Microfluidic sorting of arbitrary cells with dynamic optical tweezers

Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimens by an automated optimisation of the tweezer path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 μm small mitochondria, yeast cells, rod-shaped bacteria and 30 μm large protoplasts. Reliable sorting of yeast cells in a microfluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.     

Single molecule studies of DNA binding proteins using optical tweezers

Optical tweezers have become a versatile tool in the biological sciences. Combined with various types of optical microscopy, they are being successfully used to discover the fundamental mechanism of biological processes. Recently, the study of proteins acting on DNA was aggressively undertaken at the single-molecule level. Here, we review the most recent studies which have revealed the dynamic behavior of individual protein molecules at work on DNA, providing detailed mechanistic insight that could not be revealed, at least not easily, using bulk-phase or ensemble approaches.     

On-chip pH measurement using functionalized gel-microbeads positioned by optical tweezers

This paper demonstrates local pH measurement in a microchip using a pH-sensing gel-microbead. To achieve this, the gel-microbead made of a hydrophilic photo-crosslinkable resin was functionalized with the pH indicator bromothymol blue (BTB). The primary constituent of this photo-crosslinkable resin is poly(ethylene glycol). Gel-microbeads impregnated with BTB were obtained by stirring the mixture solution, which was composed of the resin, BTB, and an electrolyte solution. The gel-microbead is polymerized by UV illumination. The polymerized gel-microbead can be manipulated by optical tweezers and made to adhere to a glass surface. The local pH was measured from the color of the gel-microbead impregnated with BTB by calibrated color information in the YCrCb color space. We succeeded in measuring the local pH value using the pH-sensing gel-microbead by manipulating and positioning it at the desired point in the microchip.     

Mechanical property analysis of stored red blood cell using optical tweezers

The deformation of human red blood cells subjected to direct stretching by optical tweezers was analyzed. The maximum force exerted by optical tweezers on the cell via a polystyrene microbead 5 μm in diameter was 315 pN. Digital image correlation (DIC) method was introduced to calculate the force and the deformation of the cell for the first time. Force–extension relation curves of the biconcave cell were quantitatively assessed when erythrocytes were stored in Alsever's Solution for 2 days, 5 days, 7 days and 14 days respectively. Experiment results demonstrated that the deformability of red blood cells was impaired with the stored time.     

On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel

A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.     

Use of optical tweezers for colloid science

A space-borne optical tweezer apparatus for use with colloidal crystallization experiments has been characterized. The trapping force has been measured as a function of index mismatch between colloidal microspheres and the surrounding fluid and as a function of particle size. This work also presents a method to determine the refractive index of a colloidal microsphere, which is then used to calculate the applied trapping force for the case of an arbitrary background fluid. This is useful for work with dense colloidal suspensions when the usual (e.g., Stokes flow) trap force measurement methods do not apply, as well as microrheological studies of complex soft matter.     

Optical sculpture: controlled deformation of emulsion droplets with ultralow interfacial tensions using optical tweezers

We report a technique for deforming micron-sized emulsion droplets that have ultralow interfacial tensions, by the manipulation of multiple optical trapping sites within the droplets.     

Fluorescent II-VI semiconductor quantum dots in living cells: nonlinear microspectroscopy in an optical tweezers system

In this work we used a setup consisting of an optical tweezers combined with a nonlinear microspectroscopy system to perform scanning microscopy and obtain emission spectra using two photon excited (TPE) luminescence of captured single living cells labeled with core-shell fluorescent semiconductor quantum dots (QDs). The QDs were obtained via colloidal synthesis in aqueous medium with an adequate physiological resulting pH. Sodium polyphosphate was used as the stabilizing agent. The results obtained show the potential presented by this system as well as by these II-VI fluorescent semiconductor quantum dots to perform spectroscopy in living trapped cells in any neighborhood and dynamically observe the cell chemical reactions in real time.     

D3-trishomocubanetrione synthesis and optical resolution

A large-scale synthesis for the title compound rac-1 based on 11-oxahexacyclo-[5.4.1.0^2,6.0^3,10.0^4,8.0^9,12] dodecan-5-one (12) as the key intermediate and an efficient procedure for the optical resolution of rac-1 via the diastereomeric ketals with (R,R)-2,3-butandiol are reported. The absolute configuration is established by correlating the optically pure (+)-trione ([α]_D +949°) with (-)-trishomocubane.     

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.     

Interactive manipulation of blood cells using a lens-integrated liquid crystal display based optoelectronic tweezers system

This paper reports a lens-integrated liquid crystal display (LCD)-based optoelectronic tweezers (OET) system for interactive manipulation of polystyrene microspheres and blood cells by optically induced dielectrophoretic force. When a dynamic image pattern is projected into a specific area of a photoconductive layer in an OET, virtual electrodes are generated by spatially resolved illumination of the photoconductive layer, resulting in dielectrophoresis of microparticles suspended in the liquid layer under nonuniform electric field. In this study, the simple-structured OET system has been easily constructed with an OET device, an LCD and a condenser lens integrated in a conventional microscope. By using a condenser lens, both stronger dielectrophoretic forces and higher virtual electrode resolution than previously reported lens-less LCD-based OET platform are obtained. The effects of blurred LCD image and liquid chamber height on the performances of optoelectronic particle manipulation are investigated by measuring the bead velocities according to their sizes. An interactive control program for OET-based microparticle manipulation is also developed by Flash language. The integrated system is successfully applied to the parallel and interactive manipulation of red and white blood cells. Due to its simple structures, cheap manufacturing costs, and high performances, this new LCD-based OET platform may be a widely usable integrated system for optoelectronic manipulation of microparticles including living cells.     

Analysis of liposomal membrane composition using Raman tweezers

We have developed a methodology for the analysis of liposomal membranes and their contents using near-IR Raman spectroscopy on liposomes held in an optical trap. We were able to detect a variety of membrane components including lipids, cholesterol, and small molecule solutes such as ethanol, DMSO and hexafluoroisopropanol. The methodology is able to distinguish between solutes that equilibrate across the liposomal membrane from those that partition selectively into the lipid bilayer.     

Identification of biotic and abiotic particles by using a combination of optical tweezers and in situ Raman spectroscopy.

A highly versatile setup, which introduces an optical gradient trap into a Raman spectrometer, is presented. The particular configuration, which consists of two lasers, makes trapping independent from the Raman excitation laser and allows a separate adjustment of the trapping and excitation wavelengths. Thus, the excitation wavelength can be chosen according to the needs of the application. We describe the successful application of an optical gradient trap on transparent as well as on reflective, metal-coated microparticles. Raman spectra were recorded from optically trapped polystyrene beads and from single biological cells (e.g., erythrocytes, yeast cells). Also, metal-coated microparticles were trapped and used as surface enhanced Raman spectroscopy (SERS) substrates for tests on yeast cells. Furthermore, the optical gradient trap was combined with a SERS fiber probe. Raman spectra were recorded from trapped red blood cells using the SERS fiber probe for excitation.     

Retrieval of the complex refractive index of aerosol droplets from optical tweezers measurements

The cavity enhanced Raman scattering spectrum recorded from an aerosol droplet provides a unique fingerprint of droplet radius and refractive index, assuming that the droplet is homogeneous in composition. Aerosol optical tweezers are used in this study to capture a single droplet and a Raman fingerprint is recorded using the trapping laser as the source for the Raman excitation. We report here the retrieval of the real part of the refractive index with an uncertainty of ± 0.0012 (better than ± 0.11%), simultaneously measuring the size of the micrometre sized liquid droplet with a precision of better than 1 nm (< ± 0.05% error). In addition, the equilibrium size of the droplet is shown to depend on the laser irradiance due to optical absorption, which elevates the droplet temperature above that of the ambient gas phase. Modulation of the illuminating laser power leads to a modulation in droplet size as the temperature elevation is altered. By measuring induced size changes of <1 nm, we show that the imaginary part of the refractive index can be retrieved even when less than 10 × 10(-9) with an accuracy of better than ± 0.5 × 10(-9). The combination of these measurements allows the complex refractive index of a droplet to be retrieved with high accuracy, with the possibility of making extremely sensitive optical absorption measurements on aerosol samples and the testing of frequently used mixing rules for treating aerosol optical properties. More generally, this method provides an extremely sensitive approach for measuring refractive indices, particularly under solute supersaturation conditions that cannot be accessed by simple bulk-phase measurements.     

From supramolecular porphyrin tweezers to dynamic A(n)B(m)C(l)D(k) multiporphyrin arrangements through orthogonal coordination

A dynamic, supramolecular, three-component A(n)B(m)C(l) bis(zinc porphyrin) tweezer has been prepared quantitatively using the heteroleptic bisphenanthroline (HETPHEN) concept. Upon addition of nitrogenous spacers of different length, namely, the extended bipyridine 3 a, 4,4'-bipyridine (3 b), and 1,4-diazabicyclo[2.2.2]octane (DABCO; 3 c), to set up an additional orthogonal binding motif (Zn(Por)-N(spacer)), three structurally different, still dynamic, four-component A(n)B(m)C(l)D(k) assemblies were cleanly formed, as indicated by UV/Vis and NMR titrations as well as by DOSY investigations. The structures were identified as a bridged monotweezer A(2)BC(2)D, a doubly bridged double tweezer A(4)B(2)C(4)D(2), and a triply bridged double tweezer A(4)B(2)C(4)D(3), the latter resembling a porphyrin stack. Notably, the same structures were equally formed directly from a mixture of the constituents A, B, C, and D put together in any sequence if the correct stoichiometry was applied.     

Photochemistry of molecular systems for optical 3D storage memory

The photochemistry, kinetics and spectra of several spiropyran molecules have been studied. The transient species of the photochromic reactions and precursors have been identified and the rate of formation and decay measured. We also present means for the utilization of spiropyrans in writing and accessing information in 3D volume optical memory.     

Creating Complex Polyacrylamide Hydrogel Structures Using 3D Printing with Applications to Mechanobiology

Due to its favorable physical and chemical properties, including chemical inertness, low fouling by biological molecules, high porosity and permeability, optical transparency, and adjustable elasticity, polyacrylamide has found a wide range of biomedical and non‐biomedical applications. To further increase its versatility, this communication describes a simple method, using readily available reagents and equipment, for 3D printing polyacrylamide hydrogels at a resolution of 100–150 μm to create complex structures. As a demonstration of the application, the method is used for creating a lab‐on‐a‐chip cell culture surface with micropatterned stiffness, which then leads to the discovery of stiffness‐guided collective cell segregation distinct from durotaxis. The present technology is expected to unleash new applications such as the construction of biocompatible elastic medical devices and artificial organs.     

Dielectric properties under corona charging of thermoplastics for holographic optical switching

Photothermoplastics are materials which have been proposed for optical switching devices: a grating is recorded by surface deformation of the thermoplastic layer, coated on an organic photoconductive layer used, like in xerography, for an electrostatic image recording material. But first of all an electrical field is applied through a corona charging sequence and obviously dielectric properties of the abietic ester thermoplastic material are of importance. This paper discusses the behaviour of such a layered thermoplastic material in a corona set-up, by measuring the corona current and the surface voltage following thickness and temperature of the sample and charging or discharging times.