Mojmír Šerý

Optical sorting and detection of submicrometer objects in a motional standing wave

An extended interference pattern close to the surface may result in either a transmissive or an evanescent surface field for large-area manipulation of trapped particles. The affinity of differing particle sizes to a moving standing-wave light pattern allows us to hold and deliver them in a bidirectional manner and demonstrate experimentally particle sorting in the submicrometer region. This is performed without the need of fluid flow (static sorting). Theoretical predictions support the experimental observations that certain sizes of colloidal particles thermally hop more easily between neighboring traps. A generic method is also presented for particle position detection in an extended periodic light pattern and applied to characterization of optical traps and particle behavior.

Theoretical comparison of optical traps created by standing wave and single beam

Abstract We used generalised Lorenz–Mie scattering theory (GLMT) to compare submicron-sized particle optical trapping in a single focused beam and a standing wave. We focus especially on the study of maximal axial trapping force, minimal laser power necessary for confinement, axial trap position, and axial trap stiffness in dependency on trapped sphere radius, refractive index, and Gaussian beam waist size. In the single beam trap (SBT), the range of refractive indices which enable stable trapping depends strongly on the beam waist size (it grows with decreasing waist). On the contrary to the SBT, there are certain sphere sizes (non-trapping radii) that disable sphere confinement in standing wave trap (SWT) for arbitrary value of refractive index. For other sphere radii we show that the SWT enables confinement of high refractive index particle in wider laser beams and provides axial trap stiffness and maximal axial trapping force at least by two orders and one order bigger than in SBT, respectively.

Static optical sorting in a laser interference field

We present a unique technique for optical sorting of heterogeneous suspensions of microparticles, which does not require the flow of the immersion medium. The method employs the size-dependent response of suspended dielectric particles to the optical field of three intersecting beams that form a fringelike interference pattern. We experimentally demonstrate sorting of a polydisperse suspension of polystyrene beads of diameters 1, 2, and 5.2μm and living yeast cells.

Raman microspectroscopy of algal lipid bodies: β-carotene quantification

Advanced optical instruments can serve for analysis and manipulation of individual living cells and their internal structures. We have used Raman microspectroscopic analysis for assessment of β-carotene concentration in algal lipid bodies (LBs) in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have devised a simple method to measure the concentration of β-carotene in a mixture of algal storage lipids from the ratio of their Raman vibrations. This finding may allow fast acquisition of β-carotene concentration valuable, e.g., for Raman microspectroscopy assisted cell sorting for selection of the overproducing strains. Furthermore, we demonstrate that β-carotene concentration can be proportional to LB volume and light intensity during the cultivation. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on the spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.

Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy.

Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations—MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms’ “fingerprint” (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.

Optical trapping of microalgae at 735-1064 nm: photodamage assessment.

Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.

Local probe microscopy with interferometric monitoring of the stage nanopositioning

We present a system of positioning and interferometric monitoring of a sample position for measurements and calibration in the nanoscale in metrology. The positioning is based on a three-axis stage which allows replacing scanning by the probe of an atomic force microscope with a system with full interferometric displacement measurement. A stage with 200 µm × 200 µm of horizontal travel extends also the microscope range. The stage allows positioning with sub-nanometer resolution in all three axes under a closed loop control with position detection via capacitive sensors. Interferometric system monitoring all six degrees of freedom of the stage ensures full metrological traceability of the positioning to the fundamental etalon of length and improves resolution and overall precision of the displacement monitoring.

Multiaxis interferometric displacement measurement for local probe microscopy

We present an overview of design approaches for nanometrology measuring setups with a focus on interferometry techniques and associated problems. The design and development of a positioning system with interferometric multiaxis monitoring and control is presented. The system is intended to operate as a national nanometrology standard combining local probe microscopy techniques and sample position control with traceability to the primary standard of length.

Behaviour of an optically trapped probe approaching a dielectric interface

Abstract The way in which reflection of the trapping beam from a dielectric interface influences the distance of the trapped sphere from the beam waist is studied theoretically and experimentally. The reflected wave interferes with the incident wave and they create a standing-wave component in the total axial intensity distribution. This component then modulates the trapping potential and creates several possible equilibrium positions for the trapped sphere. When the beam waist approaches the surface, the potential profile changes, which consequently causes jumps of the trapped probe from its current location to a deeper potential well. We suggested theoretically and proved experimentally that the magnitude of these unwanted jumps between the neighbouring equilibrium positions can be decreased by a suitable size of the sphere.

Identification of individual biofilm-forming bacterial cells using Raman tweezers.

Abstract. A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.