Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing

Three-dimensional force measurements in optical tweezers formed with high-NA micromirrors

The three-dimensional trap stiffness of optical tweezers formed with high-NA micromirrors is investigated by back-focal-plane interferometry and power spectrum analysis. Normalized stiffness values of kappa(xy)/P-trap =1.2(mu N/m)/mW and kappa(z)/P-trap = 0.52(mu N/m)/mW in the transverse and axial directions, respectively, have been measured for polystyrene spheres with a radius of 1.03 mu m. Compared with high-NA microscope objectives, micromirrors achieve much better trapping performances, particularly in the axial direction. (C) 2009 Optical Society of Americ

SpectroCube: a European 6U nanosatellite spectroscopy platform for astrobiology and astrochemistry

SpectroCube is a CubeSat-based miniaturized in-situ space exposure platform for astrochemistry and astrobiology research. Within a 6 unit (6U, with 1U corresponding to 10 cm x 10 cm x 10 cm) nanosatellite structure, an infrared spectrometer is interfaced with a sample handling system to measure photochemical changes of organic molecules, representing important biomarkers for the detection of life in our solar system and beyond. Monitoring degradation profiles and photochemical reaction kinetics of such biomarkers allows to identify suitable search targets for current and future planetary exploration and life-detection missions. SpectroCube is designed to be launched into a highly elliptical orbit around Earth and therefore allows to expose samples to higher solar UV and energetic particle radiation levels than previous exposure platforms in low Earth orbit, as for example on the International Space Station. In-situ data will be telemetered back to Earth and compared with solar and planetary simulation experiments in ground-based laboratory. We here present the design of SpectroCube, the scientific payload and its subsystems. We demonstrate that with the miniaturisation potential of infrared spectroscopy it is possible to fit the entire optical setup plus a sample handling system for up to 60 individually contained and hermetically sealed samples within less than half of the volume of a 6U CubeSat structure. Therefore, the remaining volume can be entirely used for additional subsystems such as attitude control, propulsion, fuel, onboard computer and telemetry. The design of the scientific payload is based on a commercial off-the-shelf miniaturised Fourier-transform spectrometer consisting of an infrared light source, an interferometer and infrared detector units. The mechanical robustness and suitability of such a system for space applications was assessed. Shock and vibration testing of the mechanically most sensitive unit, the interferometer, was performed and revealed that with adequate damping the spectroscopic performance can be maintained. Additional measurements of test samples conducted with the selected commercial off-the-shelf spectrometer candidate showed that the spectroscopic range, resolution and sensitivity is capable to monitor in situ the photochemical kinetics of important classes of organic molecules and biomarkers for astrobiology and astrochemistry research

Micro-optics for multiple laser trapping in microfluidics

Optical trapping allows manipulating very small objects – varying from Angstöms to micron size particles – without physical contact by taking advantage of laser light. This technique, relying on light momentum transfer, allows manipulating biological matter (such as cells, organelles, vesicles or chemically functionalized artificial particles, etc.) and offers promising potentialities for research in biotechnology and biochemistry. Individually confining a large number of microscopic objects opens new ways for the downscaling of analysis tools for drug screening, particles sorting and the assessment of statistical data. In particular, the combination of optical trapping with microfluidics greatly enlarges the possibilities offered by both techniques. This PhD work takes place in a research aiming at developing novel bio-analytical instrumentation relying on large arrays of optical traps compatible with microfluidic systems. The dissertation focuses on the use of micro-optics for generating extended matrices of three-dimensional optical traps capable of capturing a large number of particles within microfluidic environments. The stability of optical traps is first studied with special emphasis on the conditions present in microfluidic flows, both from a theoretical and an experimental point of view, and trapping forces achievable on biological cells are investigated. A multiple optical trapping system relying on microlens arrays, adapted to work on commercial microscopes, is shown to be capable of generating arrays of more than 500 three-dimensional traps. Simultaneously trapping in multiple planes is also evidenced using matrices of microlenses, thanks to a self-imaging effect of the array of traps. Furthermore, possibilities offered by multiple optical trapping within microfluidic environments are explored. Polystyrene spheres as well as biological particles, such as native vesicles, can be trapped in arrays, manipulated inside microfluidic devices and analyzed in parallel through fluorescence microscopy while extremely small quantities of chemical reagents are flown past the array. Finally, for the sake of system miniaturization and further extension of the number of traps, multiple three-dimensional optical trapping based on arrays of miniaturized high numerical aperture parabolic mirrors is proposed, allowing the optics necessary for optical trapping, fluorescence excitation and collection to be integrated at the level of a microfluidic chip. Such miniaturized mirror matrices did not even exist before this thesis; their fabrication and characterization are detailed in this dissertation

Miniaturized optical tweezer array with an array of reflective elements for reflecting the light back to a focal area

Apparatus for forming a single or a plurality of threedimensional optical traps, the apparatus comprising: a. A collimated light source that is directed onto an array of focalizing refractive or diffractive elements providing a single or a plurality of focal areas, and b. An array of reflective elements, placed opposite to the said focalizing elements described in a), which reflect back the light into the said focal area. The invention also relates to a method for using this apparatus

Validation of a one-dimensional model of the systemic arterial tree

A distributed model of the human arterial tree including all main systemic arteries coupled to a heart model is developed. The one-dimensional (1-D) form of the momentum and continuity equations is solved numerically to obtain pressures and flows throughout the systemic arterial tree. Intimal shear is modeled using the Witzig-Womersley theory. A nonlinear viscoelastic constitutive law for the arterial wall is considered. The left ventricle is modeled using the varying elastance model. Distal vessels are terminated with three-element windkessels. Coronaries are modeled assuming a systolic flow impediment proportional to ventricular varying elastance. Arterial dimensions were taken from previous 1-D models and were extended to include a detailed description of cerebral vasculature. Elastic properties were taken from the literature. To validate model predictions, noninvasive measurements of pressure and flow were performed in young volunteers. Flow in large arteries was measured with MRI, cerebral flow with ultrasound Doppler, and pressure with tonometry. The resulting 1-D model is the most complete, because it encompasses all major segments of the arterial tree, accounts for ventricular-vascular interaction, and includes an improved description of shear stress and wall viscoelasticity. Model predictions at different arterial locations compared well with measured flow and pressure waves at the same anatomical points, reflecting the agreement in the general characteristics of the "generic 1-D model" and the "average subject" of our volunteer population. The study constitutes a first validation of the complete 1-D model using human pressure and flow data and supports the applicability of the 1-D model in the human circulation