EP1935498A1 - Dispositif et procédé destinés à la manipulation et à l'alignement sans contact de petites parties d'échantillons dans un volume de mesure à l'aide d'un champ électrique alternatif non homogène - Google Patents

Dispositif et procédé destinés à la manipulation et à l'alignement sans contact de petites parties d'échantillons dans un volume de mesure à l'aide d'un champ électrique alternatif non homogène Download PDF

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Publication number
EP1935498A1
EP1935498A1 EP06026759A EP06026759A EP1935498A1 EP 1935498 A1 EP1935498 A1 EP 1935498A1 EP 06026759 A EP06026759 A EP 06026759A EP 06026759 A EP06026759 A EP 06026759A EP 1935498 A1 EP1935498 A1 EP 1935498A1
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Prior art keywords
particles
optical
sample particles
sample
electromagnetic radiation
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EP06026759A
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German (de)
English (en)
Inventor
Moritz Kreysing
Jochen Dr. Guck
Josef Prof. Dr. Käs
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Universitaet Leipzig
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Universitaet Leipzig
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Priority to EP06026759A priority Critical patent/EP1935498A1/fr
Priority to US12/520,667 priority patent/US8076632B2/en
Priority to EP07857093A priority patent/EP2101921A1/fr
Priority to PCT/EP2007/011386 priority patent/WO2008077630A1/fr
Publication of EP1935498A1 publication Critical patent/EP1935498A1/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]

Definitions

  • the present invention relates to a device for contactless manipulation and alignment of sample particles in a measurement volume with the aid of an inhomogeneous alternating electric field according to the preamble of claim 1.
  • the invention in a second aspect, relates to a method for the contactless manipulation and alignment of sample particles in a measurement volume with the help of an inhomogeneous alternating electric field according to the preamble of claim 14.
  • the invention relates to a laser scanning microscope and to a method of operating a laser scanning microscope.
  • dielectric particles here: particles whose dielectric constant deviates from that of the surrounding medium, with a diameter of less than 1000 ⁇ m, is the dielectrophoresis, which exploits the forces exerted inhomogeneous electric fields on electrically polarizable matter. Depending on whether the particles to be manipulated follow the field gradient or migrate in the opposite direction, one speaks of positive or negative dielectrophoresis.
  • this method requires electrodes in the vicinity of the particles to be manipulated, from which electrical fields emanate.
  • a particularly practicable arrangement of these electrodes is realized in so-called field cages, in which at least four electrodes enclose a volume which is measured by the size of the particles to be manipulated.
  • field cages in which at least four electrodes enclose a volume which is measured by the size of the particles to be manipulated.
  • DC voltages prove to be unsuitable, as they can lead to unwanted side effects such as electrolysis of the medium, strong heating or currents in the medium, which, however, can not be completely ruled out even when alternating voltages are used.
  • the dielectric properties of the samples are generally a function of the frequency of the surrounding electric fields.
  • many materials embedded in common media e.g. aqueous electrolyte solutions, below a certain frequency of positive, above this frequency negative dielectrophoresis. With particles that are not fully characterized, it may therefore be necessary to adjust the frequency via a trial and error procedure to make the operation of the field cage efficient.
  • Characteristic of this rotation is that though there is a balance between the torque induced by the electric field and the torque caused by hydrodynamic friction, but the particle is generally out of balance with respect to its orientation.
  • the frequency with which the trapped particle rotates is not the frequency dictated by the field, but many orders of magnitude lower.
  • optical tweezers are optical traps which hold particles whose refractive index differs from that of the surrounding medium by means of a focused laser beam and position.
  • the basic structure is as follows: With the aid of a partially transmissive mirror, a parallel laser beam, typically monochromatic with a wavelength in the visible or near infrared spectrum and a Gaussian intensity profile, 50 mW, is coupled into the beam path of a light-optical microscope and focused through a high numerical aperture oil immersion objective into the sample space, typically: liquid film between two coverslips.
  • laser beams can be coupled into the microscope either via beam splitter optics or the laser beam is deflected by automatically controlled mirrors or acousto-optical deflectors (AOD), which jump back and forth between at least two positions, so that the resulting partial beams reach more converge as a focal point.
  • AOD acousto-optical deflectors
  • Another way to generate more than one focus is to use holographic phase plates. Such a Construction is also referred to as holographic optical tweezers, "holographic optical tweezers”.
  • Focusing glass fibers ie commercially available, light-conducting glass fibers whose end is provided with a small converging lens or suitably modified differently, can be used to keep microscopic particles stable.
  • the principle here is comparable to that of the optical tweezers, with the difference that the laser beam no longer has to be coupled into the microscope optics, but passes through the glass fiber into the sample space. Due to the elongated shape of the focus produced by the prepared fiber end, microscopic particles align with their longest axis parallel to the propagation direction of the laser beam. If one overlays the foci of several glass fibers, it is possible to reorient the particles caught by suitably switching the fiber laser on and off.
  • the laser power required for trapping and holding depends on the density difference of the particle to that of the surrounding medium, size of the particle, relative refractive indices, temperature and geometry of the trap and optionally of divergence and width of the laser beams. However, it is related to the trapping and holding of biological cells in aqueous media between 5 and 300mW of continuous power per laser beam, typical: full divergence angle in the far field in air 15 degrees, near infrared wavelength, eg 1060nm.
  • the defined turning of particles is not possible with this structure.
  • a trapped particle may be forced onto a periodic path within the trap.
  • the dynamics of this process is characterized by the alternating attack of scattering and gradient forces of the two laser beams on the particle. This can qualitatively be described as follows: The particle is located in the center of laser beam1, the scattering force acting on it pushes it in the direction of laser beam2 until the gradient force emanates from it, the particle is newly centered and the laser beam2 emanating from the scattering force back towards the laser beam1 This effect usually occurs inadvertently when the laser beams are not optimally aligned, but has no application at all.
  • optical traps have been constructed using more than two laser beams in which trapped particles are forced onto similar periodic trajectories at non-optimally aligned fiber ends.
  • elliptical particles can be rotated by one laser beam in another, since in optical traps they always align with their main ash parallel to the propagation direction of the laser beam.
  • the number of possible orientations corresponds here, as in the case of focusing optical fibers based on optical fibers, a maximum of twice the number of glass fibers used.
  • Fiber-based laser traps are also used in the field of viscoelasticity measurement on biological cells, first by J. Guck et. al. realized in a fiber-based divergent two-beam laser trap. It is exploited here that, given sufficiently high laser intensities, as a result of the relativistic energy-momentum relationship and the general principle of conservation of momentum, forces on the membrane of a cell which are able to deform it are attacked.
  • a trap operated for this purpose is also referred to as optical stretcher, or "optical stretcher”.
  • two-beam laser traps can be used to line up spherical microparticles to a size of a few micrometers equidistant.
  • optical tweezers to rotate microscopic particles is a severe limitation on the useful microscope optics, which are generally used simultaneously to observe the particles.
  • Essential here is the use of high numerical aperture lenses. This results in a very small working distance and a not always desired very high magnification.
  • optical tweezers are not to be seen as universally applicable additional modules for any microscope.
  • the integration of optical tweezers in a microscope is generally very expensive and not possible at all or only to a limited extent in many microscope types. Problematic for the combination with optical tweezers are e.g. Confocal microscopes, deconvolution microscopes, all microscopes using lenses with a numerical aperture smaller than ⁇ 1.1.
  • Optical tweezers are largely unsuitable for the direct manipulation of biological samples due to the extremely high peak intensity due to the focusing of the laser beams used. Thermal damage, as well as radiation damage to the samples can be minimized by the choice of suitable wavelengths, but not completely avoided.
  • the distance of the laser-emitting objective to the particle may not be much larger than 250 .mu.m, but lenses used for optical tweezers typically have a diameter of not less than 2 cm, the objective used for observation would have to have a working distance of at least 1 cm.
  • this constellation would significantly reduce the achievable resolution because it is essentially a function of the maximum angle at which light emitted by the sample falls into the objective.
  • Dielectric field cages typically operate on the principle of negative dielectrophoresis, i. Particles to be captured must be in a medium of higher dielectric constant. Since the field strengths required in this case are considerable, typically> 20 KV / m, small electrical currents generally flow between the electrodes in the sample chamber, which may have undesirable effects on the trapped particles. These can range from warming to structural changes or death of sensitive samples, e.g. biological samples.
  • the object of the invention is to provide an apparatus and a method with which the manipulation and alignment of sample particles in a measurement volume is facilitated.
  • the device of the type described above is further developed according to the invention in that a beam shaping device for generating an asymmetric intensity profile to a beam axis is present as part of the optical means, sample particles in the measurement volume in a generated by the asymmetric intensity profile inhomogeneous field distribution of the electric field can be caught, and in that, for carrying sample particles captured in the inhomogeneous field distribution, a rotating device is provided for rotating the asymmetrical intensity profile about the beam axis relative to the measuring volume.
  • the method of the abovementioned type is further developed according to the invention by impressing an asymmetrical intensity profile on the electromagnetic radiation into the measurement volume, which generates an inhomogeneous field distribution of the electric field in the measurement volume, in which sample particles are trapped, and in that In the inhomogeneous field distribution trapped sample particles, the asymmetric intensity profile is rotated about the beam axis relative to the measurement volume.
  • the invention also provides a laser scanning microscope, in particular a confocal laser scanning microscope, which has a device according to the invention for the contactless manipulation and alignment of sample particles in a measuring volume with the aid of an inhomogeneous alternating electric field.
  • the subject matter of the invention is also a method for operating a laser scanning microscope, in particular a confocal laser scanning microscope, in which the method steps of claim 14 are carried out.
  • the recognition can be considered that with the aid of a non-rotationally symmetric beam profile in a measurement volume an inhomogeneous field distribution of the electric field can be generated, with which an azimuthal orientation of a sample particle relative to a beam axis can be accomplished.
  • Another core idea of the invention is then to be considered that trapped or detained particles or sample particles in this way can be manipulated, aligned and rotated by simply rotating the non-rotationally symmetrical intensity profile relative to the measurement volume in the measurement volume.
  • a rotation of the field distribution is accomplished about an arbitrary axis of rotation.
  • the effect of the invention results from the behavior of specifically polarizable matter in the field of an anisotropic, for example, not rotationally symmetric radiated electromagnetic radiation.
  • laser sources are considered as radiation sources.
  • the invention allows, for example, isolated microscopic particles with a diameter between 0.2 and 5000 microns, which are already in a stable equilibrium with respect to their position or are brought into balance with the aid of the device according to the invention, to rotate without contact by defined angle.
  • the rotation can be carried out in particular in such a way that it is possible to keep a particle stable in any orientation relative to a rotation axis.
  • the device according to the invention represents in itself a unit which, with respect to its functionality, is independent of any instruments necessary for the observation of the manipulated, aligned and / or rotated particles, in particular independently of a microscope used for this purpose. Nevertheless, there are numerous advantageous and new applications in the field of microscopy. For example, the non-contact rotation of the particles can take place transversely to an optical axis, in particular perpendicular to an optical axis, of an instrument used for observation. The possible new applications go beyond the solutions described above and there existing limitations can be largely avoided.
  • the arrangement according to the invention can also be described as an electromagnetic radiation trap which makes it possible to keep microscopic particles which differ in their optical properties, in particular refractive index and absorption behavior, from those of a surrounding medium in any orientation relative to at least one axis of rotation. Also asymmetrical intensity profiles of several radiation sources overlapping one another in the measurement volume are conceivable in principle and can be used for specific applications be beneficial.
  • the refractive index of the particle to be manipulated must be greater than that of the surrounding medium.
  • the invention relates to stable non-contact alignment and rotation of particles having a typical diameter of 0.2 to 5000 microns. This is especially important for microscopy techniques to achieve high isotropic resolutions, such as e.g. light microscopic computed tomography on individual biological cells, suspended cell organelles or small cell clusters.
  • microfluidic systems such as the viscosity of very small amounts of substance, as e.g. be implemented in microreactors, to determine or to quantify smallest torques.
  • the device according to the invention which can also be referred to as a cell rotator, can also be usefully used with the "optical stretcher".
  • microfluidic flow can be prevented from inducing cell rotation while the cell is being deformed or "stretched".
  • At least one electromagnetic beam is used, which is illuminated by suitable optics, e.g. optical waveguides, mirrors or microprisms, is guided into the sample space such that its transverse extent there corresponds approximately to the particle size or is generated in the immediate vicinity of the sample space with suitable geometry, e.g. from a laser diode.
  • suitable optics e.g. optical waveguides, mirrors or microprisms
  • the sample particles are thus oriented with respect to at least one axis.
  • An orientation relative to several axes is basically possible.
  • a plurality of radiation sources can be used.
  • a special feature of the guidance of the electromagnetic radiation used is that, unlike optical tweezers, it is completely decoupled from microscope optics possibly used to observe the sample.
  • the purpose of the electromagnetic radiation used is first, as in laser traps, to bring the particles to be manipulated in a stable equilibrium with respect to its position and to compensate for possible other forces acting on the particle. If only one beam is used for this purpose, then it is necessary for it to be convergent or else a force directed counter to the propagation direction of this beam, eg
  • the point of the stable position of the particle in the trap is characterized by the disappearance of the sum of all attacking forces, as well as the occurrence of restoring forces for no deflections from the equilibrium position.
  • the use of at least one electromagnetic beam having a non-rotationally symmetrical profile results in a potential for the orientation of trapped particles that are not completely homogeneous or asymmetrically shaped with respect to their optical properties with respect to the rotation about the propagation direction of this beam. The asymmetry of this beam can affect the intensity profile, its polarization and the modulation of the phase across the steel cross-section.
  • the method according to the invention consists of the following steps, some of which, depending on the nature of the sample, are to be regarded as optional.
  • the particles to be examined for the implementation of the method according to the invention can be prepared in the following manner.
  • the particles to be examined are separated and particle aggregates broken up.
  • various methods are suitable for this purpose, which depend on the gross mechanical effects on the sample, such as Crushing in a mortar, ranging from ultrasonic methods to methods in which the sample is suspended in liquid media with the addition of suitable chemicals.
  • enzymatic treatment of the sample may also be needed to resolve intercellular structures.
  • the sample may be prepared using conventional techniques, such as e.g. Sedimentation, centrifugation, chemical treatment, to be freed of impurities.
  • the separated particles are now placed in their medium in the sphere of action of the radiation trap relating to the invention.
  • the use of microfluidic transport systems, micropipettes and optical tweezers are suitable.
  • gases as well as in vacuum for this transport e.g. Micro-probes, electric fields, optical tweezers or atomizers, the latter being only partially suitable in vacuum can be used.
  • the medium make sure that it does not react chemically with the particles.
  • the medium should be a good conductor of heat.
  • the power of the laser beams used can be reduced until all particles except for a single driven by thermal fluctuations or directed flow of the medium leave the area of action of the trap to have.
  • the rotation of the trapped particle occurs via the rotation of at least one asymmetric beam profile.
  • this asymmetry may mean the distribution of intensity, polarization state and / or a modulation of the phase position over the radiation cross section.
  • the hydrodynamic coupling can be used on a rotating waveguide brought into the vicinity of the particle for rotation of the particle.
  • the particle When the measurement on the particle for which the rotation has been performed is completed, the particle can be sorted according to the measurement result using a transport mechanism known per se.
  • the rotation of microscopic particles is coupled to their potential.
  • Such a rotation is, depending on the expression of the. Asymmetries of electromagnetic beam and particles, the viscosity of the medium surrounding the particle and the intensity of the laser beam and the relative average refractive indices can be carried out very quickly.
  • this allows the invention in the case of particularly sensitive particles, e.g. biological cells to be rotated for the purpose of computed tomography, for which angular velocities of 360 ° / sec are sufficient to operate at relatively low powers, e.g. Laser beams each with 10 - 100 mW. This corresponds to the use of divergent laser beams much lower loads on the cells than they occur in the manipulation by optical tweezers.
  • a trapped particle can be stably held in any passable orientation without the need for a feedback mechanism. All angles are traversable between 0 ° and 360 ° with respect to at least one axis of rotation. This is useful, for example, in the long-term observation of biological, non-adherent cells, in which one is dependent on a random, for example, by Brownian motion-related rotation of the cell to prevent to keep the view of the cell constant.
  • Embodiments of the described invention for aligning and rotating microparticles are seen as a functional unit decoupled from any microscope optics used for observation. This offers the following advantages:
  • the invention enables the rotation of microscopic particles perpendicular to the optical axis of a microscope.
  • This can e.g. be used for light microscopic computed tomography or other microscopy method to achieve high isotropic resolutions of isolated, suspended, biological cells and smaller cell clusters.
  • a microscope used to observe the trapped particles can be operated independently of the invention. It is e.g. it is possible to vary the focal plane of the microscope with respect to trapped particles, which is of great importance for confocal and deconvolution microscopy, among other things.
  • Microscopes used for observation purposes require no or at most slight modifications.
  • the invention can be combined as desired with optical tweezers.
  • a combination of the invention with a laser microbeam that can cut and microinject is possible.
  • the invention may also be combined with a microfluidic chamber that allows renewal of a cell medium and thus can be used for long-term observation of cells.
  • the use of high numerical aperture lenses is optional. This allows e.g. the use of lenses with a greater working distance.
  • the invention makes no particular demands on the medium surrounding the particles.
  • it is possible to capture biological cells in any cell media, ie in particular in all standard in medicine and biology standard media and rotate to orient.
  • the only requirement for the media to use is that its refractive index is lower than that of the cell to be examined. This is usually the case.
  • a special feature of the invention is that it can be realized in a very space-saving manner using laser-guided glass fibers.
  • these have an outer diameter of 80 ⁇ m, optionally 125 ⁇ m, and are thus well integrated into an arrangement that can be easily adapted to the sample holders conventional light microscopes.
  • Fiber-based embodiments are conceivable, which manage entirely without free-beam optics.
  • the feeding of the electromagnetic radiation trap, here: a laser trap can thus be extremely flexible, which makes it possible to move the trap with respect to laser source and microscope, without recalibration would be necessary.
  • laser sources diode-pumped glass fiber lasers can be used.
  • a concrete application is the measurement of the viscosity of very small amounts of substance, as e.g. in chemical microreactors, by measuring the maximum angular velocity with which a known test object can be rotated.
  • the invention offers the possibility to quantify extremely small torques, as e.g. in the movement of the flagella of a bacterium, in which the maximum achievable by an active rotation of the particle by the invention angular velocity is compared with the behavior of the particle in the stationary trap.
  • an asymmetric intensity profile can be achieved by phase modulators of any type.
  • the device according to the invention basically works without the use of optical lenses, but can also be realized or combined with optical lenses.
  • the beam shaping device has optical components with an asymmetrical to an optical axis Transmission characteristic on.
  • asymmetric transmission characteristic is to be understood here in its broadest meaning, for example, this should also be understood situations in which electromagnetic radiation is coupled asymmetrically into an optical fiber.
  • the asymmetric transmission characteristic may be provided by a transition region at which two optical fibers adjoin one another with a radial offset.
  • the coupling of the light into a fiber leading to the sample space can also be effected in another way eccentrically.
  • a slight radial displacement of the focus also results in the generation of higher modes.
  • the asymmetrical transmission characteristic is provided by an asymmetrical termination of an optical fiber.
  • the glass fiber can also allow by their construction an asymmetric, correlated with the orientation of the fiber beam profile.
  • the glass fiber may have an elliptical core.
  • the asymmetric beam profile can also be generated, for example, by targeted squeezing of the glass fiber.
  • Rotation of the asymmetrical intensity profile can be accomplished by rotation of glass fibers.
  • astigmatic lenses or mirrors, asymmetric diaphragms, and / or variable aperture diaphragms can be used to provide the desired asymmetric transmission characteristic.
  • a variable asymmetrical intensity profile of the laser radiation can be achieved in variants in which the beam shaping device has electronically controllable lenses or a spatial light modulator (SLM).
  • SLM spatial light modulator
  • any method in which at least one asymmetrical laser mode is superposed with a symmetrical laser fundamental mode is suitable for generating an asymmetrical beam profile.
  • waveguides or even photonic crystals can be used as optical means for guiding the electromagnetic radiation into the measurement volume.
  • the optical means for guiding the electromagnetic radiation into the measurement volume comprise optical fibers.
  • the rotation of the asymmetrical intensity profile according to the invention can in principle be carried out in any desired manner.
  • the beam shaping device is mechanically rotated relative to the measuring volume with the aid of the rotating device.
  • an asymmetrical termination of an optical fiber extending into the measurement volume can be rotated with a simply constructed rotating device.
  • an already asymmetrically emitting radiation source can also be mechanically rotated relative to the optical means for guiding the radiation into the measurement volume.
  • This variant can be selected if the optical means for directing the radiation into the measurement volume itself have a negligible influence on the intensity profile.
  • an asymmetrically emitting light source for selectively rotating the asymmetrical intensity profile can also be controlled. In this case, practically no moving parts are necessary, so that such an arrangement is particularly advantageous in mechanical terms.
  • Another group of variants of the device according to the invention and of the method according to the invention is likewise characterized in that the rotation of the anisotropic intensity profile does not take place mechanically.
  • rotation of the asymmetrical intensity profile can be accomplished by rotation of the plane of polarization.
  • the device may have an active polarization device, in particular a Faraday cell.
  • other components such as birefringent and / or non-linear optical components can be achieved by rotation of the plane of polarization and rotation of a non-symmetrical intensity profile.
  • the polarization plane is also rotated when the intensity profile is rotated.
  • optical fibers with non-rotationally symmetrical profile can also be used.
  • the electromagnetic radiation enters the measurement volume from one end of an optical fiber, wherein the end of the optical fiber can either be planar, can be in the form of a diaphragm or can have a defined asymmetry.
  • the electromagnetic radiation can in principle originate from any sources, it being advantageous to use lasers.
  • these can be pulsed lasers, which may be advantageous, for example, if non-linear optical components are used.
  • continuously operated radiation sources are used.
  • the sample particles to be manipulated must first be transported in some way into the effective range of the electromagnetic radiation in the measuring volume.
  • the sample particles are introduced by means of a capillary to a suitable position in the measuring volume.
  • the sample does not have to leave the capillary.
  • a microfluidic transport system can be used with a glass capillary which has a square cross-section and through the walls of which the electromagnetic radiation radiates onto the sample particles.
  • the particles can be brought into the sphere of action of the radiation with a microfluidic system.
  • the device according to the invention is particularly advantageous and the method according to the invention can be used if biological samples, in particular cells, cell organelles and / or pieces of tissue, are examined as sample particles.
  • biological samples in particular cells, cell organelles and / or pieces of tissue
  • the sample particles are preferably suspended in aqueous media.
  • An essential advantage of the invention compared to manipulation methods which are known in the prior art, is that it has the greatest possible freedom to rotate the sample particles continuously at high angular velocity or very slowly or in defined steps, in particular abruptly.
  • sample particles for microscopy can be selectively rotated in order to achieve a specific, in particular isotropic, resolution.
  • the beam axis of the device according to the invention can be selected completely independently of the optical axis of a light microscope.
  • the sample may be rotated and imaged in steps for the purpose of computed tomography.
  • the isotropic resolution results from the computation of multiple images of the sample at varying angles with the help of a computer.
  • sample particles for microscopy can be positioned and aligned with different contrasting principles, in particular phase contrast, fluorescence microscopy, ultrasound microscopy, confocal microscopy, CARS and / or light microscopic manipulations, for example FRAP, un-caging.
  • phase contrast fluorescence microscopy
  • ultrasound microscopy confocal microscopy
  • CARS CARS
  • / or light microscopic manipulations for example FRAP
  • the possibility is taken advantage of, the sample particles in principle with a selectable speed in the surrounding medium to rotate.
  • the rotation of the particles can also take place arbitrarily slowly, in the limiting case of small angular velocities in stable equilibrium with respect to position and / or orientation.
  • the method according to the invention can be used to measure forces and torques acting on the particles positioned in the anisotropic radiation field. Accordingly, elasticity measurements are possible.
  • At least one further radiation source is present to compensate for forces which are exerted on the sample particles by pulse transmission of photons of the electromagnetic radiation.
  • Such other radiation sources can also be used to perform elasticity measurements on the aligned sample particles.
  • the rotation of one or more sample particles can also be used to set a surrounding sample medium in rotation.
  • the method according to the invention can also be used for processing and for targeted external manipulation, for example for aligning a sample particle for exposure to a micro-tool, such as an optical scalpel, a micropipette or a patch clamp.
  • a micro-tool such as an optical scalpel, a micropipette or a patch clamp.
  • a viscosity of the surrounding medium for example as the aqueous medium in which the particle moves, can be determined.
  • a measured maximum angular velocity for a given viscosity for example of water, can also say something about the sample, in particular the sample shape. For example, clues can be obtained as to whether a nucleus is dividing.
  • a glass-fiber-based two-beam laser trap modified in accordance with the invention will be described below.
  • the structure shown schematically in Fig. 1 , consists of a ceramic body 1, which ensures the alignment of laser-beam guiding glass fibers 6 and 7 by a precise guide through holes, two plain bearings, consisting of the ceramic sleeves 3 and 13 and the guided ceramic cylinders 2 and 11, a twist-free rotation of the right in allow the sample chamber 10 guided glass fiber 6.
  • the entire assembly is mounted on a commercially available light microscope with an indicated objective 16, so that samples can be observed in the laser trap 10 through the slide 15.
  • the left-hand optical fiber 7 is a so-called "single mode” fiber, ie a glass fiber which radiates the laser light guided through it with a Gaussian rotationally symmetrical intensity profile
  • the laser beam emitted by the right-hand optical fiber 6 does not possess this symmetry.
  • the reason for this is the slightly offset transition 8 from a "single mode” fiber 5 to a glass fiber 6, which at the wavelength of the laser used, due to the relative to the "single mode” fiber 5 larger fiber core, excited in higher vibration modes and therefore also as “multi mode” fiber is called.
  • the extension of the "single mode" fiber 5 coupled in the region of the glass fiber transition 8 to the glass fiber 6 is provided with the reference numeral 9.
  • This glass fiber 9 is an extension of the glass fiber 5, but is mechanically decoupled from the glass fiber 5 mechanically at the transition point 14.
  • the glass fiber 9 is the same "single mode" fiber as in the case of glass fiber 5.
  • the laser profile generated in this way, within the glass fiber piece 6 guided from the right into the sample space 10, is still dominated by the fundamental, ie Gaussian, laser mode , however, no longer possesses rotational symmetry due to the superimposition of higher modes, which generally have only discrete symmetries.
  • the beam shaping device is thus provided by the transition 8 between the fiber 5 to the fiber 6 here.
  • the rotation of the fiberglass piece 6 containing the source of asymmetry of the laser profile, guided from the right into the sample space, can be done manually and using a motorized drive.
  • the components 2, 4, 6, 8, 9 and 11 form a rigid unit that is rotatable with respect to the rest of the system.
  • the glass fibers used are commercially available step index fibers, ie glass fibers, whose refractive index varies abruptly in the region of the transition from the fiber core to the fiber cladding surrounding this core, or fiber cladding.
  • the numerical aperture of the fibers (NA) is about 0.14.
  • the "multi mode" glass fiber by additional structural elements around the region of the fiber core polarization maintaining and thus enables a particularly stable transport of the laser profile, which receives its shape in the region of the offset splice or glass fiber transition 8.
  • Both "multi mode” fiber and “single mode” fibers have an outer diameter of 125 ⁇ m after the removal of the acrylic protective sheath surrounding them first and can thus be optimally guided and aligned through the bores of the ceramics used, which have a diameter of 126 ⁇ m , Furthermore, the core diameter of the "multi-mode” fiber 6 is chosen so that the propagation of only a few vibration modes in the fiber is possible.
  • the so-called "V parameter" characteristic of the wave propagation in the optical fiber assumes for the "multi mode” fiber at the used wavelength of 1060 nm, a value between 2.405, transition to the "single mode” range, and about 4.
  • the glass fibers are fed by fiber laser modules, which, depending on the sample to be manipulated, have a Output power can be operated between a few milliwatts and several watts.
  • the attenuation of the laser beam intensity in the glass fiber is negligible here because of the short fiber lengths. Losses in the area of the fiber transition 8, however, can amount to 5-10%.
  • the mode of operation of this arrangement is as follows:
  • the gradient forces and scattering forces which are typical of optical double-jet traps attack particles centered in the region of the laser beams emitted by the glass fibers and center them in the trap.
  • the rotation of the asymmetric laser profile emitted by glass fiber piece 6 coupled to the rotation of the fiber itself causes the rotation of the particle in the trap parallel to the optical axis of the glass fibers.
  • the rotation of the particle is thus directly correlated with the rotation of the glass fiber and only slightly delayed in the case of highly viscous media.
  • FIG Fig. 2 is shown schematically.
  • the structure of the system is similar to that in Embodiment 1.
  • the main differences are the use of only one laser beam and the generation of its profile.
  • the construction consists of a "single mode" glass fiber piece 28 oriented by a ceramic guide 21, the twist-free rotation of which is glued by two plain bearings consisting of the ceramic sleeves 22 and 24 glued to the ceramic guide 21 and the ceramic cylinder 25, respectively, and to the ceramic cylinder 23. which together with glass fiber piece 28 forms a rigid unit rotatable relative to the rest of the assembly.
  • the mechanical decoupling of the glass fiber piece 28 from the "single mode" glass fiber 26 is ensured by the transition region 27 in which the planar-polished ends of the glass fibers 26 and 28 touch.
  • the laser beam used is not divergent by glass fiber 28 as in the arrangement example 1, but by the miniature lens 32 (rounding of the fiber end) focused and also has a slight astigmatism.
  • miniature lens 32 is meant here a rounding of one end of the glass fiber 28, which begins at the transition region 27 and leads into the sample space.
  • the preparation of the glass fiber end is carried out as follows: First, the core of the glass fiber 28 is exposed in the region of the end with hydrofluoric acid, which decomposes the cladding glass. The resulting tapered tail of the optical fiber 28 is then exposed in a so-called "arc fusion splicer" (a device normally used to connect glass fibers) to an arc between two needle tips for about 0.2 seconds. In this case, the fiber end is rounded off due to the surface tension of the glass and thus forms after cooling the miniature lens 32. This lens 32 has a slight astigmatism because of the preferred direction of the arc, which causes the laser beam emitted from the optical fiber 28 an elliptical profile has.
  • the arrangement is usually fixed on a slide 31 via the ceramic guide 21 in such a way that particles trapped in the focus of the laser beam 29 can be viewed by means of a light microscope whose objective 30 is indicated in the figure.
  • Zebrafish embryos represent an interesting field of research for developmental biology and genetics, as they are easy to handle and, because of their transparency, their development can be followed up to a high stage by light microscopy.
  • the embryo can be continuously rotated as it moves in steps around the optic axis of the trap by rotating the asymmetric profile of one of the laser beams used. This allows tracking of embryo development in three dimensions by imaging any slices parallel to the spin axis through the sample.
  • the rotation of the beam profile is done manually or motorized with a resolution less than a degree.
  • the use of fluorescence or other microscopy techniques is optional and possible.
  • the procedure consists of the following steps:
  • a microfluidic system is integrated. This essentially consists of a glass capillary of square cross section, through which the cells are transported into the effective range of the optical trap. Regulation of the flow through this capillary is by an electric syringe pump.
  • the desired cells are removed from the culture or an organism and prepared appropriately.
  • Adherent cells are detached from their substrate and optionally suspended in a cell medium with the addition of enzymes (e.g., trypsin) and chemicals.
  • the cells are diluted in their medium to a concentration of 10,000 cells / ml or enriched by centrifugation.
  • the cells are injected in their medium by means of a syringe into the microfluidic transport system.
  • the cells are transported through the microfluidic system into the area of action of the laser trap using a syringe pump.
  • the cell is then rotated 360 ° in 5 ° increments as a result of the rotation of the asymmetric profile of one of the laser beams used and photographed in any orientation by a camera connected to the phase contrast microscope used for observation.
  • the pictures are read in and digitized by a computer immediately or after completing the series of pictures.

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  • Electrochemistry (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Optical Measuring Cells (AREA)
EP06026759A 2006-12-22 2006-12-22 Dispositif et procédé destinés à la manipulation et à l'alignement sans contact de petites parties d'échantillons dans un volume de mesure à l'aide d'un champ électrique alternatif non homogène Withdrawn EP1935498A1 (fr)

Priority Applications (4)

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EP06026759A EP1935498A1 (fr) 2006-12-22 2006-12-22 Dispositif et procédé destinés à la manipulation et à l'alignement sans contact de petites parties d'échantillons dans un volume de mesure à l'aide d'un champ électrique alternatif non homogène
US12/520,667 US8076632B2 (en) 2006-12-22 2007-12-21 Device and method for the contactless manipulation and alignment of sample particles in a measurement volume using a nonhomogeneous electric alternating field
EP07857093A EP2101921A1 (fr) 2006-12-22 2007-12-21 Dispositif et procede consistant a manipuler et orienter sans contact des particules d'echantillon dans un volume de mesure au moyen d'un champ alternatif electrique non homogene
PCT/EP2007/011386 WO2008077630A1 (fr) 2006-12-22 2007-12-21 Dispositif et procédé consistant à manipuler et orienter sans contact des particules d'échantillon dans un volume de mesure au moyen d'un champ alternatif électrique non homogène

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EP06026759A EP1935498A1 (fr) 2006-12-22 2006-12-22 Dispositif et procédé destinés à la manipulation et à l'alignement sans contact de petites parties d'échantillons dans un volume de mesure à l'aide d'un champ électrique alternatif non homogène

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EP07857093A Withdrawn EP2101921A1 (fr) 2006-12-22 2007-12-21 Dispositif et procede consistant a manipuler et orienter sans contact des particules d'echantillon dans un volume de mesure au moyen d'un champ alternatif electrique non homogene

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Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0813090D0 (en) * 2008-07-17 2008-08-27 Univ St Andrews Optical trap
HU0800781D0 (en) * 2008-12-31 2009-03-02 Femtonics Kft Focusing system comprising acousto-optic deflectors for focusing an electromagnetic beam
JP5484981B2 (ja) * 2010-03-25 2014-05-07 東京エレクトロン株式会社 基板載置台及び基板処理装置
DE102010003750A1 (de) 2010-04-08 2011-10-13 Trumpf Laser- Und Systemtechnik Gmbh Verfahren und Anordnung zum Verändern der Strahlprofilcharakteristik eines Laserstrahls mittels einer Mehrfachclad-Faser
EP2446703B1 (fr) 2010-05-03 2015-04-15 Goji Limited Placement d'antenne(s) dans des cavités modales dégénérées d'un système de transfert d'énergie électromagnétique
DE102010036082B4 (de) * 2010-08-26 2015-07-23 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Mikrofluidischer Messaufbau und optisches Analyseverfahren zur optischen Analyse von Zellen
GB201209837D0 (en) * 2012-06-01 2012-08-29 Univ Bristol Orbital angular momentum
KR101566587B1 (ko) * 2013-11-05 2015-11-05 포항공과대학교 산학협력단 베셀 빔 생성용 광 섬유 및 이를 사용하는 광학 이미징 장치
US9734927B2 (en) * 2015-04-09 2017-08-15 International Business Machines Corporation Optical capture and isolation of circulating tumor cells in a micro-fluidic device utilizing size selective trapping with optical cogwheel tweezers
GB201508376D0 (en) * 2015-05-15 2015-07-01 Univ St Andrews Light sheet imaging
US9784899B2 (en) * 2015-06-01 2017-10-10 U-Technology Co., Ltd. LED illumination apparatus
US10168501B2 (en) * 2016-05-27 2019-01-01 Nxgen Partners Ip, Llc System and method for transmissions using eliptical core fibers
US10187171B2 (en) * 2017-03-07 2019-01-22 The United States Of America, As Represented By The Secretary Of The Navy Method for free space optical communication utilizing patterned light and convolutional neural networks
WO2018187610A1 (fr) 2017-04-05 2018-10-11 The Regents Of The University Of California Dispositif de focalisation continue et de rotation de cellules biologiques et son utilisation pour la cytométrie en flux par électrorotation à haut débit
US11933707B2 (en) * 2018-04-17 2024-03-19 Chemometec A/S Depicting of objects
CN109261361B (zh) * 2018-08-08 2020-02-07 青岛大学 一种同轴型介电微米纳米粒子连续分离器
CN112461830B (zh) * 2020-11-05 2022-09-06 山东建筑大学 一种组合透明介质微球小型光镊装置及应用
EP4112171A1 (fr) 2021-06-29 2023-01-04 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procede et appareil de manipulation spatiale d'au moins une particule dans un fluide, produit de programme informatique et moyen de memoire lisible par ordinateur
CN113436777B (zh) * 2021-08-27 2022-01-14 之江实验室 基于探针的双向电泳力光阱起支方法及装置与应用
CN114453038A (zh) * 2022-01-24 2022-05-10 中南大学 一种基于双光纤和对撞流的光流控分选微纳颗粒芯片

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002043870A1 (fr) * 2000-11-29 2002-06-06 Evotec Oai Ag Microsysteme permettant la manipulation dielectrique et thermique de particules
US20040011650A1 (en) * 2002-07-22 2004-01-22 Frederic Zenhausern Method and apparatus for manipulating polarizable analytes via dielectrophoresis
WO2004098777A2 (fr) * 2003-05-09 2004-11-18 Evotec Technologies Gmbh Procedes et dispositifs permettant de traiter des particules en suspension au moyen d'un liquide
US20060196772A1 (en) * 2005-02-12 2006-09-07 Kim Sook-Young Microfluidic device including membrane having nano- to micro-sized pores and method of separating polarizable material using the same

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2057506C (fr) 1990-12-13 2003-05-13 Keiji Sasaki Piegeage laser et methode d'application de cette technique
JP3292515B2 (ja) * 1992-09-07 2002-06-17 オリンパス光学工業株式会社 顕微鏡観察のための微調整方法及び微調整装置
DE19758745C5 (de) 1997-01-27 2008-09-25 Carl Zeiss Jena Gmbh Laser-Scanning-Mikroskop
US6067859A (en) * 1999-03-04 2000-05-30 The Board Of Regents, The University Of Texas System Optical stretcher
DE102004023466B4 (de) 2004-05-12 2008-11-13 Evotec Technologies Gmbh Verfahren und Vorrichtung zur Sammlung von suspendierten Partikeln

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002043870A1 (fr) * 2000-11-29 2002-06-06 Evotec Oai Ag Microsysteme permettant la manipulation dielectrique et thermique de particules
US20040011650A1 (en) * 2002-07-22 2004-01-22 Frederic Zenhausern Method and apparatus for manipulating polarizable analytes via dielectrophoresis
WO2004098777A2 (fr) * 2003-05-09 2004-11-18 Evotec Technologies Gmbh Procedes et dispositifs permettant de traiter des particules en suspension au moyen d'un liquide
US20060196772A1 (en) * 2005-02-12 2006-09-07 Kim Sook-Young Microfluidic device including membrane having nano- to micro-sized pores and method of separating polarizable material using the same

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WO2008077630A1 (fr) 2008-07-03
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US20100282984A1 (en) 2010-11-11

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