EP1935498A1 - Device and method for contactless manipulation and alignment of sample particles in a measurement volume with the aid of an inhomogeneous electrical alternating field - Google Patents

Abstract

A device has a radiation source for transmission of electromagnetic radiation, and an optical device (6,5) for conducting the electromagnetic radiation into the measurement volume. Part of the optical device (6,5) is a beam shaping device (8) for generating an intensity profile asymmetric to one beam axis, in which the sample particles in the measurement volume (10) are captured in an inhomogeneous field distribution (of the electric field) caused by the asymmetrical intensity profile. The captured particles are carried along via a rotational device (2,4,11). Independent claims are given for the following: (1) (A) A method for contactless manipulation. (2) (B) A laser scanning microscope (3) (C) A method for operating a laser scanning microscope.

Description

In a first aspect, 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.

In a second aspect, the invention 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.

In other aspects, the invention relates to a laser scanning microscope and to a method of operating a laser scanning microscope.

A generic device and a generic method are described in: Arthur Ashkin, Optical trapping and manipulation of neutral particles using lasers, 1997; Volume 94; Pages 4853 - 4860 PNAS , Laser scanning microscopy and applications thereof in biology are described in: James B. Pawley's Handbook of Biological Confocal Microscopy, 1995, Plenum Press, New York , A confocal laser scanning microscope is also disclosed in DE 197 02 753 A1 ,

The following are the arrangements and methods for aligning and rotating particles known.

i) One possibility for the positioning and alignment of dielectric particles, here: particles whose dielectric constant deviates from that of the surrounding medium, with a diameter of less than 1000 μm, 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.

In concrete terms, 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. For generating the electric field distributions around the electrodes, one applies to these alternating voltages of defined amplitude, frequency and phase. 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.

Furthermore, the dielectric properties of the samples are generally a function of the frequency of the surrounding electric fields. For example, 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.

By suitable geometries of the field cage and appropriate voltages at the electrodes, it is possible to generate local extremes of electric field strength that can be used to capture dielectric particles, ie, to hold them stable with respect to their spatial position. Furthermore, it is possible to transmit a continuous torque to trapped particles by means of a rotating electric field generated by the geometry of the cage adapted phase positions of the voltages applied to the individual electrodes. This may have different orientations depending on the cage, so that it is possible to rotate a trapped dielectric particle by more than one axis solely via phase adjustments. Depending on the characteristics of the concrete overall system, rotational speeds of over 100 rotations per second can be achieved. 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. In particular, the frequency with which the trapped particle rotates is not the frequency dictated by the field, but many orders of magnitude lower.

The adaptation of the rotational speed of the particles to a desired value is carried out in the general case of ignorance of the complete structure of the respective particle via feedback mechanisms according to the principle "trial and error". Thus, for example, the rotation of biological cells in suspension can be observed through a microscope and, if appropriate, accelerated or slowed down by suitable adaptation of the alternating electric fields. As a result, the rotation of particles that can not be fully characterized, such as biological cells, is only possible at small angles at best via accompanying measurements on the system. Literature: Christoph Reichle, Torsten Müller, Thomas Schnelle and Günter Fuhr: "Electro-rotation in octopole micro cages", J. Phys. D: Appl. Phys. 32 (1999) 2128-2135 ; DE 100 59 152 C2 . DE 10 2004 023 466 A1 and DE 103 20 869 A1 ,

ii) Another possibility for rotating microscopic particles are so-called optical tweezers. 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. As the field energy of the electromagnetic wave is reduced when entering higher refractive index media, visually denser particles, which come randomly or otherwise deliberately into the area of the finally extended focus due to molecular motion, experience a force in the direction of its center (gradient force) ). Furthermore, as a result of the light scattering on the particles, a so-called scattering force acts on the particles, which stabilizes them in the axial direction. The scattering force alone, the sample particle slides away from the laser. A stabilizing effect results together with the gradient forces.

With regard to the laser steel, there is therefore an equilibrium for the position of the particle in the focus, which is characterized in that stray and gradient forces acting on the trapped particle just compensate or particles in the case of small deflections from the equilibrium position driven back into this become.

This can for example be exploited to fix microparticles or to move them by changing the entrance angle of the laser beam into the lens. In the case of manipulation of biological cells, it is necessary to attach microparticles of about the size of the cells themselves, eg small latex beads, to the cells by suitable methods and then to attack them with the optical tweezers, since the laser intensity due to the focusing of the used Laser beam is too high in the area to be used for holding microparticles for biological cells to ensure their integrity. Literature: A. Ashkin, JM Dziedzic, JE Bjorkholm, and Steven Chu: "Observation of a single-beam gradient force optical trap for dielectric particles", OPTICS LETTERS / Vol. 5 / May 1986 ; DE 691 13 008 T2 ,

1. a) Bei doppelbrechende Proben ändert sich der Polarisationszustand des Laserlichtes derart, dass diese ein Drehmoment erfahren. Dieser Drehmomentübertrag führt zu einer kontinuierlichen Drehung um die Laserachse und kann durch die Änderung der Intensität sowie Polarisation des einfallenden Laserstahls reguliert werden. Eine Anwendung dieses Prinzips sind lichtgetriebene Zahnräder mit einem Durchmesser von unter 20µm, die in sogenannten Mikromaschinen Einsatz finden. Literatur: M. E. J. Friese, T. A. Nieminen, N. R. Heckenberg & H. Rubinsztein-Dunlop: "Optical alignment and spinning of laser-trapped microscopic particles", Nature 394, 348-350 (1998) , E. Higurashi, R. Sawada, and T. Ito: "Optically induced angular alignment of trapped birefringent micro-objects by linearly polarized light", NTT Opto-electronics Laboratories, 3-9-11 , M. E. J. Friese and H. Rubinsztein-Dunlop: "Optically driven micromachine elements", Applied Physics Letters -- January 22, 2001 -- Volume 78, Issue 4, pp. 547-549 .
2. b) Proben, deren Geometrie und Brechungsindexverteilung dazu führt, dass der bei einer optischen Pinzette verwendete Laserstrahl derart asymmetrisch gestreut wird, dass aufgrund der für Photonen gültigen Impulserhaltung ein Drehmoment auf die Probe übertragen wird, werden aufgrund dessen in Rotation versetzt. Bekannt ist dieser Effekt als Windmühleneffekt und tritt üblicherweise bei speziell gefertigten Mikropartikeln auf, deren Form an die eines Propellers erinnert. Im weitesten Sinn handelt es sich hierbei auch um eine Form der Doppelbrechung des Partikels, da sowohl Spin als auch Bahndrehimpuls des verwendeten Laserstrahls verändert werden kann. Ebenfalls erfolgt die Drehung kontinuierlich. Literatur: E. Higurashi, O. Ohguchi, T. Tamamura, H. Ukita, R. Sawada: "Optically induced rotation of dissymmetrically shaped fluorinated polyimide micro-objects in optical traps", J. Appl. Phys., Vol. 82, No. 6, 15 September 1997 .
3. c)"Optical Spanners": Hierzu wird der oben beschriebene Aufbau der optischen Pinzette dahingehend modifiziert, als dass der in die Mikroskopoptik eingekoppelte Laserstrahl zuvor so polarisiert wird, dass der mittlere Gesamtdrehimpuls der Photonen stark von Null abweicht. Dies geschieht durch räumliche Lichtmodulatoren, die Licht über Modulation der Phasenlage über die Wellenfront mit einem Bahndrehimpuls ausstatten. Durch Streuung und Absorption dieses Laserlichts an gefangenen Partikeln findet ein kontinuierlicher Drehimpulsübertrag auf selbige statt, woraus eine Rotation des gefangenen Partikels um die Laserachse resultiert. Ebenso ist es möglich, Mikropartikel auf Kreisbahnen zu schicken, die sie periodisch durchlaufen, ohne dass es einer Führung der einzelnen Partikel z.B. über Auslenkung des einfallenden Laserstrahls bedürfte. Literatur: M. E. J. Friese, J. Enger, H. Rubinsztein-Dunlop, and N. R. Heckenberg: "Optical angular momentum transfer to trapped absorbing particles", Physical Review A 54, 1593-1596, (1996) , J Leach, M. R. Dennis, J. Courtial and M. J. Padgett: "Vortex knots in light", New J. Phys. 7 (2005) 55 .
4. d) Außerdem gibt es Ansätze, Objekte mit mehreren optischen Pinzetten gleichzeitig zu halten und durch Variation der relativen Lage der Foki zueinander asymmetrische Partikel um die optische Achse des Mikroskops zu drehen.

In order to turn particles with this structure, there are various possibilities.

1. a) In birefringent samples, the polarization state of the laser light changes so that they experience a torque. This torque transfer results in a continuous rotation about the laser axis and can be regulated by the change in intensity and polarization of the incident laser beam. An application of this principle are light-driven gears with a diameter of less than 20μm, which find use in so-called microengines. Literature: MEJ Friese, TA Nieminen, NR Heckenberg & H. Rubinsztein-Dunlop: "Optical alignment and spinning of laser-trapped microscopic particles", Nature 394, 348-350 (1998) . E. Higurashi, R. Sawada, and T. Ito: "Optically induced angular alignment of trapped birefringent micro-objects by linearly polarized light", NTT Opto-electronics Laboratories, 3-9-11 . MEJ Friese and H. Rubinsztein-Dunlop: "Optically Driven Micromachine Elements", Applied Physics Letters - January 22, 2001 - Volume 78, Issue 4, pp. 547-549 ,
2. b) Samples whose geometry and refractive index distribution cause the laser beam used in optical tweezers to be so asymmetrically scattered that Due to the momentum valid for photons, a torque is transmitted to the sample, due to which are set in rotation. This effect is known as a windmill effect and usually occurs in specially manufactured microparticles whose shape is reminiscent of that of a propeller. In the broadest sense, this is also a form of birefringence of the particle, since both spin and orbital angular momentum of the laser beam used can be changed. Also, the rotation is continuous. Literature: E. Higurashi, O. Ohguchi, T. Tamamura, H. Ukita, R. Sawada: "Optically induced rotation of dissymmetrically shaped fluorinated polyimide micro-objects in optical traps", J. Appl. Phys., Vol. 82, no. 6, 15 September 1997 ,
3. c) "Optical Spanners": For this purpose, the above-described construction of the optical tweezers is modified to the effect that the coupled into the microscope optics laser beam is previously polarized so that the average total angular momentum of the photons deviates greatly from zero. This is done by spatial light modulators that provide light via modulation of the phase angle over the wavefront with a train angular momentum. By scattering and absorbing this laser light on trapped particles, a continuous angular momentum transfer takes place thereon, resulting in a rotation of the trapped particle about the laser axis. Likewise, it is possible to send microparticles on circular paths, which they pass through periodically, without the need for a guide of the individual particles, for example via deflection of the incident laser beam. Literature: MEJ Friese, J. Enger, H. Rubinsztein-Dunlop, and NR Heckenberg: "Optical angular momentum transfer to trapped absorbent particles", Physical Review A 54, 1593-1596, (1996). . J Leach, MR Dennis, J. Courtial and MJ Padgett: "Vortex knots in light", New J. Phys. 7 (2005) 55 ,
4. d) There are also approaches to hold objects with multiple optical tweezers simultaneously and by varying the relative position of the foci to each other to rotate asymmetric particles about the optical axis of the microscope.

For this purpose, several 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. 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".

Rotations perpendicular to the optical axis of the microscope were realized on a dumbbell-shaped microparticle made especially for this purpose, consisting of two partially fused glass beads, each about 5 μm in diameter, in a "trial and erorr" experiment. It has also been shown in laboratory experiments that it is possible to modify solid-state lasers by introducing a suitable aperture stop into the resonator chamber, or "cavity", so that the laser beams emitted by them are focused by an objective on more than one point. Each of these focuses can thus be used as optical tweezers. Literature: V. Bingelyte, J. Leach, J. Courtial, and MJ Padgett: "Optically Controlled Three-dimensional Rotation of Microscopic Objects," APPLIED PHYSICS LETTERS VOLUME 82, NUMBER 5, 3 FEBRUARY 2003 ; Amiel Ishaaya, Nir Davidson, and Asher Friesem: "Very high-order pure Laguerre-Gaussian mode selection in a passive Q-switched Nd: YAG laser", Optics Express # Vol. 13, Iss. 13 - June 2005 pp: 4952-4962 ; Enrico Santamato, Antonio Sasso, Bruno Piccirillo, and Angela Vella: "Optical angular momentum transfer to transparent isotropic particles using zero beam angular momentum", Optics Express Vol. 10, Iss. 17 - August 2002 pp: 871-878 ,

iii) 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. They align within a short time in each case parallel to the optical axis of the respective active fiber. This method allows particles to be rotated in steps from one equilibrium position to the next, provided that the geometry of the devices involved in the other construction and the flexibility and dimension of the glass fibers allow it to be rotated. The number of stable orientations corresponds to at most twice the number of fibers. Literature: K. Taguchi, H. Ueno, T. Hiramatsu and M. Ikeda: "Optical Trapping of the Radical Particle and Biological Cell Using Optical Fibers", ELECTRONICS LETTERS 27th February 1997 Vol. 33 ; K. Taguchi, H. Ueno and M. Ikeda: "Rotationally manipulating a yeast cell using optical fibers", ELECTRONICS LETTERS 3rd July 1997 Vol. 14 ; Taguchi, M. Tanaka, K. Atsuta and M. Ikeda: "Three Dimensional Optical Trapping Using Plural Optical Fibers", Proc. of CLEO2000, pp.CtuK19, (2000-9 ); Taylor, RS; Hnatovsky, C .: "Particle trapping in 3-D using a single fiber sample with an annular light", Optics Express, vol. 11, Issue 21, p.2775 ,

iv) Two-beam laser traps and methods for the manipulation of microparticles: This type of laser trap was first realized in 1970 by A. Ashkin using freely propagating laser beams. The now more common, technically slightly modified form makes use of the guidance of the laser beams through glass fibers into the sample space. However, the principle of both designs is the same. Two divergent laser beams, which are Gaussian in shape from their intensity profile, are directed against each other so that their optical axes coincide. Similar to the optical tweezers, two types of forces also attack optically denser particles with respect to the surrounding medium, which come into the area of the laser beams. Gradient forces that draw the particle in the range of maximum laser intensity, ie radially centering, and scattering forces in the propagation direction of the laser beams, which provide alignment along the optical axis. As a result, with the same condition of the two laser beams, the particle is centered between the two laser beams after a relatively short time in a stable equilibrium position. If the intensity of one of the laser beams is increased, this equilibrium position of the trapped particle on the optical axis is shifted somewhat in the propagation direction of this laser beam. The diameter of the laser beams should not significantly exceed the size of the particles in the equilibrium position for trapped particles to make the trap efficient. The full divergence angle of the laser beams is typically 10-20 degrees in the far field. 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. However, by slightly tilting the laser beams toward each other, 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.

Furthermore, similar to the principle of the two-beam laser trap, 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.

Furthermore, by varying the relative laser intensities, 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".

Furthermore, two-beam laser traps can be used to line up spherical microparticles to a size of a few micrometers equidistant. Literature: A. Ashkin: "Acceleration and Trapping of Particles by Radiation Pressure", Phys. Rev. Lett. 24, 156-159 (1970) ; SD Collins, RJ Baskin, and DG Howitt, "Microinstrument gradient force optical trap," Applied Optics 38, 6068-6074 (1999) ; Guck, J., R. Ananthakrishnan, TJ Moon, CC Cunningham, and J. Käs: "Optical deformability of soft dielectric materials," Phys. Rev. Lett., 84 (23), 5451-5454 (2000) ; Guck, J., R. Ananthakrishnan, TJ Moon, CC Cunningham and J. Käs: "The Optical Stretcher - A Novel, non-invasive tool to manipulate biological materials", Biophys. J., 81, 767-784 (2001) ; W. Singer, M. Frick, S. Bernet, and M. Ritsch-Marte: "Self-organized array of regularly spaced microbeads in a fiber-optic trap", J. Opt. Soc. At the. B 20, 1568 (2003 ).

All the solutions described above have at least one of the following shortcomings with respect to the field of application of the invention:

The rotation of the particles results from a continuous angular momentum transfer. As a result, trapped, not fully characterized particles can only be rotated through defined angles using a trial and error method using a feedback mechanism. In the case of the dielectric field cages and "optical tensioners" this means concretely: The rotation of microscopic particles by defined angles is only possible by stopping a continuously induced rotation shortly before passing through the desired orientation and taking into account the ratio of inertia occurring Friction forces is braked. In order to judge whether the desired orientation has already been reached, it is generally necessary to carry out a measurement, which is typically carried out with a light microscope.

It is not possible to perform the rotation of microscopic particles in the limit of small angular velocities in equilibrium. This means that the orientation of a particle after completion of a rotation is generally not stable. It is therefore not possible with those mentioned under i), ii) a), ii) b), iic), iii) and iv), to keep a particle stable in any orientation with respect to at least one of the possible axes of rotation. If a certain orientation is to be maintained, it is necessary to counteract dynamically, ie necessarily using feedback mechanisms, against torques acting on asymmetric particles due to structural asymmetries. In the case of field cages, the rotational symmetry becomes of the system broken by the finite number of electrodes used. In the case of the "optical tensioners", it is the direction of polarization of the laser beam used to hold the particle, which provides for preferential orientation of asymmetric particles.

As a result of the previous point, rotations can only be performed via constant angular velocity feedback mechanisms. To assess whether, e.g. a biological cell rotating at a constant angular velocity, is problematic in particular if the structure of the cell is still largely unknown and should be clarified only by the rotation.

The use of 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. Furthermore, 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.

In most cases, the birefringence of microscopic particles is far too small for them to experience torque in a linearly polarized laser trap which causes them to rotate. Exceptions are specially made micro gears and optically active crystals.

The turning of microscopic particles by means of optical tweezers generally takes place about the optical axis of the microscope optics used to guide the laser beam, which is also commonly used to observe the particle. That is, turning the particle under observation gives no additional information gain. This method is thus completely unsuitable as a basis for tomographic examinations. Although it is theoretically conceivable to observe the particle from the side with a second microscope, it is simply impractical due to the functionality of subordinate geometry of commercially available microscopes. For example, since 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. However, 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 manipulation of biological samples therefore requires the use of special, low-conductivity media that are incompatible with many cell types or whose effects on the integrity of the cells are unknown.

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.

This object is achieved in a first aspect of the invention by the device having the features of claim 1.

In a second aspect of the invention, the object is achieved by the method having the features of claim 14.

Preferred embodiments of the device according to the invention and advantageous variants of the method according to the invention are the subject of the dependent claims.

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.

Finally, 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.

As a first core idea of the invention, 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. By the variation of the electromagnetic radiation, 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. In particular, 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.

In particular, 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.

Another application is the use in 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". In the combination, microfluidic flow can be prevented from inducing cell rotation while the cell is being deformed or "stretched".

In the device according to the invention, 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. The sample particles are thus oriented with respect to at least one axis. An orientation relative to several axes is basically possible. For this purpose, 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

Gravitation, frictional forces caused by the flow of the medium, the particles attacked to compensate for stray forces occurring.

If several jets are used, they can be directed against each other so that scattering forces acting on them from the trapped particle cancel each other out. In general, 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. Furthermore, 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. Even the smallest deviations of the particle shape of bodies of revolution, which are practically always present in real samples, suffice here for the formation of a potential for the angular orientation. As a result of this potential, there is a preferential orientation of the particle in the trap, which is captured during capture and then kept stable. Turning now the profile of the asymmetric beam and thus the potential for the angular orientation of a trapped particle, this rotates with. This rotation occurs in the limit of small angular velocities in equilibrium, i. in the minimum of the potential. The rotation of the asymmetric steel profile responsible for the orientation of the particle is realized, most simply by the rotation of a waveguide emitting the beam asymmetrically. Other ways of rotating the beam profile, e.g. those using astigmatic lenses or mirrors are possible.

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.

First, 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. Depending on the sensitivity and nature of the sample, 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. In the case of biological cells, enzymatic treatment of the sample may also be needed to resolve intercellular structures.

If necessary, the sample may be prepared using conventional techniques, such as e.g. Sedimentation, centrifugation, chemical treatment, to be freed of impurities.

After preparation of the particle, they can be treated as follows.

The separated particles are now placed in their medium in the sphere of action of the radiation trap relating to the invention. In the case of liquid media, the use of microfluidic transport systems, micropipettes and optical tweezers are suitable. In 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. When choosing the medium, make sure that it does not react chemically with the particles. Also, in the case of the radiation of absorbing particles used, the medium should be a good conductor of heat.

In the unfavorable for the further process, that several non-contiguous particles are in the trap, 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.

In cases of heavily underdamped or over-damped systems, e.g. large particles in dilute gases or in vacuum or small particles in high viscosity media, it is necessary to wait until the trapped particle arrives in a stable position in the trap. Usually, this process takes only a few hundredths of a second

In the case of greatly varying particle sizes, it may also be advantageous to adapt the geometry of the trap, insofar as it works with divergent electromagnetic radiation, to the size of the particular trapped particle.

The rotation of the trapped particle occurs via the rotation of at least one asymmetric beam profile. In this case, this asymmetry may mean the distribution of intensity, polarization state and / or a modulation of the phase position over the radiation cross section. Likewise, the hydrodynamic coupling can be used on a rotating waveguide brought into the vicinity of the particle for rotation of 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 arrangement according to the invention and the method are associated with a number of advantages.

The rotation of microscopic particles is coupled to their potential. In particular, this means that a trapped particle can be rotated by the inventive arrangement without the use of feedback mechanisms to defined, any angle. This is especially important if the spatial structure of the particles to be rotated is not fully characterized or even, as in the use of rotation for the purposes of computer tomography, to be informed by the rotation.

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. On the other hand, 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.

Unlike, for example, dielectric field cages and optical tensioners, 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. In addition, a combination of the invention with a laser microbeam that can cut and microinject is possible. In addition, 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.

Unlike optical tweezers, the use of high numerical aperture lenses is optional. This allows e.g. the use of lenses with a greater working distance.

Furthermore, the invention makes no particular demands on the medium surrounding the particles. Thus, 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.

Further advantages result from the configuration of the arrangement and the method according to the invention.

A special feature of the invention is that it can be realized in a very space-saving manner using laser-guided glass fibers. Typically, these have an outer diameter of 80μm, optionally 125μm, 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. As laser sources diode-pumped glass fiber lasers can be used.

Due to the minimal size of conceivable embodiments of the invention, their use for measuring microfluidic systems is conceivable. 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.

Also, 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.

In principle, 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.

In preferred embodiments of the device according to the invention, the beam shaping device has optical components with an asymmetrical to an optical axis Transmission characteristic on. The term 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. For example, the asymmetric transmission characteristic may be provided by a transition region at which two optical fibers adjoin one another with a radial offset.

In principle, the coupling of the light into a fiber leading to the sample space can also be effected in another way eccentrically. For example, when focusing an initially parallel beam by means of a converging lens onto a clean-cut end of a glass fiber, a slight radial displacement of the focus also results in the generation of higher modes.

In a particularly easy-to-implement variant of the device according to the invention, 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. For example, 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.

Alternatively, 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). In principle, 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.

In principle, waveguides or even photonic crystals can be used as optical means for guiding the electromagnetic radiation into the measurement volume. In particularly preferred variants of the invention, 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. In embodiments that are easy to implement, the beam shaping device is mechanically rotated relative to the measuring volume with the aid of the rotating device. For example, an asymmetrical termination of an optical fiber extending into the measurement volume can be rotated with a simply constructed rotating device.

This already results in an advantageous development of the method according to the invention, in which a rotation of the sample particles by a hydrodynamic coupling to a rotating in the region of the measuring volume optical element, in particular the end of an optical fiber is at least supported.

Accordingly, for rotating the intensity profile, 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. One then has the advantage that an intervention in the measuring volume is practically not necessary, in particular no rotating parts are present there.

As an alternative to mechanically rotating an anisotropically emitting radiation source, 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. For example, rotation of the asymmetrical intensity profile can be accomplished by rotation of the plane of polarization. For this purpose, the device may have an active polarization device, in particular a Faraday cell. Along with 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.

Conversely, if, for example, the entire light source is rotated and this already emits polarized light, the polarization plane is also rotated when the intensity profile is rotated.

For this purpose, optical fibers with non-rotationally symmetrical profile can also be used.

In particularly advantageous variants, 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.

In principle, these can be pulsed lasers, which may be advantageous, for example, if non-linear optical components are used. In simple variants, 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.

This can be done for example with the aid of the optical tweezers described above and additionally or alternatively with the aid of dielectrophoretic forces.

If spatially possible, 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. For example, 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. In general, 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. In this case, 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.

A particularly advantageous application arises in conjunction with microscopy, in which the resolution in the lateral direction differs from that in the axial direction. With the aid of the device according to the invention and the method according to the invention, sample particles for microscopy can be selectively rotated in order to achieve a specific, in particular isotropic, resolution. This is possible because the beam axis of the device according to the invention can be selected completely independently of the optical axis of a light microscope. For example, 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.

In addition, there are still further advantageous applications in the field of microscopy.

For example, 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. A combination of the method according to the invention with methods of cell microinjection and long-term observation of cell balls and cells is also possible.

Particularly advantageous applications also arise in the field of laser scanning microscopy and tomographic methods.

In further applications of the method according to the invention, which are fundamentally independent of a possible observation of the measuring volume with the aid of a microscope, the possibility is taken advantage of, the sample particles in principle with a selectable speed in the surrounding medium to rotate. In principle, 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.

With the help of suitable calibrations to be performed, 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.

The passage of the photons through the sample particles, if they have a deviating from the environment refractive index, leads to a momentum transfer, and thus to a force on the sample particle. This force can be compensated, for example, with proper positioning of the radiation source by gravity.

In particularly preferred variants, 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.

Finally, from a maximum possible angular velocity of a sample particle, 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.

Fig. 1

ein erstes Ausführungsbeispiel einer erfindungsgemäßen Vorrichtung; und

Fig. 2

ein zweites Ausführungsbeispiel einer erfindungsgemäßen Vorrichtung.

Further advantages and features of the invention will be described with reference to the accompanying figures. Hereby shows:

Fig. 1

a first embodiment of a device according to the invention; and

Fig. 2

A second embodiment of a device according to the invention.

Embodiment 1

As a first exemplary embodiment, 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.

While 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 this intensity profile via the twist-free rotation of the last centimeter of the right glass fiber 6 in front of the sample chamber 10, starting at transition point 14 together with the ceramic cylinders 2 and 11, in the central holes of the glass fiber 6 is glued, and the protective sheath 4 of the fiber transition 8, which simultaneously serves as a mechanically rigid coupling of the ceramic cylinder 2 to the ceramic cylinder 11. In the region of the transition point 14, aligned by a substantially consisting of two ceramic cylinders 11 and 12 and a ceramic guide 13 sliding bearing, two plane cut, polished glass fiber ends, so that on the one hand, the rotation of the two fibers is made possible relative to each other, on the other hand from the Glass fiber 5 emitted laser light virtually lossless in glass fiber 9 can couple. 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. Furthermore, 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 μm 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 μm , 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.

Embodiment 2

In the following, a glass-fiber-based individual jet trap corresponding to the invention will be described, which in 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. By the term 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.

In the focus 29 of the thus modified glass fiber 28, it is possible to catch and orient microscopic particles. Rotation of trapped particles is again effected by the rotation of the laser profile coupled to the optical fiber 28.

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.

Other embodiments are possible, e.g. those generated by the laser beams of laser diodes in close proximity to the sample space and are prepared via suitable optics.

Process Example 1 - Method for the long-term study of zebrafish embryos

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.

However, as the extent of these embryos exceeds the depth of field of conventional microscopes, other methods are required to obtain spatially high resolution images of the samples. Confocal microscopy, which scans the sample into layers by means of a laser beam in order to subsequently combine these into a three-dimensional model, and the use of deconvolution techniques, in which a stack of light-microscopic individual images from parallel focal planes is widespread, are widespread a three-dimensional image can be calculated. Disadvantage of this method is that it sometimes takes several minutes until a picture stack is recorded and can be displayed by a computer. An "on-line screening" of embryo development is therefore not possible.

• Das Verfahren besteht aus den Schritten:
  • Präparation der Zweistahlfalle: Fixierung der die Glasfasern führenden Keramik auf dem Objektträger eines Mikroskops, Adaption des Abstands der Glasfaserenden auf ca. 2mm, Speisung der Glasfasern durch Faserlaser (Ausgangsleistung etwa 2W pro Faser, Wellenlänge 1064nm)
  • Entnahme eines oder mehrerer Embryonen aus der Kultur
  • Gegebenenfalls weitere Vorbehandlung, wie Exposition von Zellgiften, Medikamenten oder anderen Einflüssen gemäß dem Zweck der Untersuchung
  • Großzügige Benetzung der Glasfaserenden mit dem den Anforderungen des Experiments entsprechenden Medium
  • Zugabe eines oder mehrerer Embryonen mit einer weiten Pipette
  • Einfangen eines Embryos in der Falle: In den wenigsten Fälle befindet sich ein Embryo sofort in der Falle. Meistens ist es notwendig, ihn mittels von Mikropipetten ausgehendem Fluss in die Falle zu spülen. Alternativ kann dieser Fluss von einer Sonde verursacht sein, die durch das Medium bewegt wird, den Embryo jedoch nicht berührt.

The method example given below describes how the arrangement described in arrangement example 1 can be used to examine the three-dimensional development of a zebrafish embryo using a conventional light microscope:

• The procedure consists of the steps:
  • Preparation of the two-wire trap: Fixation of the glass fibers leading ceramic on the microscope slide, adaptation of the distance of the glass fiber ends to about 2mm, feeding of the glass fibers by fiber laser (output power about 2W per fiber, wavelength 1064nm)
  • Collection of one or more embryos from the culture
  • If necessary, further pretreatment, such as exposure to cytotoxins, medicines or other influences according to the purpose of the study
  • Generous wetting of the glass fiber ends with the medium corresponding to the requirements of the experiment
  • Add one or more embryos with a wide pipette
  • Trapping an embryo in the trap: In very few cases, an embryo is immediately trapped. Mostly, it is necessary to flush it into the trap by means of micropipettes. Alternatively, this flow may be caused by a probe that is moved through the medium but does not touch the embryo.

Once captured, 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.

For long-term examinations (longer than 30 minutes), it may be useful to replace the medium used continuously using a microfluidic system operated with a syringe pump or to add distilled water to counteract an evaporation-related increase in the concentration of solutes in the medium.

Process Example 2

Rotating suspended, discrete, biological cells for the purpose of computed tomography using a microfluidic system integrated with Example 1 together with a phase-contrast microscope.

The procedure consists of the following steps:

In the arrangement described in Example 1, 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.

• Abstand der Faserenden ca. 250µm
• Laserleistung etwa 100 mW je Glasfaser (nicht gepulst)
• Wellenlänge der verwendeten Laser im nahen Infrarot (z.B. 1064nm)

The preparation of the optical double-jet trap is based on the parameters:

• Distance of the fiber ends approx. 250μm
• Laser power about 100 mW per fiber (not pulsed)
• Wavelength of the used laser in the near infrared (eg 1064nm)

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.

Possible contaminants as well as other cell types are removed from the sample by methods such as density gradient centrifugation or flow cytometry.

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.

If a cell is in the trap, the flow stops.

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.

Software-based, a three-dimensional model of the cell is calculated from the individual images.

LIST OF REFERENCE NUMBERS

1

keramische Glasfaserführung mit zylindrischem Fortsatz

2

Keramikzylinder

3

Keramikhülse verklebt mit (1) als Führung für (2)

4

Schutz des Übergangsstücks (8), sowie mechanisch starre Kopplung von (2) an (11)

5

"single mode" Glasfaser gespeist von Faserlasermodul

6

"multi mode" Glasfaser

7

"single mode" Glasfaser gespeist von Faserlasermodul

8

etwa 2µm versetzter Übergang von Glasfaser (5) zu Glasfaser (6)

9

"single mode" Glasfaser

10

eigentliche Laserfalle, Probenraum

11

Keramikzylinder verklebt mit (9) und (4)

12

Keramikzylinder

13

Keramikhülse oder -führung verklebt mit (12)

14

drehbarer Übergang von (9) zu (5)

15

Objektträger (dünne Glasplatte)

16

Objekt (als Bestandteil eines Mikroskops, optional)

Erläuterung: Die Bauteile 2, 4, 6, 8, 9 und 11 bilden eine starre Einheit, die in Bezug auf den Rest des Systems drehbar ist

21

Keramische Glasfaserführung mit zylindrischem Fortsatz

22

Keramikhülse verklebt mit (21) als Führung für (23)

23

Keramikzylinder, drehbar, darin eingeklebt Glasfaser: (28); mechanisch starre Kopplung von (22) an (31)

24

Keramikhülse verklebt mit (25) als Führung für (23)

25

Keramikzylinder, darin eingeklebt: Glasfaser (26)

26

"single mode" Glasfaser gespeist von Faserlasermodul

27

drehbarer Übergang von (26) zu (28)

28

"single mode" Glasfaser mit asymmetrisch abgerundetem Ende, eingeklebt in (23)

29

aus der Glasfaser austretender, fokussierter Laserstrahl mit leichtem Astigmatismus (eigentliche Laserfalle, Probenraum)

30

Objektiv eines Lichtmikroskops (optional)

31

Objektträger (dünne Glasplatte)

Erläuterung: Die Bauteile (23) und (28) bilden eine starre, in Bezug auf den Rest des Systems drehbare Einheit

32

Miniaturlinse am Ende der Glasfaser (28)

1

ceramic glass fiber guide with cylindrical extension

2

ceramic cylinder

3

Ceramic sleeve glued with (1) as a guide for (2)

4

Protection of the transition piece (8), as well as mechanically rigid coupling of (2) to (11)

5

"single mode" fiber fed by fiber laser module

6

"multi mode" fiber

7

"single mode" fiber fed by fiber laser module

8th

about 2μm staggered transition from glass fiber (5) to glass fiber (6)

9

"single mode" fiber

10

actual laser trap, sample space

11

Ceramic cylinder glued with (9) and (4)

12

ceramic cylinder

13

Ceramic sleeve or guide glued with (12)

14

rotatable transition from (9) to (5)

15

Slide (thin glass plate)

16

Object (as part of a microscope, optional)

Explanation: The components 2, 4, 6, 8, 9 and 11 form a rigid unit which is rotatable with respect to the rest of the system

21

Ceramic fiberglass guide with cylindrical extension

22

Ceramic sleeve bonded with (21) as a guide for (23)

23

Ceramic cylinder, rotatable, glued inside Glass fiber: (28); mechanically rigid coupling of (22) to (31)

24

Ceramic sleeve glued with (25) as a guide for (23)

25

Ceramic cylinder glued into it: fiberglass (26)

26

"single mode" fiber fed by fiber laser module

27

rotatable transition from (26) to (28)

28

"single mode" fiberglass with asymmetric rounded end, glued in (23)

29

Focused laser beam with light astigmatism emerging from the glass fiber (actual laser trap, sample space)

30

Lens of a light microscope (optional)

31

Slide (thin glass plate)

Explanation: The components (23) and (28) form a rigid unit rotatable relative to the rest of the system

32

Miniature lens at the end of the glass fiber (28)

Claims (35)

Device for non-contact manipulation and alignment of sample particles in a measurement volume with the help of an inhomogeneous alternating electric field,
in particular for carrying out the method according to one of claims 14 to 33,
with a radiation source for emitting electromagnetic radiation and
with optical means (6, 5, 26, 28) for guiding the electromagnetic radiation into the measuring volume (10, 29),
characterized,
that as part of the optical means (6, 5; 26, 28) there is a beam-shaping device (8; 28) for generating an asymmetric intensity profile to a beam axis, sample particles in the measuring volume (10; 29) being inhomogeneous by the asymmetrical intensity profile Field distribution of the electric field are trappable, and
that for carrying of trapped in the inhomogeneous field distribution of sample particles, a rotating means (2, 4, 11; 23, 28); is present for rotating the asymmetric intensity profile around the beam axis relative to the measuring volume (29 10). Device according to claim 1,
characterized,
in that the beam-shaping device (8; 28) has optical components with a transmission characteristic which is asymmetrical with respect to an optical axis. Device according to one of claims 1 or 2,
characterized,
in that the optical means for guiding the electromagnetic radiation into the measurement volume (10; 29) comprise optical fibers (6, 5; 26, 28). Device according to one of claims 2 or 3,
characterized,
in that the asymmetrical transmission characteristic is provided by a transition region (14) on which two optical fibers (5, 9) adjoin one another with a radial offset. Device according to one of claims 2 to 4,
characterized,
that the asymmetric transmission characteristic is provided by an asymmetrical completion of an optical fiber (28). Device according to one of claims 1 to 5,
characterized,
that the beam shaping means comprises at least one optical fiber having nichtrotationssymmetrischem profile. Device according to one of claims 2 to 6,
characterized,
that the asymmetric transmission characteristic is provided by astigmatic lenses or mirrors. Device according to one of claims 2 to 6,
characterized,
that the asymmetric transmission characteristic is provided by an asymmetrical panel and / or by variable aperture stops. Device according to one of claims 1 to 8,
characterized,
that the beam shaping device electronically controllable lenses or a spatial light modulator (SLM) having. Device according to one of claims 1 to 9,
characterized,
in that the beam-shaping device (8; 28) can be rotated with respect to the measuring volume (29) with the aid of the rotary device (2, 4, 11; Device according to one of claims 1 to 10,
characterized,
that the rotary device comprises an active polarization device, in particular a Faraday cell. Device according to one of claims 1 to 11,
characterized,
that the electromagnetic radiation from an end of an optical fiber (6; 28) in the measurement volume (10; 29) occurs. Device according to one of claims 1 to 12,
characterized,
in that at least one further radiation source is present to compensate for forces exerted by momentum transfer of photons of the electromagnetic radiation onto the sample particles. Method for the contactless manipulation and alignment of sample particles in a measurement volume with the aid of an inhomogeneous electric field, in particular using the device according to one of claims 1 to 13, in which electromagnetic radiation is conducted into a measurement volume (10; 29) of a sample and
in which sample particles in the measurement volume (10; 29) align themselves in an inhomogeneous electric field of the introduced electromagnetic radiation,
characterized,
in that the electromagnetic radiation introduced into the measurement volume (10; 29) is impressed with an asymmetrical intensity profile to a beam axis which generates an inhomogeneous field distribution of the electric field in the measurement volume (10; 29) in which sample particles are trapped and
in that the asymmetrical intensity profile about the beam axis relative to the measuring volume (10; 29) is rotated to carry sample particles trapped in the inhomogeneous field distribution. Method according to claim 14,
characterized,
that laser light is used as electromagnetic radiation. Method according to claim 14 or 15,
characterized,
that the radiation source is operated continuously. Method according to one of claims 14 to 16,
characterized,
in that, for rotating the intensity profile, an asymmetrically emitting radiation source is rotated relative to the optical means for guiding the radiation into the measurement volume. Method according to one of claims 14 to 17,
characterized,
that a rotation of the asymmetrical intensity profile is accomplished by a rotation of the plane of polarization. Method according to one of claims 14 to 18,
characterized,
in that, to rotate an asymmetrical intensity profile, an asymmetrically emitting light source is selectively modulated. Method according to one of claims 14 to 19,
characterized,
that the sample particles are transported by means of optical tweezers into the effective range of the electromagnetic radiation in the measuring volume. Method according to one of claims 14 to 20,
characterized,
that the sample particles are transported by means of dielectrophoretic forces into the effective range of the electromagnetic radiation in the measuring volume. Method according to one of claims 14 to 21,
characterized,
that the sample particles are introduced by means of a capillary into the measuring volume. Method according to one of claims 14 to 22,
characterized,
that the sample particles are rotated continuously or in defined steps, in particular by leaps and bounds. Method according to one of claims 14 to 23,
characterized,
that as sample particles biological samples, in particular cells, cell organelles or pieces of tissue, are captured and rotated or aligned for examination purposes. Method according to one of claims 14 to 24,
characterized,
that the sample particles are suspended in aqueous media. Method according to one of claims 1 to 25,
characterized,
that sample particles are specifically rotated for microscopy in order to achieve a certain, in particular isotropic, resolution. Method according to one of claims 14 to 26,
characterized,
that a sample particles for processing and / or for the targeted external manipulation, especially for exposure to a micro-tool such as a optical scalpel, a micropipette or a patch clamp, is purposefully manipulated and aligned. Method according to one of claims 14 to 27,
characterized,
that the sample particles for microscopy with different contrasting principles, in particular phase contrast, fluorescence microscopy, ultrasound microscopy, confocal microscopy, CARS and / or light microscopic manipulations, in particular FRAP, un-caging, are selectively positioned and aligned. Method according to one of claims 14 to 28,
characterized,
that one or more particles are rotated to move the surrounding sample medium in rotation. Method according to one of claims 14 to 29,
characterized,
that forces and torques acting on a sample particle positioned in the anisotropic radiation field are measured. Method according to one of claims 14 to 30,
characterized,
that rotation of the sample particles by a hydrodynamic coupling to a in the area of the measuring volume (10; 29) is at least supported rotating optical element, in particular the end of an optical fiber. Method according to one of claims 14 to 31,
characterized,
that elasticity measurements are carried out on aligned sample particles. Method according to one of claims 14 to 32,
characterized,
that a viscosity of the medium surrounding the particle is determined from a maximum possible angular velocity of a particle. Laser scanning microscope, in particular a confocal laser scanning microscope, in particular for carrying out the method according to Claim 35,
which has a device according to one of claims 1 to 13, in particular coupled to such a device. Method for operating a laser scanning microscope, in particular a confocal laser scanning microscope, in particular according to claim 34,
in the sample to be examined particles in a measuring volume using the method according to one of claims 14 to 33 selectively manipulated contactless and aligned and
in which the sample particles to be examined in the measuring volume are examined with the laser scanning microscope.

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