Optical nano-printing provides a versatile platform to print various nanoparticles into arbitrary configurations. Optical printing, the use of light to direct the formation of a desired structure, has been of significant interest in the last two decades. For particles much smaller than the laser wavelength, optical forces can be well described in the dipole approximation. For a focused laser beam, two main optical force components are identified: the gradient force, which attracts particles toward the high-intensity focal spot, and the scattering force, which tends to push particles along the beam propagation direction. When the wavelength light is close to the particle localized surface plasmons resonance, a scattering force is dominant and can be used to efficiently push nanoparticles along the beam optical axis onto a substrate. In this context, optical forces can be applied to optically print nanoparticles into patterns aggregated on surfaces such as glass. Here, we report on use optical nanoprinting of plasmonic nanoparticles to create an active aggregate in a solution containing dyes or nanoplastics. The active aggregate, produced by optical forces, serves as a sensitive sensor which is used to detect dyes in concentrations below the limit of detection for Raman spectroscopy and/or to detection of plastic nanoparticles.
Optical control of mechanical motion of solid-state objects weakly interacting with the environment, referred to as optomechanics, continues to enable new, ground-breaking methods and applications in the area of ultra- weak force sensing and quantum technologies. The platform based on optically levitated nanoparticles in vacuum (referred to as levitated optomechanics) constitutes an entirely new type of light-matter interface, which provides a broad and an easy tunability of all the system parameters. However, the majority of the previously reported experimental achievements in this area have only dealt with a single levitated object. Here, we demonstrate for the first time scalability of the levitated optomechanics to systems containing up to tens of nanoparticles and provide a unique methodology for characterizing the system parameters and non-linear inter-particle interactions. This work represents the first and crucial step in accessing many-body dynamical effects in the classical and quantum regimes. In particular, it opens the door to the experimental studies of many-body stochastic thermodynamics and to the preparation of mesoscopic entangled states between relatively massive objects.
We report on the development of a set of Raman based techniques to monitor a large variety of biological and chemical analytes, such as various microorganisms, gels of hyaluronic acid and selected halogenated hydrocarbons using Raman spectroscopy, Raman tweezers and surface-enhanced Raman spectroscopy (SERS). We analyzed individual microbial cells with Raman tweezers to provide solutions for fast and label-free identification of specific bacterial or yeast species. We designed an optofluidic SERS platform for quantification of sub-millimolar concentrations of halogenated environmental pollutants such as 1,2,3-trichloropropane and chloroform. We also examined the gel structure of hyaluronic acid by Raman spectroscopy.
We developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The device is based on a silicon chip, on which structures were fabricated using electron lithography and wet etching to achieve a pattern of inverted pyramids on the surface, which was then covered by gold layer of defined thickness and roughness. The geometry of the surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system from silicone polymer and glass, which allowed transport and precise mixing of fluids entering the chip, while preventing contamination or abrasion of the highly sensitive substrate. We used this device as a platform for quantitative detection of halogenated hydrocarbons such as 1,2,3-trichloropropane (TCP) in water in submillimolar concentrations. TCP is used in industry and it is a persistent environmental pollutant. The presented sensor allows fast and simple quantification of such molecules and it could contribute to environmental monitoring disciplines as well as enzymologic experiments with genetically engineered dehalogenases, which are potentially useful for bioremediation. This research is supported by Czech Science Foundation (CSF) 16-07965S, infrastructure was supported by MEYS (LO1212, LM2015055) and EC (CZ.1.05/2.1.00/01.0017).
Multimode fiber imaging using transmission matrix (TM) is a promising way of deep inspection of living objects. These techniques, however, face a problem of obtaining the transmission matrix using phase-shifting interferometry with external reference beam, which requires additional instrumentation and increases space requirements of the experiment. We suggest a method employing input mode represented by a focal spot at the proximal end of the fiber as an internal reference wave. Due to speckle nature of the output, it is necessary to cover blind spots in the transmission matrix arising from the lack of interference signal by several measurements with different input modes used as a reference. The effect of optimized selection of internal references will be analyzed and compared with the external reference approach.
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Because of the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We used laser tweezers Raman spectroscopy (LTRS) in a microchamber chip to manipulate and analyze individual E. coli cells. We subjected the cells to antibiotic cefotaxime, and we observed the changes by the time-lapse microscopy and Raman spectroscopy. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments [18].
Optofluidics, a research discipline combining optics and microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g. for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.
In the present days light emitting diodes (LED) based light sources become very common in most areas of life and scientific applications. Due to the nature characteristic of the LED’s, angular and spectral distribution of the emitted light must be improved for specific purposes of light source. It could be done by standard bulk optics, reflective and refractive, or in modern way by integrating planar diffractive structures. Measuring system of spatial distribution of illumination was developed, which could be used in general but with advantage mainly for improved progressive diffraction optics components. The measurement system consists of three main parts. The first one is rigid optical bench with goniometric light source holder driven by stepper motor, which operates in three degrees of freedom. The source holder has ability to correct thickness of the light source with respect to the plane of the radiation. Maximal size of the light source is 500 by 500 mm and maximal length between the source and detector part is over 3 meters. The second detection part includes fiber spectrometer, photopic photodiode and elevation laser. Latter ensures correct repeatable setting of the light source. The third part - control and acquisition hardware is crucial for proper measurement of spectral and intensity data sets. The whole system is driven by custom software, which is able to generate most types of output reports, charts and file formats.
We investigate motion of particle pairs optically bound in tractor beam. The tractor beam can exert a negative force on a scatterer, in contrast to the pushing force associated with radiation pressure, which can pull the scatterer towards the light source. The particle movements can be enhanced by long-range interaction between illuminated objects, called optical binding. We study optical binding of two micro-particles in various geometrical configurations and investigate their motional behaviour in tractor beam. We demonstrate that motion of two optically bound objects strongly depends on their mutual distance and spatial orientation. Such configuration-dependent optical forces add an extra flexibility to our ability to control matter with light. Understanding these interactions opens the door to new applications involving the sorting or delivery of colloidal self-organized structures.
Emulsion droplets of liquid crystals (LC) suspended in water and labeled with a suitable fluorescent dye can serve as active optofluidic microcavities, since the contrast of refractive index between the LC droplets and the surrounding aqueous medium allows excitation of whispering gallery modes (WGMs) in the droplets. In addition, such emulsion droplets can be also stably trapped in three-dimensions using optical tweezers which stabilizes the droplets while investigating their spectral characteristics. We explore various combinations of fluorescently dyed LC droplets and host liquid - surfactant systems and show that the WGM emission spectrum of an optically trapped LC droplet-based cavity can be largely and (almost) reversibly tuned by controlled changes of the ambient temperature that induce phase transitions in the LC droplets. Our results indicate feasibility of this approach for creating miniature tunable sources of coherent light.
KEYWORDS: Raman spectroscopy, Microorganisms, Yeast, Biofuels, Spectroscopy, Microscopes, Statistical analysis, Chemical analysis, Iron, Process control
A biomass of yeast strains has been studied using Raman spectroscopy due to their potential applications in the field of biofuel generation, food industry and biotechnological applications. In order to utilize biomass for efficient industrial/biotechnological production, the optimal cultivation parameters have to be determined which in turn lead to high production of desired substances such as oil, carotenoids, and pigments in the selected cell line of yeast. Therefore, we focused on different cultivation conditions (the effects of temperature regime and medium composition) and their influence on microorganisms growth and metabolic changes.
Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate large variety of reactions, while ensuring appropriate catalytic activity and high selectivity. These properties make enzymes attractive biocatalysts for a wide range of industrial and biomedical applications. Over the last two decades, directed evolution of enzymes has transformed the field of protein engineering. We have devised microfluidic systems for directed evolution of haloalkane dehalogenases in emulsion droplets. In such a device, individual bacterial cells producing mutated variants of the same enzyme are encapsulated in microdroplets and supplied with a substrate. The conversion of a substrate by the enzyme produced by a single bacterium changes the pH in the droplet which is signalized by pH dependent fluorescence probe. The droplets with the highest enzymatic activity can be separated directly on the chip by dielectrophoresis and the resultant cell lineage can be used for enzyme production or for further rounds of directed evolution. This platform is applicable for fast screening of large libraries in directed evolution experiments requiring mutagenesis at multiple sites of a protein structure.
Two-photon photopolymerization is used to create structures with sub-micron details or tiny particles by solidifying monomer liquid in pre-defined locations with focused laser beam. The process is time consuming, especially when high volumes are processed, because the building blocks are typically 200 nm in diameter. Possible way how to speed up the technique is to use multiple focal points created by dynamically generated hologram. Corrections of the focal spot shape and even distribution the laser beam intensity is described together with examples of structures created with this method.
The main goal of our investigations is to focus on the basic physiological mechanisms of microorganisms (yeast and bacteria), exposed to different conditions, by time-resolved Raman spectroscopy. This study provides an insight into the mechanism of targeted stress factors or the influence of different cultivation times on species metabolism in vivo, in realtime and label free. We also focused on time-course study of physico-chemical properties of bacterial cells and cell cytoplasm with respect to the intracellular content of polyhydroxyalkanoates and to the production of yeast lipids or carotenoids.
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.
We introduce tunable optofluidic microlasers based on optically stretched or thermally modified, dye-doped emulsion droplets of liquid crystals (LC) confined in a dual-beam optical trap. Droplets were created in microfluidic chips or by shaking. Optically trapped microdroplets emulsified in water and stained with fluorescent dye act as an active ultrahigh-Q optical resonant cavity hosting whispering gallery modes (WGMs). Tuning of the laser emission wavelength was achieved by a controlled deformation of the droplet shape using light-induced forces generated by dual-beam optical trap and by thermal changing of the order in the LC.
We report on Raman spectroscopy measurements - separated by a given time intervals - for the selected yeast strains (biofilm positive and biofilm negative) on colonies grown directly on the Petri dishes or on the well-plate. Chemometric principal component analysis of these spectra sets generated clusters of data points, from which the reproducibility of the measurement could be analysed. Consequently, these resulted in clusters coinciding well with the biofilm positive and biofilm negative strains measurement of a particular sample dish, suggesting good reproducibility of our measurement procedure, even when the samples were prepared and measured days up to months apart. This suggests the potential of Raman spectroscopy in routine clinical diagnostic.
We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.
We have built a complex apparatus for optical trapping, stretching, heating and concurrent whispering gallery mode (WGM) lasing excitation of liquid crystal (LC) emulsion micro-droplets doped with various fluorescent dyes. We have explored the changes of WGM lasing wavelength when the LC droplets were optically stretched or electrically heated beyond the transition to the isotropic phase. We have found that the range of lasing wavelengths was in some cases considerably higher than when we optically stretched ordinary fluorescent oil droplets in our previous experiments.
Super-paramagnetic particles are used extensively in diagnostics and other research applications for the purification of cells, biomolecules and assays. Here, we demonstrate full 3D optical manipulation of such sub-micrometer sized particle using optical tweezers. We report three types of anomalous behavior of such a trapped particle. Firstly, the analysis of particle motion recorded by the quadrant photodiode reveals spikes in the total detected intensity that are formed by a set of very fast oscillations. Secondly, the dependence of trap stiffness on the laser power deviates from the expected linear increase for higher trapping powers. And further, such a particle cannot be optically trapped above certain trapping power threshold. We speculate that these effects are caused by particle internal structure and by its heating.
Raman tweezers represents a unique method for identification of different microorganisms on the basis of Raman scattering. Raman tweezers allows us to fix and sterile manipulate with the trapped object and in the same time check the growth, viability, response to the external environment etc. by Raman signal evaluating. The investigations presented here include distinction of bacteria in general (staphylococcal cells), identification of bacteria strains (biofilm-positive and biofilm-negative) by using principal component analysis (PCA) and monitoring the influence of antibiotics.
Here we report on combination of the data obtained from MICs (minimum inhibitory concentrations) with infor- mation of microoragnisms fingerprint provided by Raman spectroscopy. In our feasibility study we could follow mechanisms of the bacteriostatic versus bactericidal action on biofilm-positive Staphylococcus epidermidis simply by monitoring Raman bands corresponding to DNA translating the changes introduced by selected antibiotics. The Raman spectra of Staphylococcus epidermidis treated with a bacteriostatic agent show little effect on DNA which is in contrast with the action of a bactericidal agent where decreased in dedicated Raman spectra signal strength suggests DNA fragmentation. Moreover, we demonstrate that Raman tweezers are indeed able to distinguish strains of biofilm-forming (biofilm-positive) and biofilm-negative Staphylococcus epidermidis strains using principal component analysis (PCA).
We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment in order to identify and sort biological objects, such as living cells of various prokaryotic and eukaryotic organisms. Our main objective was to create a robust and universal platform for non-contact sorting of microobjects based on their Raman spectral properties. This approach allowed us to collect information about the chemical composition of the objects, such as the presence and composition of lipids, proteins, or nucleic acids without using artificial chemical probes such as fluorescent markers. The non-destructive and non-contact nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used differently treated cells of algae to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.
We introduce tunable optofluidic microlasers based on active optical resonant cavities formed by optically stretched, dye-doped emulsion droplets confined in a dual-beam optical trap. To achieve tunable dye lasing, optically pumped droplets of oil dispersed in water are stretched by light in the dual-beam trap. Subsequently, resonant path lengths of whispering gallery modes (WGMs) propagating in the droplet are modified, leading to shifts in the microlaser emission wavelengths. We also report lasing in airborne, Rhodamine B-doped glycerolwater droplets which were localized using optical tweezers. While being trapped near the focal point of an infrared laser, the droplets were pumped with a Q-switched green laser. Furthermore, biological lasing in droplets supported by a superhydrophobic surface is demonstrated using a solution of Venus variant of the yellow fluorescent protein or E. Coli bacterial cells expressing stably the Venus protein. Our results may lead to new ways of probing airborne particles, exploiting the high sensitivity of stimulated emission to small perturbations in the droplet laser cavity and the gain medium.
We introduce tunable optofluidic microlasers based on optically stretched, dye-doped emulsion droplets confined
in a dual-beam optical trap. Optically trapped microdroplets of oil emulsified in water and stained with
fluorescent dye act as active ultrahigh-Q optical resonant cavities hosting whispering gallery modes (WGMs)
which enable dye lasing with low threshold pump powers. In order to achieve tunable dye lasing, the droplets are
pumped with a pulsed green laser beam and simultaneously stretched by light in the dual-beam trap. For a given
stretching power, the magnitude of the droplet deformation is dictated by the interfacial tension between the
droplet and the host liquid which is adjustable by adding surfactants. Increase of power of the dual-beam trap
causes a directly proportional change of the droplet stretching deformation. Subsequently, resonant path lengths
of different WGMs propagating in the droplet are modified, leading to shifts in the corresponding microlaser
emission wavelenghts. Using this technique, we present all-optical, almost reversible spectral tuning of the lasing
WGMs and show that the direction of wavelength tuning depends on the position of the pump beam focus on the
droplet, consistent with the deformation of originally spherical droplet towards a prolate spheroid. In addition,
we study the effects of changes of the droplet and immersion medium temperature on the spectral position of
lasing WGMs and demonstrate that droplet heating leads to red-tuning of the droplet lasing wavelength.
The main goal of our investigation is to use Raman tweezers technique so that the responce of Raman scattering on
microorganisms suspended in liquid media (bacteria, algae and yeast cells in microfluidic chips) can be used to identify
different species. The investigations presented here include identification of different bacteria strains (biofilm-positive
and biofilm-negative) and yeast cells by using principal component analysis (PCA). The main driving force behind our
investigation was a common problem in the clinical microbiology laboratory - how to distinguish between contaminant
and invasive isolates. Invasive bacterial/yeast isolates can be assumed to form a biofilm, while isolates which do not
form a biofilm can be treated as contaminant. Thus, the latter do not represent an important virulence factor.
We have constructed a device for active optical manipulation and Raman spectral analysis in a microfluidic channel for efficient, nondestructive and contactless sorting of biological samples based on the Raman spectroscopic characteristics of living cells. In our previous work, we have linked such Raman spectral characteristics of microalgal lipid bodies with the unsaturation or carotene concentration via a calibration curve. As the sorting platform we have used a combination of fast galvano-optic laser steering system and specially designed microfluidic chips. We used X shaped channels with two input and output ports, and also several differently shaped variants. The steerable trapping laser beam was designed to move the cell to the specified locations and confine the cell for the time period needed for the analysis.
Advanced optical instruments are useful for analysis and manipulation of individual living cells and their internal structures. We have employed Raman microspectroscopic analysis for assessment of algal lipid body (LB) volume 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 detected proportionality between the Raman vibrations of β-carotene and the LB volume. This finding may allow fast acquisition of LB volume approximation valuable e.g. for Raman microspectroscopy assisted cell sorting. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on 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.
The ability to identify and characterize microorganisms (algae, bacteria, eukaryotic cells) from minute sample volumes
in a rapid and reliable way is the crucial first step in their classification and characterization. In the light of this
challenge related to microorganisms exploitation Raman spectroscopy can be used as a powerful tool for chemical
analysis. Raman spectroscopy can elucidate fundamental questions about the metabolic processes and intercellular
variability on a single cell level. Moreover, Raman spectroscopy can be combined with optical tweezers and with
microfluidic chips to measure nutrient dynamics and metabolism in vivo, in real-time, and label free. We demonstrate
the feasibility to employ Raman spectroscopy-based sensor to sort microorganisms (bacteria, algae) according to the
Raman spectra. It is now quite feasible to sort algal cells according to the degree of unsaturation (iodine value) in lipid
storage bodies.
Raman spectroscopy is a powerful tool for chemical analysis. This technique can elucidate fundamental questions about
the metabolic processes and intercellular variability on a single cell level. Therefore, Raman spectroscopy can
significantly contribute to the study and use of microalgae in systems biology and biofuel technology. Raman
spectroscopy can be combined with optical tweezers. We have employed microfluidic system to deliver the sampled
microalgae to the Raman-tweezers. This instrument is able to measure chemical composition of cells and to track
metabolic processes in vivo, in real-time and label-free making it possible to detect population variability in a wide array
of traits. Moreover, employing an active sorting switch, cells can be separated depending on input parameters obtained
from Raman spectra. We focus on algal lipids which are promising potential products for biofuel as well as for nutrition.
Important parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids. We
demonstrate the capacity of our Raman tweezers based sensor to sort cells according to the degree of unsaturation in
lipid storage bodies of individual living algal cells.
The use of an ultra low expansion cavity plays a crucial role in laser stabilization, and in atomic or ion clocks.
We propose an easy method of precise monitoring of optical path distance in Fabry-Perot interferometer. The
spacing of mirrors of the Fabry-Perot interferometer in ambient air represents the optical path distance referenced
to stable optical frequency of the femtosecond mode-locked laser. With the help of highly selective optical filter it
is possible to get only a few of separate spectral components of laser comb. Optical path distance is transfered to
optical frequency of the comb component and through the repetition frequency of the laser to the radio-frequency
domain. Repetition frequency of the laser can be monitored with the uncertainty referenced to the any local
oscillator or through the GPS to the atomic clock standard. By using this mehod we are able to measure and
lock the Fabry-Perot cavity to a selected single component of optical frequency comb an to measure the optical
path distance directly in rf domain.
Active contactless optical sorting of microobjects represents very useful technique in many areas of biology,
chemistry, and medicine. We suggest here a configuration that combines optical sorting, trapping, excitation,
and detection paths and provides efficient sorting of biological samples according to their various parameters
(fluorescence, Raman spectrum, CCD image, motion etc.). This approach is based on the shape of the laser
beam and we succeeded in sorting of several types of living microorganisms.
In this contribution we propose a scheme of Fabry-Perot interferometer measuring the absolute distance in
atmosferic conditions using a femtosecond laser comb. The spacing of mirrors of the Fabry-Perot interferometer
represents the length standard referenced to stable optical frequency of the femtosecond mode-locked laser. With
the help of highly selective optical filter it is possible to get only a few of separate spectral components. By tuning
and locking of the Fabry-Perot cavity to a selected single component it is possible to get a mechanical length
standard with the uncertainty of the repetition frequency of the femtosecond laser. If the interferometer measures
distance in atmospheric conditions, the absolute value of the laser wavelength fluctuates with a refractive index
of air. Compairing the measurement in evacuated chamber with measurement in ambient air leads to enhanced
precision in measurement of refrective index of air.
Need of precise definition of the calibrated length is of great importance in industrial application in these days.
The Fabry-Perot interferometer or etalon with very high stable laser produces length etalon sensitive in nanometer
scale with linear response to its change. Fabry-Perot interferometer (etalon) with length L represents a set of
equidistant frequencies that could be transmitted through the length etalon. Each frequency could be described
as multiple of free spectral range of Fabry-Perot etalon which depends inversely to the mirror spacing. Tuning
DFB diode covering the tuning frequency range of hundreds of GHz is used as laser source for detection of
transmitted light. Found DFB diode laser wavelength transmitted through the Fabry-Perot etalon is measured
by wavelengthmeter. Train of femtosecond laser pulses produces an optical frequency spectrum (optical comb)
of separate equidistant frequencies with an offset frequency. Stabilized optical comb generates a very precise
frequency rule. Frequency beat between DFB laser source and the closest femtosecond laser line is detected to
find the exact frequency. This procedure is done in whole DFB laser diode tuning range. Such method produce
about hundred of reproductive and well defined measured points in DFB laser diode tuning range. Measured
points are treated by computer algorithm. Moreover the Fabry-Perot mirror distance changes could be precisely
analyzed by this method.
We describe and characterize an experimental system for Raman microspectroscopy of micro- and nanoobjects
optically trapped in aqueous suspensions with the use of a single-beam gradient optical trap (Raman tweezers).
This system features two separate lasers providing light for the optical trapping and excitation of the Raman
scattering spectra from the trapped specimen, respectively. Using independent laser beams for trapping and
spectroscopy enables optimizing the parameters of both beams for their respective purposes. Moreover, it is
possible to modulate the position of the trapped object relative to the Raman beam focus for maximizing the
detected Raman signal and obtaining spatially resolved images of the trapped specimen. Using this experimental
system, we have obtained Raman scattering spectra of individual optically confined micron and sub-micron sized
polystyrene beads and baker's yeast cells. Sufficiently high signal-to-noise ratio of the spectra could be achieved
using a few tens of milliwatts of the Raman beam power and detector integration times on the order of seconds.
Optical interference of two or more waves with different wave vectors generates a periodic, harmonic spatial
profile of the optical intensity. This well known property combined with mechanical effect of light offers an
excellent route to organize and manipulate large ensembles of microobjects in a controllable manner. Since
the sub-micron size objects dispersed in liquid suffer from Brownian motion,such fields may assist to control its
influence at will. In our experiments the periodic field is obtained by interference of co-propagating non-diffracting
beams and counter-propagating non-diffracting and even evanescent fields. These types of trapping fields enable
spatial organization of submicrometer-sized objects into one-dimensional arrays containing even thousands of
objects, their controlled delivery over a distance of 1 mm, their sorting according to the size of refractive index.
Moreover, the particle tracking enables to study the Brownian dynamics, jumps between neighboring optical
traps and interactions between the objects.
We present an experimental way how to produce extremely narrow polymer fibers using photopolymerization. Such fibers
solidify in the high intensity regions of nondiffracting zero-order Bessel beam from the solution of UV cured resin. Bessel
Beams are particularly suitable for this purpose because of their narrow high intensity core and long axial region of uniform
intensity. The width of the created polymer fiber is strongly dependent on the properties of the Bessel beam, used laser
power and curing time. Fibers wide 2 μm were created and their length exceeded 1 cm. This length several times exceeded
the axial region of the Bessel beam existence due to self-writing waveguide mechanism.
The photopolymerization in focused laser light is a modern way how to create three-dimensional microstructures with even sub-micron details. We present how this method can be utilized even in very narrow non-diffracting beams. Combination of the self-healing property of these beams and the narrow core of the non-diffracting beam enables the generation of a very uniform fiber of diameter less than 2 micrometers and lengths of several millimeters. Manufacturing conditions and dimensions of the generated fibers are studied.
KEYWORDS: Particles, Optical tracking, Motion analysis, CCD cameras, Fusion energy, Nondestructive evaluation, Space reconnaissance, In vivo imaging, Visualization, Thin films
Tracing of foreign objects inside living cells is very exciting way how to study interior of living objects in nondestructive way. We imported fluorescent submicron particles into the living cells using liposomes as carriers to study the local mechanical heterogeneity of the cell cytoplasm. Thermal motion of these probes within the cell is tracked using fluorescent video-microscopy. The time-records of the probe positions reveal their trajectories and accessible space to
the probes inside the cytoplasm of living cells. Further analyses of the thermal motion of the probes can reveal the mechanism of sub-cellular transport and properties of the cytoplasm in vivo.
The polymerization by focused laser light is a modern way how to create easily three-dimensional microstructures with even sub-micron details. We present how this method can be combined with non-classical laser beams to get a unique tool for generation of long and narrow polymer fiber.
The influence of size of the trapped object on its position near
the dielectric interface is studied experimentally. The trapping
beam is reflected on a surface and creates weak standing wave
component in resulting field distribution. This component causes
unwanted jumps of the trapped particle, when the beam waist moves
axially in the surface vicinity. Particles of different sizes are
more and less influenced by the standing wave, respectively. The
position of the trapped particle is measured with quadrant
photodiode and photomultiplier tube at the same time.
We study the transfer of the cell nucleus and individual chromosomes from one living cell to the other one during their fusion. To achieve this, the nuclei of the two fused cells are stained with different fluorescent dyes which serve as identification markers. The fusion itself is done in an inverted optical microscope by combined system that uses optical tweezers to bring two living cells into contact and optical scalpel to punctuate their membranes at the contact point.
This process initiates a fusion of both cells into one hybrid cell containing two nuclei. If the fusion product is viable, these nuclei tend to mix together. The dynamics of the fusion process is then visualized by exciting the fluorescently labeled fusion product with a suitable light source. The time evolution of the mutual position of the fused cell nuclei and their final orientation is traced from a video record of the experiment. The spatial distribution of the nuclear material in the resulting hybrid nucleus is studied by analysis of positions of FISH (fluorescent hybridization in situ) signals of specific genetic loci in automated fluorescence microscope (high resolution cytometer). The obtained results are compared to the signals distribution of FISH in the original cells.
We present two methods for surface profiles measurement using optically trapped probe in tightly focused laser beam (optical tweezers). The first method is based on a continuous contact of the probe with the surface (contact mode) and the second one employes the alternating contact (tapping mode). The probe deviations are detected by two-photon fluorescence excited by the trapping beam and emitted by the trapped dyed probe.
It is studied theoretically and experimentally how a wave reflected from the surface influences the distance of the trapped sphere from the beam waist. The reflected wave interferes with the incident one and they create a standing wave component in the final axial intensity distribution. This component modulates the trapping potential and creates several equilibrium positions for the trapped sphere. When the beam waist approaches the surface, the potential profiles changes and causes jumps of the trapped objects to deeper potential well. We proved that these unwanted jumps of the trapped objects between the neighboring equilibrium positions can be eliminated by proper size of the sphere.
In this article we describe a combined system that uses optical tweezers to bring two living cells into contact and optical scalpel to punctuate their membranes at the contact point. This process initiates a fusion of both cells into one hybrid cell containing two nuclei. If the fusion product is viable, these nuclei tend to mix together. The spatial distribution of the nuclear material in the resulting hybrid nucleus is studied by analysis of positions of FISH (fluorescent hybridization in situ) signals of specific genetic loci in automated fluorescence microscope (high resolution cytometer). The obtained results are compared to the signals distribution of FISH in the original cells.
KEYWORDS: Luminescence, Optical tweezers, Particles, Geometrical optics, Laser beam diagnostics, Near field optics, Microscopes, Signal detection, 3D metrology, Calibration
We present a method which uses two-photon fluorescence excited in an optically confined fluorescent-labelled probe by the trapping beam for the study of topology of transparent surfaces. We demonstrate that for probe diameter of 200 nm, the vertical resolution of the method is better than 25nm.
The basic behavior of microparticles placed in the Gaussian standing wave is studied theoretically in this article. It is shown that the optical force depends periodically on the particle size and, as the consequence, the equilibrium object position is alternating between the standing wave antinodes and nodes. It is presented that the particle confinement is disabled for certain particle sizes. Simplified theoretical description giving analytical formulae for weak dielectric spherical objects of micrometer sizes is presented. Coincidence with the generalized Lorenz- Mie theory is studied here. Experimental confirmation of the theoretical results is briefly discussed.
In this article we describe a system which enables creation of several optical traps by splitting the laser beam in two parts and by using an acoustooptical deflector in one of these parts. This system is combined with a UV pulse laser so that a complex apparatus for optical trapping and cutting is obtained. Movable lenses ensure independent 3D positioning of a beam focus of the trapping and cutting beam without power losses at the back objective aperture. We present several applications of this system.
Plasma polymers were deposited on various surfaces using Ar/n-hexane/water working gas mixture. In most cases a test surface consisting from glass substrate coated by plasma polymer of Ar/n-hexane was used. It has been found that wettability of this test surface improves with the increased water concentration in a working gas mixture or with the increased power to the discharge. The increased concentration of polar groups was observed by FTIR absorption in the films stored in humid environment. Rate of the water absorption in dependence on power used for the film deposition is discussed.
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