WO2009065519A1

WO2009065519A1 - Method for measurement of the force which acts on an object which is held in optical tweezers/an optical catch, and optical tweezers/an optical catch

Info

Publication number: WO2009065519A1
Application number: PCT/EP2008/009582
Authority: WO
Inventors: Andy Sischka; Dario Anselmetti
Original assignee: Universität Bielefeld
Priority date: 2007-11-20
Filing date: 2008-11-13
Publication date: 2009-05-28
Also published as: DE102007055598A1; WO2009065519A8

Abstract

The invention relates to a method for measurement of the force which acts on an object which is held in optical tweezers/an optical catch, in which the light which is scattered back from the held object through the objective of the optical tweezers/catch is detected by a detector and the detector signal is used to determine the force in the case of which a laser beam (1) which is used to produce the optical tweezers/catch and is polarized linearly in a first direction passes through a polarizing beam splitter (2) then passes through a polarization-rotating element (8) and an objective (3), wherein the laser beam (1) is circular-polarized by the polarization-rotating element (8) and is focussed by the objective (3) and holds an object in a focus area, and wherein, furthermore, light which is scattered back from the held object through the objective (3) from the same laser beam (1) passes through the polarization-rotating element (8) in the opposite direction, is linear-polarized by this polarization-rotating element (8) in a second direction at right angles to the first direction, and passes through the polarizing beam splitter (2), wherein the polarizing beam splitter (2) separates the illuminating laser beam (1) and the back-scattered laser light, and the back-scattered laser light is detected by means of the detector (7). The invention also relates to optical tweezers/an optical catch having an objective (3) for focussing of laser radiation (1) for holding an object in the focus area of this laser radiation (1) and to a detector (7) for detection of a detector signal produced by the laser light which is scattered back onto the detector (7) from an object through the objective (3), wherein a force which acts on an object can be determined on the basis of the detector signal, which force comprises, in a common area of the beam path of the incident laser beam and the back-scattered laser beam, a polarizing beam splitter (2) and a polarization-rotating element (8) which follows the beam splitter (2) in the direction of the illuminating laser beam (1), in order to form an optical switch between incident laser radiation (1) and the laser light which is scattered back on the object through the objective (3), wherein the detector (7) is arranged in the beam path of the back-scattered light, which beam path is separate from the incident laser light (1).

Description

Method for measuring the force acting on an object caught in an optical tweezer / trap and optical tweezers / trap

The invention relates to a method for measuring the force acting on an object trapped in an optical tweezers / trap in which light scattered back from the object being trapped by the optical tweezer / trap lens is detected by a detector and determines the force from the detector signal becomes.

The invention furthermore relates to an optical tweezers / trap, comprising an objective for focusing a laser radiation for capturing an object in the focal region of this laser radiation and having a detector for detecting a detector signal which generates the laser light backscattered by an object through the objective onto the detector; wherein based on the detector signal acting on an object force can be determined.

When using optical traps or optical tweezers, which are essentially synonymous terms for the same arrangement, objects for observation or manipulation are caught in a strong laser light, here in particular in the focal range of a laser beam, the focus being produced by a correspondingly suitable objective. In this case, it is of interest to measure the forces acting on the trapped object by measurement. It is known to perform a metrological detection by means of the backscattered at the trapped object light.

Such a measurement in back reflection is known in the art, wherein in a possible application of the original laser beam in back reflection is also used as a detector beam. This method is based on the fact that the laser beam first passes through a beam splitter, which is intended to deflect the backscattered light from the direction of backscattering. In this case, known conventional beam splitters are used, usually with a beam splitting ratio of 50 to 50, so that on the one hand, the irradiated to generate the optical trap laser beam is attenuated, since only the transmitted portion is available for the generation of the trap and also has the disadvantage in that the backscattered light in the beam splitter is also attenuated, since only the reflected component is reflected by the backscattered component in accordance with the beam splitter ratio of the beam splitter used, that is, for example, also only 50%. Thus, for measurement purposes, only very low beam intensities are available at the detector used, so that usually only force measurements in one or two dimensions were possible by means of such arrangements.

In order to perform force measurements in up to three dimensions in reflective geometry, a further device or method is known, according to which a laser beam generating the trap is beamed into the optical trap in an exactly parallel manner into this optical path, with a very low intensity Object backscattered light of this additional laser beam is detected and evaluated.

This can for example take place in that a separate laser beam is used with a shorter wavelength, this laser light, for example, by means of a dichroic filter on and the backscattered light from the beam path of the laser beam forming the trap can also be decoupled again. However, this separate laser beam, which serves as a separate detection beam, must also pass through a beam splitter in order to be directed to the dichroic mirror and also to transmit the returning light on the return path so that this light can fall on the detector and not into the generating laser runs back. The disadvantage of such an arrangement or this known method is that a very precise adjustment relative to the first laser beam must be made, since usually deflections in the nanometer range must be detected and can also be seen in that the second laser beam, which serves as a detection beam, may only have a very low intensity so as not to form an optical trap itself.

The object of the invention is therefore to provide a method for measuring the force by evaluating the backscattered on an object in the case laser light of the illuminating laser beam and thus with a so-called single-beam optical tweezers, which does not have the aforementioned features, provides higher overall intensities , provides a high trap quality and requires less adjustment accuracy compared to the prior art. The object is furthermore to provide a suitable optical tweezers or an arrangement of optical elements for forming such an optical tweezers or trap.

According to the invention this object is achieved in that a used for generating the optical tweezers / trap, in a first direction linearly polarized laser beam passes through a polarizing beam splitter, then passes through a polarization rotating element and an objective, the laser beam through the polarization rotating element circularly polarized and through the lens is focused and captures an object in the focus area and further wherein the object trapped by the object backscattered light of the same laser beam, the polarization rotating element in the reverse direction is linearly polarized by this polarization rotating element in a second direction perpendicular to the first direction and the polarizing beam splitter happens, wherein the polarizing beam splitter the illuminating laser beam and the backscattered laser light separates and the backscattered laser light is detected by the detector. The object is further achieved by an optical tweezers / trap of the aforementioned type, which in a common region of the beam path of incident and backscattered laser beam a polarizing beam splitter and this in the direction of the illuminating laser beam subsequent polarization rotating element umfas'st to form an optical switch between incident laser radiation and the laser light scattered back on the object by the objective laser light of the same laser beam, wherein the detector is arranged in the separate from the incident laser light beam path of the backscattered light.

The essential core idea of this method or of the named device is therefore to use the original laser beam used to form the trap as well as a detection beam without using a necessarily attenuating beam splitter, which is realized here in that the used beam splitter compared to Prior art has polarizing properties, ie the laser light of an excellent linear polarization direction can pass through the beam splitter without being deflected and laser light of a perpendicular linear polarization is reflected by the beam splitter out of the beam direction.

Thus, such a beam splitter, which may be formed for example as a beam splitter cube, serve as an optical switch for laser beams of mutually perpendicular Poiarisationsrichtungen.

In order to achieve that the illuminating laser beam incident in the trap has a different linear polarization than the light scattered back from the trap at the object at the location of the beam splitter, it is further provided according to the invention, in the direction of the incident illuminating laser beam after this beam splitter, a polarization-rotating element in that, for example, a lambda quarter plate is used to move the original illuminating laser light from the first linear polarization into a circular polarization corresponding to the direction of rotation of the polarization-rotating element, ie For example, to convert a left or a right circular polarization. Accordingly, according to the invention, circularly polarized light in the optical trap or tweezers in the focus area is used for holding an object by the following objective, whereas linearly polarized light is used in the usual way in the prior art.

This results in a particular advantage over the usual known methods, since the circularly polarized light generates an optical trap having a fully isotropic trapping strength laterally. In contrast, an optical trap or tweezers, based on linearly polarized light, as used in the prior art, parallel to the polarization direction have a different trap strength than perpendicular to the polarization direction.

The backscattered at the trapped object light has now due to its reverse propagation direction just as reversed circularity of the polarization, this backscattered light after passing through the lens in the rearward direction again the aforementioned polarization-rotating element, ie in particular a quarter-wave plate passes through and due to the inverted circular Polarization is not zurücksondern but further rotated, so that after passing this element, the backscattered light has a linear polarization, which is arranged with exact adjustment exactly 90 degrees relative to the linear polarization of the incident laser beam.

Thus, the backscattered light is separated from the originally incident laser light by the polarizing beam splitter, which now follows the polarization-rotating element in the rear beam path and can fall in a separate beam path on the detector, where this backscattered laser light detected and a corresponding signal is formed, which with regard to the force, in particular with regard to at least two, preferably of three dimensions can be evaluated. Thus, in this arrangement, the polarizing beam splitter and the polarization-rotating element form an optical switch, which separates the beam path of the incident laser light from the beam path of the backscattered laser light at the location of the beam splitter.

This can be provided in a preferred embodiment such that the linearly polarized in the first direction laser light passes through the polarizing beam splitter in transmission and the backscattered laser light is reflected by the polarizing beam splitter. Of course, it is possible in an analogous manner to align the arrangement inversely with respect to the two beam paths.

The polarizing beam splitter has the particular advantage over conventional beam splitters that, in the aforementioned embodiment of the beam splitter in transmission for the illuminating laser beam, this laser light substantially passes through the beam splitter to a proportion of greater than 95% and the backscattered laser light becomes one Share of greater than 95%, optionally greater than 98% reflected. Intensity losses due to such a polarizing beam splitter element are therefore negligible in the method according to the invention or the optical tweezers according to the invention, so that, compared with the arrangements in the prior art, significantly higher intensities of the backscattered light are present at the detector.

The intensity of the incident light on the detector is therefore only a few percent less than the proportion of the light scattered back to the trapped object, especially where the losses are typically in the range of a few percent, whereas in the prior art methods the intensities would be attenuated to less than a quarter, namely half of the first passage through the beam splitter and again by half in the repeated passage of the backscattered light plus any further reflection and absorption losses. In a method or the device according to the invention it can be provided that in the beam path both the illuminating laser beam and the backscattered laser light, a telescope, in particular one and the same telescope is used, which may be constructed, for example, of lenses or from concave mirrors. Such a telescope is intended to widen the incident laser beam to produce the optical trap before passing through the lens. In the case of optical tweezers, the laser beam should have a diameter which is approximately 20-50% larger than the rear aperture of the trapping objective used. For this purpose, the laser beam must be widened in typical cases to about 10 millimeters, which is to be understood here only as an example and not limiting for the invention.

In this case, provision may be made for the polarizing beam splitter to be introduced into the beam path where the laser beam has already been widened, i. after such a telescope arrangement. In such a case it can be provided that a four-quadrant detector is used as the detector for detecting the backscattered light in order to carry out the measurement of the force.

In a contrast preferred embodiment, however, it may also be provided to arrange the polarizing beam splitter between the generating laser and the expanding telescope. In this case, the telescope arrangement thus acts for the incident, forming the trap laser beam straightening while reducing the divergence, whereas for the backscattered light telescope this arrangement reduces the beam while increasing the deflections that has experienced the backscattered light on the trapped object.

Thus, this telescope arrangement thus amplifies the lateral deflection of the backscattered light by the magnification factor of the expanding system in the incident laser light beam path, which increases the sensitivity of the detector to the smallest deflections of the trapped object and thus also the force resolution. The light spot of the backscattered light incident on the detector in this case is reduced almost exactly by the magnification factor of the beam widening, but the lateral deflection is increased by almost exactly this factor as well.

Therefore, it is preferably provided in such an arrangement, instead of a quadrant detector to use a linear detector, which detects the usual lateral deflections of the backscattered beam with its sensitive detector surface and preferably generates over its entire detection range a signal that is proportional to the lateral deflection of the beam. This signal can be used to deduce the force in terms of magnitude and direction.

In order to avoid disturbing residual reflections on the surface of the polarization-rotating element facing the lens, for example the lambda quarter plate, it can preferably be provided to slightly tilt this element in the beam path, at least so that back reflections are outside the detector range.

With the inventive method or apparatus for measuring the force acting on a trapped object as described herein, the force in the Z direction, i. in the direction of the optical axis of the incident laser light of a trapped object. For this purpose, for example, the total intensity of the incident light on the detector can be measured and compared with a reference, if no external force in the Z direction acts on the captured object.

For example, if a Z-direction force deflects the lens somewhat up or down out of the trap, the angle of the beam cone of the backscattered light will be somewhat reduced or increased, resulting in the trap lens being more or less well-scattered light As a result, less or more scattered light through the edges of the objective lenses and the rear aperture of the lens dimmed. This results in an increase or even reduction in the intensity of the backscattered light, which can be evaluated to infer the force or direction of the acting force.

In addition to the advantages set out, the method according to the invention or the device is also very unadjustable to adjustment, since the adjustment is maintained even during a movement of the objective, for example during a focusing, as in an inverted microscope.

Another particular advantage of the arrangement of the polarizing beam splitter between the laser light generating laser and the elements for beam expansion of the incident laser beam is that in such an arrangement in a preferred embodiment, the distance of the detector to the lens or focus can be set the same as the distance of the lens or focus to a pivot point of a pendulum motion of the illuminating laser beam.

Thus, it is known that a laser beam generated by a laser does not have an exactly constant propagation direction, but performs a pendulum motion, this pendulum motion being around a pivot point. This movement is also referred to as the so-called beampoint instability of the laser. Such a pendulum movement can therefore also lead to a falsification of the measurement results.

By the distance symmetry mentioned here can be achieved that the fulcrum of the pendulum movement of the backscattered light comes to rest exactly on the detector, which makes the beampoint instability of the generating laser no longer noticeable and thus the resolution and quality of force measurement are significantly increased can. The advantages of the method mentioned here and the optical tweezers are summarized that according to this invention no longer a second, difficult to be adjusted detection beam is required, therefore, no second optical trap is generated and it no longer to a significant agreement of the used Laser beams due to the beam splitter used, since this is performed according to the invention polarizing.

In addition, a method of the type described herein for evaluating the backscattered light also has the advantages that compared to other methods that make measurements on the basis of the light transmitted to the trapped object, now need no adjusting measuring devices on the optical trap. The space above an optical trap thus remains free for arbitrary experimental setups.

In the case of the invention proposed here, the movement of the trapping objective no longer leads to readjustment, which would otherwise be required if, in the case of a force measurement in transmission after the object, another objective provided for the measurement would have to be used, which is always confocal with the other Lens to arrange would be. Thus, with respect to such a confocal arrangement eliminates any impairment, for example, due to mechanical vibrations.

Falsifications of the measurement result by Schmutzteiichen, which adversely affect in a forward scattered force measurement, omitted, since these dirt particles, which usually collect at the top of the captured object due to the radiation pressure, for a measurement with backscatter irrelevant. Just the symmetrical structure, as mentioned above, with respect to the pivot point of the laser or its Beampointinstability thereby decisively improves the force resolution and thus both the measurement of the direction of the force in up to three dimensions as well as the measurement of the amount of forces in these dimensions. An embodiment of the invention in comparison with a known prior art show the following figures. Show it

Figure 1 shows an arrangement for measuring the force with backscattered

Light of a second separate detection beam Figure 2 shows an arrangement for carrying out the inventive

process

FIG. 1 shows a typical structure of an optical trap formed by a laser beam 1, which here passes through a lens 3 through a beam splitter 2 in order to be focused through it and in the focal region 4 to an optical trap for capturing an object not shown here form. By a lateral beam path, a laser beam 5 is first coupled in parallel by a beam splitter 6 and the beam splitter 2 mentioned in the beam path of the laser beam generating the trap 1 and also passes through the trap lens 3 and thus through the formed optical trap, where this detection beam 5 as well meets the captured object and is back-scattered at this.

The backscattered light is in turn reflected back after passing through the trap lens at the beam splitter 2, passes through the beam splitter 6, optionally a subsequent filter and incident on a detector 7 for receiving a detection signal and evaluation for determining the forces. The arrangement shown here clearly shows the disadvantages of two beam splitters 2 and 6, at each of which significant reflection losses occur, wherein furthermore the additional detection laser beam 5 optionally generates a second optical trap which falsifies the measurement result. In addition, the beam path of the detection beam 5 must be adjusted very accurately with respect to the beam path of the laser beam 1 in order to operate this measuring device. In contrast, Figure 2 shows a structure for forming an optical tweezers or case according to the invention and for carrying out the method according to the invention.

It can be seen here that a laser beam 1, the only laser beam in this structure, which passes through the polarizing beam splitter 2 unhindered with an exemplary P polarization and passes through a subsequently arranged lambda quarter plate 8 with this P polarization, which is adjusted so that the incident Laser beam is right circular polarized now in this embodiment.

The circularly polarized laser light 1 then passes through the trap lens 3, is thereby focused and thus forms the optical trap for capturing an object, not shown here in the focal plane F. The backscattered on a trapped object light also passes through in a rear beam path falling Objective 3, is now polarized left circular due to the exact reverse propagation direction, passes through the lambda quarter plate 8 in the rearward optical path, so that thereafter results in an S-polarized linear polarization of the backscattered light.

The backscattered light and the originally irradiated laser light of the same laser therefore have polarizations which are perpendicular to one another, which means that the backscattered light which is now polarized in S polarization is reflected at the polarizing beam splitter 2 out of the beam path in the direction of the right side in this illustration In this case, the backscattered light can also pass through a filter 9 for blocking ambient light, which only allows the backscattered light to pass.

Visible here is the particular advantage of the arrangement according to the invention, according to which the polarizing beam splitter in conjunction with the lambda quarter plate forms an optical switch for separating the originally incident Laser light for the formation of the optical trap and the backscattered light, without significant reflection losses, which amount to only a few percent at most due to surface losses. Thus, such an arrangement of Figure 2 over that of Figure 1 in the prior art has a lower adjustment sensitivity and offers higher intensities, which can be used for the measurement.

Claims

claims

A method of measuring the force acting on an object caught in an optical tweezer / trap wherein light scattered back from the trapped object by the optical tweezer / trap lens is detected with a detector and the force is determined from the detector signal characterized in that a laser beam (1) linearly polarized in a first direction for generating the optical tweezers / trap passes a polarizing beam splitter (2), then passes through a polarization rotating element (8) and a lens (3), the laser beam (1) is circularly polarized by the polarization-rotating element (8) and focused by the objective (3) and captures an object in the focal region and further wherein light from the trapped object through the lens (3) backscattered light of the same laser beam (1) the polarization-rotating element (8) passes in the reverse direction, through this polarization rotating element (8) in a second direction is linearly polarized perpendicular to the first direction and passes the polarizing beam splitter (2), wherein the polarizing beam splitter (2) the illuminating laser beam (1) and the backscattered laser light separates and the backscattered laser light is detected by the detector (7).

2. The method according to claim 1, characterized in that in the first direction linearly polarized laser light (1) passes through the polarizing beam splitter (2) in transmission and the backscattered laser light from the polarizing beam splitter (2) is reflected.

3. The method according to any one of the preceding claims, characterized in that in the beam path of the illuminating laser beam (1) after the polarizing beam splitter (2) a laser beam expanding optical system is arranged, which is traversed in the reverse direction of the reflected laser light.

4. The method according to any one of the preceding claims, characterized in that a linear detector is used as the detector (7) whose signal depends linearly on a deflection of the reflected laser light, in particular with respect to an undeflected reference position.

5. The method according to any one of the preceding claims, characterized in that the total intensity of the backscattered laser light is measured by the detector (7) and from this a force in or against the direction / Z direction of the illuminating laser beam in the case / tweezers is determined.

6. The method according to any one of the preceding claims, characterized in that the distance of the detector (7) to the lens (3) or focus is set equal to the distance of the lens (3) or focus to a pivot point of a pendulum movement of the illuminating laser radiation.

7. Optical tweezers / trap comprising a lens (3) for focusing a laser radiation (1) for capturing an object in the focal region of this laser radiation (1) and with a detector (7) for detecting a detector signal, which of an object through the Lens (3) to the detector (7) backscattered laser light generated, wherein based on the detector signal acting on an object force can be determined, characterized in that they in a common region of the beam path of incident and backscattered laser beam, a polarizing beam splitter (2) and a polarization - rotating element (8) which follows this in the direction of the illuminating laser beam (1) comprises an optical switch between incident laser radiation (1) and the laser light scattered back at the object by the objective (3), the detector (7) is arranged by the incident laser light (1) separate beam path of the backscattered light.