Using multiphoton microscopy, we can image the tissue with sub-cellular resolution at depths down to 1.5 mm. Beyond this reach, the light must be delivered to the target region by optical relay elements inserted into the tissue – the endoscopes. Focusing light through a step-index multimode optical fibre (MMF) using wave-front control enables minimally-invasive endoscopy. With fibre micro-endoscopes, we can reach any depth with negligible tissue disruption and keep the resolution below 1 micrometre, sufficient for in vivo microscopy. We designed a novel custom-terminated multimode fibre probe. This work will focus on optical fibre probe manufacturing methods (etching, polishing and coating) and their impact on imaging quality.
A beam of light may possess both spin and orbital angular momentum. In non-paraxial conditions part of the spin converts into orbital angular momentum through the spin-orbit angular momentum conversion phenomenon. This effect has important consequences at the nanoscale, particularly in nano-manipulation and nano-photonics. In this work, we thoroughly analyze the rotation of microscopic beads subjected to a tightly focused Laguerre-Gaussian beam. Particularly, we observe the rotation of particles along circular trajectories that will depend strongly on the combination of topological charges and the state of polarization. Based on Richard and Wolf theory for non-paraxial beam focusing, we found a very good agreement between the experimental results and the theoretical model based on calculation of the optical forces using the generalized Lorenz-Mie theory.
Optically levitated nanoparticles in vacuum are a promising tool aiming for the extremely sensitive force measurements in reaching up to the order of zN•Hz-1/2 and for the potential investigation in the field of quantum physics. In contrast to other mechanical oscillators, the optically trapped nanoparticle in vacuum has no clamping losses, its motion is influenced only by a laser beam and its potential profle and therefore the mechanical quality factor of such oscillator is very high. In water immersion the optical trap is almost exclusively considered as harmonic but in vacuum the optical potential anharmonicity starts to play an important role. This can be observed in power spectrum density profile where the oscillation peak is asymmetric. Here we demonstrate on the simulated trajectories of the levitated particle how the standard power spectral density method provides strongly biased values of parameters describing the optical trap and its surrounding properties.
Particles held in optical tweezers are commonly thought to be at thermodynamic equilibrium with their environment. Under this assumption the elastic energy of the trap is equal to the thermal energy. As a result the variance of the particle position is completely independent of viscosity and inversely proportional to the optical power in the trap. Here we show that these conditions only hold for very high symmetry cases e.g. perfectly spherical particles in unaberrated, linearly polarized Gaussian traps. Here we show that any reduction in symmetry leads to asymmetrically coupled degrees of freedom. The associated force field is linearly non-conservative and the tweezer is no longer at equilibrium. In overdamped systems the effect is a underlying systematic bias to the Brownian motion. In underdamped systems, this systematic component can accumulate momentum, eventually destabilizing the trap. We illustrate this latter effect with reference to two systems, (i) an isotropic sphere in a circularly polarized trap, and (ii) a birefringent sphere in a linearly polarized trap. In both cases the instability can be approached either by decreasing air pressure or by increasing optical power. Close to instability, the trapped particle executes increasingly coherent motion that is highly sensitive to external perturbations. Potential applications to weak force sensing are discussed.
When using multimode fibers as high-resolution endoscopes, advanced adaptive optics is needed to overcome the modal dispersion which scrambles the image. Additionally, for non-linear imaging methods, all the wavelengths of a femtosecond laser pulse must be simultaneously focused at the sample plane, with appropriate dispersion compensation, that might vary across the sample area. We investigate the bandwidth of the focused spot for a graded index fiber used as a point scanning imaging device. We demonstrate that with proper compensation for the dispersion of the spatial light modulator this can be <45 nm. We also measure the spectral phase at the sample plane, and demonstrate that this does not vary substantially with spot position.
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.
Optically levitated nanoparticles in vacuum have gained much attention for their ultrasensitivity to forces of zN·Hz-1/2 orders and for the potential investigation in the field of quantum physics. In contrast to other nano-and micromechanical oscillators, the optically trapped nanoparticle in vacuum has no clamping losses, its motion is influenced only by a laser beam and its potential profile and therefore the mechanical quality factor of such oscillator is very high. In water solution, an optical trap can be considered as harmonic but in vacuum the optical potential anharmonicity starts to play an important role. This can be observed in power spectrum density profile where the oscillation peak is asymmetric. Here we show that commonly used method employing power spectrum density fitting for revealing the system parameters is not sufficient and other approach has to be developed.
Description of stochastic motion of a particle in an unstable potential is a challenging topic since even small number of diverging trajectories leads to undefined statistic moments of particle position. This breaks down the standard statistical analysis of unstable mechanical processes and their applications. Therefore, we employ a different approach taking advantage of the local characteristics of the most-likely particle motion instead of the average motion. We experimentally verify theoretical predictions for a Brownian particle moving near an inflection in a cubic optical potential. Notably, the most-likely position of the particle atypically shifts against the force despite the trajectories diverge in opposite direction. In this work we study the influence of the analytical formula used for quantification of the most likely particle position parameters in the case where only limited number of trajectories is available.
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 present a few of our recent theoretical and experimental results related to the behavior of micron-scale particles placed into nonlinear optical potentials. The two-dimensional optical ratchet can rectify motion of Brownian particles in any direction in the plane and unstable cubic optical potential results in noise-induced particle motion. Action of optical spin-force was demonstrated in a novel geometry where it is responsible for particle orbiting.
The noise-to-signal transitions are considered as interesting processes in physics as they might transform environmental noise to useful mechanical effects. Previously, we theoretically analyzed stochastic noise-to-signal transition, first passage times, and other stochastic quantities of overdamped Brownian motion of a nanoparticle in the cubic potential. Here we present a feasibility study showing that the cubic potential can be successfully obtained in a pair of overlapping optical tweezers even in the case of a dielectric microparticle having radius comparable to the trapping wavelength.
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.
Larger golden nanoparticles grow into several preferred forms. Some of those may be easily approximated by ellipsoids. In this paper we examine the rotational dynamics of spheroidal particles in an optical trap comprising counter-propagating Gaussian beams of opposing helicity. Isolated spheroids undergo continuous rotation with frequencies determined by their size and aspect ratio. We study the rotational frequencies and stability of these golden nano-particles theoretically by the means of T-Matrix.
A spatial light modulator (SLM) is a versatile device capable of real-time generation of diffractive phase gratings. Employing the SLM in an optical setup opens the possibility of dynamic modification of properties of the incident laser beam, such as its splitting into an arbitrary number of diffracted beams, changing its convergence or its modification into non-traditional laser beam profiles. Advanced feedback procedures enable resolving complex phase masks correcting aberrations inherent to the whole optical system, such as imprecisions of manufacturing process, inhomogeneity of refractive index of materials used or misalignment of optical elements. In this work, generation of Bessel beams (BB) using the SLM is presented. The BB quality is very sensitive to the optical aberrations of the system, especially when higher topological charge is applied to create so-called optical vortices. Therefore, the method compensating those aberrations is applied and the corrected beam is inspected by a CCD camera and optical micro-manipulations of micro-particles. Our experimental results demonstrate successful trapping, rotation and translation of micrometer-sized particles purely by optical forces.
We study theoretically the angular momentum transfer between strongly focused laser vortex beam and a dielectric oblate spheroidal particle (OSP). We find sets of geometrical parameters of the particle and the beam for which the particle is stably trapped on the beam axis in a uniform rotating state, thereby serving as a possible test probe of the global beam angular momentum as well as its spin and orbital parts.
Micro-particles with higher refractive index than the surrounding medium irradiated by a laser beam are pushed by optical forces towards places having the highest local optical intensity. These intensity maxima are usually created by focusing a laser beam using a microscope objective with high numerical aperture. A convenient alternative offers usage of light patterns created by an interference of collimated beams. This way tens or hundreds of optical traps are created in a spatially well-organized structure (also called as optical lattice) which is well-suited for studies of quasi-crystalline structures, targeted delivery of living cells or particle sorting and fractionation. Hereby, we investigate theoretically and experimentally properties of optical traps organized in hexagonal, rectangular and calleidoscopic structures created by interference of 3 up to 8 collimated laser beams.
Holographic Raman tweezers (HRT) manipulates with microobjects by controlling the positions of multiple optical traps via the mouse or joystick. Several attempts have appeared recently to exploit touch tablets, 2D cameras or Kinect game console instead. We proposed a multimodal “Natural User Interface” (NUI) approach integrating hands tracking, gestures recognition, eye tracking and speech recognition. For this purpose we exploited “Leap Motion” and “MyGaze” low-cost sensors and a simple speech recognition program “Tazti”. We developed own NUI software which processes signals from the sensors and sends the control commands to HRT which subsequently controls the positions of trapping beams, micropositioning stage and the acquisition system of Raman spectra. System allows various modes of operation proper for specific tasks. Virtual tools (called “pin” and “tweezers”) serving for the manipulation with particles are displayed on the transparent “overlay” window above the live camera image. Eye tracker identifies the position of the observed particle and uses it for the autofocus. Laser trap manipulation navigated by the dominant hand can be combined with the gestures recognition of the secondary hand. Speech commands recognition is useful if both hands are busy. Proposed methods make manual control of HRT more efficient and they are also a good platform for its future semi-automated and fully automated work.
We investigated the behavior of an oblate spheroidal polystyrene microparticle trapped in a focused vortex beam when the beam vorticity and polarization were modified. We demonstrated that such particles can be trapped in three dimensions, spin in a circularly polarized beam and an optical vortex beam around the axis parallel to the beam propagation. We compared the immediate frequencies and showed that contribution from the circularly polarized beam is one order of magnitude weaker comparing to the beam angular orbital momentum. Using a phase-only spatial light modulator we generated several vortex beam traps with well-defined parameters. Measuring the rotations of trapped spheroids we observed hydrodynamic phase and frequency locking for certain sets of beam parameters.
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).
Single beam optical tweezers hold particles behind the focal plane due to the highly focused laser beam. However, even moderately focused beams may stably con ne particles of high refractive index contrast and sizes up to half wavelength. This is caused by the fact that the light structure near focus is complex even for such moderately focused beams. The intensity pro le consists of several axial oscillations as well as off-axis lobes or rings which enhance trapping. Moreover, side lobes provide another multiple trapping sites for small sized particles. The properties of these traps will be analyzed theoretically by means of Generalized Lorenz-Mie Scattering theory.
Optical force acting upon a dielectric microparticle illuminated by a non-di racting vortex beam is expressed using the Generalized Lorenz-Mie theory (GLMT). Numerical results are presented for di erent widths and topological charges of the vortex beam. We show that such particle may be stably trapped either in the dark center of the vortex beam, in one of two stable positions placed o the optical axis, and as the third option it may circulate along almost circular trajectory having its radius smaller or equal to the radius of the smallest high intensity vortex ring.
While the behavior of spherical particles confined in light beams is well-studied, the dynamics of confined nonspherical particles may be qualitatively different, but remain largely unexplored. We studied the rotation of microscopic dielectric discs induced by the incident angular momentum of an elliptically polarized Laguerre-
Gaussian beam. These flat particles are confined in three dimensions by the beam and are oriented naturally with its long axis along the direction of the propagation of the beam. Due to the rotationally asymmetric shape of the particles, we were able to induce a constant rotation of the particles and control it by changing the vorticity
and ellipticity of the beam. We also showed a strong dependence on the induced rotation respect to size of the particles. These results provide a new approach to generate or study flows in the microscopic realm as an alternative to the former techniques based on birefringent, absorbent or chiral particles.
We demonstrate optical manipulation and sorting of micrometer-sized dielectric particles using one-dimensional
periodic interference pattern created by interference of two beams in a sample space. These beams are generated
by a combined phase grating applied on the spatial light modulator which allows to set dynamically the position
and spatial period of the interference pattern. If a microparticle of fixed size is placed into such pattern, the
optical forces acting upon it vary according to the spatial period of this optical lattice. We show how to use this
property for sorting of mixtures of particles by moving either the interference pattern or the sample chamber.
The mechanism is examined both theoretically and experimentally.
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.
We present a thorough experimental and theoretical study of micrometer-sized particle dynamics in optical
lattices. The spatial parameters of the lattices are set by configuration of the interfering beams with computercontrolled
spatial light modulator. The behaviour of particle confined in the interference pattern is monitored
by fast CCD camera. Analyses of the particle trajectory and Brownian motion reveal force interaction between
the particle and the lattice. We use polymer microspheres as the experimental objects. The experimental results
are compared with the theoretical predictions based on the generalized Mie theory.
We demonstrate optical manipulation with micrometer-sized dielectric particles. Instead of well-known optical tweezers
we use complex interference pattern (so-called optical landscape) to trap and confine polymer microspheres in maxima and
minima of optical intensity. The optical pattern is created by interference of two or three beams, respectively, which results
in interference fringes or hexagonal interference structure. Multiple beam setup uses spatial light modulator to divide
incoming laser beam into several directions by diffraction on the phase grating. Small particles are trapped in maxima of
intensity, while larger particles are attracted by neighbouring maxima and are confined in minima of intensity with their
center.
Rapid development of micromanipulation and experimental techniques increases the need for easy-to-handle tools, which
are suitable for routine operations. Our new concept represents a very suitable platform for incorporating such new experimental
techniques into standard laboratory microscopy equipment. Implementing of microscopy methods such as optical
trapping, cutting, fluorescence excitation, photopolymerization and most spectroscopic techniques is very expensive and
time consuming. In most cases, microscope parts and body must be adapted to insert additional lasers, detectors etc. We
overcome these difficulties by inserting source of radiation directly between microscope body and microscope objective.
Our concept allows using arbitrary source of radiation (laser diodes, solid-state lasers, gas lasers). Laser diode source can
be incorporated inside the adapter. Optical fiber is used to deliver other sources of radiation. Light microscopy adapter
(LMA) can be easily mounted on most types of microscopes including special types as confocal microscope.
We demonstrate ability of LMA for stable optical trapping of particle in the range of hundreds of nanometers to tens
of micrometers, bleaching (FRAP, FLIP) and microdissection experiments. Mechanical and optical construction of LMA
addresses spatial constrains of commercially available microscopes, mainly the fact that optical parts of microscopes are
optimized for visible spectrum of light. Our solution preserves all microscope properties unaffected and it is possible
modern microscopy techniques to many types of microscopes.
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.
In this article we present laser diode based tool for optical manipulation with microobjects. This tool is very
suitable for micromanipulations with large spectrum of speciments in the diameter range 0.5 - 30 μm. Adapter
is directly mounted to the microscope without any aditional improvements and fits to many comercially available
microscopes. Key feature of this adapter is compactness, usability and simple handling. With this adapter
user takes advantage of wide spectrum of comercially available laser diodes with different wavelengths. For this
reason the tool can be used in many areas such as biology, medicine and measurements.
We demonstrate how the simple one-dimensional optical lattices can be
used to sort micron-sized particles. Experimentally we focused on the simplest case where the periodical structure is created by an interference of two co-propagating beams and third non-interfering beam is used to compensate the radiation pressure caused by the previous two beams. We proved, that this geometry can be used for fast and efficient static sorting of microscopic objects without fluid flow.
An optical conveyor belt created by a standing wave can be used to deliver Brownian particles in a controlled way. The dependence of the particle speed on the speed of traveling standing wave is not simple but two basic modes can be distinguished. For very slow standing wave motion the particle is tightly coupled to the potential, i.e. it "surfs along with the potential wave" and therefore it is called the "Brownian surfer". For bigger standing wave velocities the particle behaves like a swimmer afloat on the surface of the ocean and thus it is called the "Brownian swimmer". The mutual speed of the particle and the standing wave is studied experimentally and theoretically.
We demonstrate the use of interference between non-diffracting Bessel beams (BB) to generate a system of optical traps. They offer sub-micron particle confinement, delivery and organization over a distance of hundreds of μm. We analyze system of two identical counter-propagating BBs and the case of two co-propagating BBs with different propagation constants separately. In both of these cases, the interference results in periodic on-axis intensity oscillations involving particle confinement. Altering the phase of one of the interfering beams, the whole structure of optical traps can be shifted axially. Implementing this conveyor belt enables the particle delivery over the whole distance where the optical traps are strong enough for particle confinement. Experimentally we succeeded with generation of both of these systems. In case of two counter-propagating BBs we observed a strong sub-micron particle confinement, while in case of co-propagating BBs the confinement was observed only with help of fluid flow against the radiation pressure of both beams.
Scanning probe microscopy with multiple optically held probes is presented. Acousto-optical deflectors are employed to rapidly switch the optical trap between two positions so that the trapped probes are not allowed to leave the trap region. The probes are fluorescently labelled and their vertical position is acquired from the level of two-photon fluorescence. This particle position detection technique is very sensitive and allows obtaining surface details with resolution better than 10 nm. Using two probes simultaneously accelerates the measurement process and allows scanning of larger
regions.
We present an optical conveyor belt that provides trapping and subsequent precise delivery of several sub-micron particles over a distance of tens of micrometers. This tool is based on a standing wave (SW) created from two counter-propagating evanescent beams where the phase of one of the beams can be changed. Therefore, the whole structure of SW nodes and antinodes moves and deliver confined microobjects to specific regions. Based on the theoretical calculations we confirm experimentally that certain sizes of polymer particles can be more easily delivered but other sizes do not feel the SW motion at all.
When a probe confined in a single focused laser beam approaches the surface, it is getting more influenced by the retroreflected beam. This beam interferes with the incident one and a weak standing wave (SW) is created, which slightly modulates the incident beam. We studied experimentally how this phenomena influences the optical trap properties if SW is created using surfaces of two different reflectivities. We used polystyrene probes of diameters 690 nm and 820 nm, tracked their positions with quadrant photodiode (QPD) and analysed their thermal motion to get the axial trap stiffness along optical axis.
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 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.
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.
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.
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.
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 harmonic nature of the potential well for the small displacement of the probe from its equilibrium position allows us to classify the trap characteristics with three independent spring constants. These can be obtained from the spectral analysis of the thermal noise of the particle position. Probe position in all three dimensions is monitored with a single quadrant photodiode placed in the back focal plane of the microscope condenser. Experimental results of the trap stiffness measurements are presented.
KEYWORDS: Ferroelectric materials, Objectives, Calibration, Near field optics, Nanoprobes, Photomultipliers, Actuators, Near field, Microscopes, Sensors
An experimental method for the measurement of the profile of the laser beam focused by a high NA lens is presented. A homemade PZT driven stage is used to scan a near-field optical microscope probe through the beam profile. The probe position is detected via strain gauges which were calibrated by laser interferometer and the intensity collected by the probe is measured by photomultiplier. The stage positioning accuracy +/- 50 nm enables the measurement of the intensity distributions within submicron-sized beam spots. As an example, intensity profiles of a TEM00 laser beam focused by a water immersion objective are presented.
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