective lens (NA=1.49). Our results show that, besides of its main aim for the 3D velocimetry, the modulated illumination is also potentially useful for: (1) estimating the actual illumination depth directly in the channel as well as (2) detecting and quantitatively measuring a thin layer of air trapped at the nano-structures, which may explain the previously reported enhanced slip length values at nano-structured surfaces. We also discuss how the fluorescence emission efficiencies of the tracer particles may depend on each particle's location, particularly relative to the nano-structured surfaces and to the neighbouring particles.

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument - opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.

This is the pre-peer reviewed version of the following article: 3D correlative single-cell imaging utilizing fluorescence and refractive index tomography, which has been published in final form at https://dx.doi.org/ 10.1002/jbio.201700145. Cells alter the path of light, a fact that leads to wellknown aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, quantifying the intracellular RI distribution and colocalizing it with fluorescence in 3D. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.

Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it i...