WO2015127940A1

WO2015127940A1 - A detection system for an optical manipulation system for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps

Info

Publication number: WO2015127940A1
Application number: PCT/DK2015/050040
Authority: WO
Inventors: Lene Broeng ODDERSHEDE; Dino OTT; Seyed-Nader SEYED-REIHANI
Original assignee: Københavns Universitet
Priority date: 2014-02-28
Filing date: 2015-02-27
Publication date: 2015-09-03

Abstract

A detection system for detecting light (3a, 4a) comprising a signal from each of at least two optical traps (61, 62) obtained in a sample plane (6), the detection system comprising a detector (11),and a filtering device (9) adapted for spatially filtering the light (3a, 4a) such as to separate the signal (3a) from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps, and wherein the filtering device is arranged in a focal plane being a conjugate plane of the sample plane (6) and an optical manipulation system (1, 100) comprising such a detection system.

Description

A detection system for an optical manipulation system for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps

The present invention relates in a first aspect to a detection system for an optical manipulation system, for manipulating micro-particles or nano- particles of a sample by means of at least two optical traps and to an optical manipulation system with such a detection system. In a second aspect the invention furthermore relates to a method for separating signals in an optical manipulation system for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps.

It is noted that as used herein, the term "optical manipulation system" is intended to include in principle any kind of system employing two or more optical traps for manipulating micro-particles or nano-particles of a sample, but in particular such optical manipulation systems for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps commonly known as dual-trap optical tweezers or multiple-trap optical tweezers.

It is noted that as used herein, the terms "particles", "micro- particles" and "nano-particles" are intended to encompass all types of microscopic and nanoscopic objects and particles and particularly both organic, e.g. cells, and inorganic objects and particles.

Standard dual-trap optical tweezers comprise two single optical traps formed by focusing two laser light beams. The light beams may have two orthogonal polarization states such as to facilitate independent control and detection of each of the traps.

Such optical manipulation systems are advantageous in that they allow for more advanced trapping geometries as compared to an optical ma- nipulation system with only one optical trap, commonly known as a single- trap optical tweezers. This in turn allows for more advanced manipulation and measurements, such as position and force measurements, than possible with single-trap systems. It is important to note that the forces, relative positions and changes in position of the trapped object that it is desired to perform measurements on by means of such optical manipulation systems are extremely small as they relate to micro- or nano-particles and/or systems of such particles. In practice it is desired to measure forces as small as in the order of 10 ¹² Newton (piconewton) or even 10 ¹⁵ Newton (femtonewton) and distances as small as one or a few nanometres with a high degree of precision and accuracy. Consequently even very small errors occurring in the detection process may result in large deviations in the measurement results. In particular systematic errors compromise the accuracy. Therefore a high level of accuracy and precision in the detection process is essential.

It is well known that in optical manipulation systems with at least two optical traps, the measurement results generally comprise a considerable amount of noise, deviation, or systematic errors stemming from spill-over effects or crosstalk between the signals from the individual optical traps. In other words, the signals from the individual optical traps are, to some extent, mixed with each other. This compromises the precision and accuracy of the detection process. It is also well known that a complete separation of the signals from individual traps is quite problematic.

The problems related to spill-over or crosstalk have been attempted solved in several ways.

The most common approach is the use of polarization optics, such as polarizing beamsplitters, to split the signals from two or more individual optical traps if they are formed by orthogonally polarized laser beams. This ap- proach, however, has at least two drawbacks, namely that the splitting of the signals is imperfect, and that a partial depolarization of the light beams may occur. Both of these effects result in a parasitic and erratic signal from one or more optical traps while detecting the other. Especially the partial depolarization may, typically due to the required use of high numerical aperture optics, be a serious source of error resulting in large deviations in force and distance measurements. It has been attempted to rectify the polarization errors described above by so-called beam back propagation, i.e., by leading the signals back through the objective or other focusing device used to focus the beams to form the optical traps.

Other approaches are time-multiplexing the signals and detecting the time-multiplexed signals by fast sequential detection and improved signal processing after detection.

An example of the latter approach is described in WO 2008/145110 Al, in which it is proposed to evaluate measurement data relating to the indi- vidual optical traps of a dual-trap optical tweezers by means of correlation. This particular approach requires, according to the document, a system comprising, i.a., a detector device for each optical trap as well as an evaluation device adapted for evaluation based on correlation.

These approaches, however, necessitate complicated and expensive equipment with many additional components and/or complicated and time- consuming data evaluation processes.

Therefore, there is a desire to provide an alternative and improved system and method for removing or effectively suppressing spill-over or crosstalk, which employs fewer components, which is simple and inexpensive, and which allows for a more straight-forward data evaluation process.

It is the object of the invention to provide a detection system for an optical manipulation system for manipulating micro-particles or nano- particles of a sample by means of at least two optical traps with which spillover or crosstalk may be removed or suppressed, and which is simple and inexpensive and allows for an effective, precise and accurate signal detection and a more straight-forward data evaluation process.

According to the invention, this is obtained by means of a detection system for detecting light comprising a signal from each of at least two optical traps obtained in or adjacent to a sample plane, the detection system comprising a detector and a filtering device adapted for spatially filtering the light such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps, and wherein the filtering device is arranged in a focal plane being a conjugate plane of the sample plane.

By providing a filtering device adapted for spatially filtering the light such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps and by arranging the filtering device in a focal plane being a conjugate plane of the sample plane, a detection system is provided with which spill-over or crosstalk may be removed or suppressed by means of a single component, which is simple and inexpensive and which allows for an effective, precise and accurate signal detection and a more straight-forward data evaluation process.

Furthermore, a detection system is thereby provided with which the precision and accuracy of measurements made by means of an optical manipulation system with at least two optical traps are increased considerably, thus enabling forces as small as in the order of 10 ¹² Newton (piconewton) or even 10 ¹⁵ Newton (femtonewton) and distances as small as one or a few nanometres to be measured with a high degree of precision and accuracy.

Also, a detection system is thereby provided with which the one and same detector may be used for detection and measurement of the separated signal irrespective of which of the at least two optical traps the signal originates from, thus providing a further simplification of the detection system.

In an embodiment the light comprising a signal from each of at least two optical traps is collected by means of a collecting device and the focal plane is created by the collecting device.

Thereby the focal plane is created in a particularly simple way by mans of a single component.

In an embodiment the light comprising a signal from each of at least two optical traps is collected by means of a collecting device, wherein the collecting device comprises a back focal plane, the optical manipulation system further comprises an imaging device adapted for imaging the back focal plane of the collecting device onto the detector, the imaging device providing the focal plane and the filtering device is arranged between the imaging de- vice and the detector in the focal plane created by the imaging device.

Thereby a detection system is provided which allows for a further improvement of the efficiency, precision and accuracy in the signal detection and a particularly straight-forward data evaluation process.

In an embodiment the light comprises a signal from each of at least three optical traps and the filtering device is adapted for spatially filtering the light such as to separate the signal from one of the at least three optical traps at least partially from the signals from the other ones of the at least three optical traps.

In an embodiment the filtering device comprises an opening, the size of the opening being adjustable. Thereby a simplified detection system is provided as it is not needed to change the filtering device if an adjustment of the size of the opening in the filtering device is necessary.

In some embodiments the filtering device is adapted for being mov- able in at least one direction perpendicular to the direction of propagation of the light at the filtering device and/or in a direction parallel to the direction of propagation of the light at the filtering device.

Thereby a detection system is provided with which it is enabled to choose which signal, and thereby which optical trap, is to be detected and measured without having to change the filtering device.

In an embodiment the filtering device comprises an opening having a diameter being 150 μηι or less, a diameter of 100 μηι or less, a diameter of 50 pm or less, a diameter of 20 μηι or less, a diameter of 10 μηι or less or a diameter of 5 μηι or less.

Thereby a detection system is provided with which a particularly efficient signal separation and thus a particularly high degree of crosstalk suppression is obtained. The smaller the pinhole, the more efficient the crosstalk suppression at small trap separation distances d.

In an embodiment the detection system further comprises a further filtering device adapted for polarization-based filtering of the light originating from the sample plane.

Thereby a detection system is provided with which two different techniques for signal separation and thus for crosstalk suppression are combined. This results in the crosstalk being substantially or nearly completely removed. Particularly, as will appear from the detailed description, measurements have shown that with such a detection system crosstalk levels of below 0.5 % and even below 0.2 % may be obtained, depending on the distance d separating adjacent optical traps of the at least two optical traps from one another and on the spatial filtering device chosen.

In an embodiment the further filtering device is arranged close to or in the back focal plane of the collecting device or following the collecting device as seen in the direction of propagation of the light.

Thereby a detection system is provided with which a particularly efficient polarization-based filtering may be obtained.

In an embodiment the detection system further comprises at least one duplicating device adapted for duplicating light originating from the sample plane, at least one further detector, and at least one further filtering device adapted for spatially filtering the light originating from the sample plane such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

In further embodiments comprising an imaging device, the at least one duplicating device is arranged between the collecting device and the imaging device, and the detection system furthermore comprises at least one further imaging device adapted for imaging the back focal plane of the collecting device onto the detector, the at least one further imaging device providing a focal plane being a conjugate plane of the sample plane and the at least one further filtering device is arranged between the at least one further imaging device and the at least one further detector in the focal plane cre- ated by the at least one further imaging device.

By any of these two embodiments a detection system is provided with which simultaneous detection and measurement of signals from different optical traps of the system is enabled.

According to the invention, the above advantages are also obtained by means of an optical manipulation system for manipulating micro-particles or nano-particles of a sample comprising at least one light source adapted for, in operation, emitting light, a focusing device adapted for focusing at least one light beam obtained from the light emitted by the at least one light source onto a sample plane to obtain two or more optical traps in or adjacent to the sample plane, a collecting device, the collecting device being adapted for collecting light originating from the sample plane, the light originating from the sample plane comprising a signal from each of the at least two optical traps, and the optical manipulation system further comprising a detection system comprising a detector and a filtering device adapted for spatially fil- tering the light originating from the sample plane such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps, and the filtering device being arranged between the collecting device and the detector in a focal plane created by the collecting device.

It is noted that the focusing device and the collecting device may be one and the same component or may be two different components.

The detection system may be a detection system according to any of the above-mentioned embodiments.

It is noted that as used herein, the term "sample plane" is intended to refer to the focal plane of the focusing device, the sample to be investigated being placed such that the sample plane extends through the sample.

In an embodiment the focusing device is adapted for focusing at least two light beams obtained from the light emitted by the at least one light source onto a sample plane to obtain two or more optical traps in the sample plane.

According to a second aspect of the invention the object is achieved by a method for separating signals from an optical manipulation system for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps obtained in or adjacent to a sample plane, the method comprising the steps of providing a filtering device, arranging the filtering device in a focal plane being a conjugate plane of the sample plane and, by means of the filtering device, spatially filtering the light such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

According to a third aspect of the invention the object is achieved by a use of a filtering device in detection of signals from an optical manipulation system for manipulating micro-particles or nano-particles of a sample, the optical manipulation system comprising at least two optical traps, for signal separation by spatially filtering light comprising a signal from each of the at least two optical traps such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

The invention will be described in more detail below by means of a non-limiting example of a presently preferred embodiment and with reference to the schematic drawings.

Fig. 1 shows a diagram illustrating a setup of an embodiment of an optical manipulation system for manipulating micro-particles or nano- particles within a sample by means of two optical traps and comprising a detection system according to the first aspect of the invention with a filtering device for spatially filtering light originating from the sample plane. Fig. 2A shows an enlarged view of the sample plane of the optical manipulation system according to Fig. 1.

Fig. 2B shows an enlarged view of a focal plane created by an imaging device, the focal plane being a conjugate plane of the sample plane, and of the filtering device for spatially filtering light originating from the sample plane of the optical manipulation system according to Fig. 1.

Fig. 3 shows two graphs of which graph A illustrates the amount of crosstalk or spill-over between two optical traps of an exemplary setup of an optical manipulation system according to Fig. 1 as a function of the distance between the optical traps in the sample plane when employing a filtering device for spatially filtering light originating from the sample plane comprising openings of different sizes and without polarization-based filtering. Graph B illustrates the -3 dB decay distance, Γ, as a function of the opening diameter p of the filtering device.

Fig. 4 shows two graphs of which graph A illustrates the amount of crosstalk or spill-over between two optical traps of an optical manipulation system according to Fig. 1 as a function of the distance between the optical traps in the sample plane when employing filtering devices for spatially filtering light originating from the sample plane comprising openings of different sizes and for polarization-based filtering simultaneously. Graph B illustrates the improvement obtained by means of the optical manipulation system and the detection system according to the invention for the curves shown in graph A.

Fig. 5 shows a diagram illustrating a setup of an embodiment of an optical manipulation system for manipulating micro-particles or nano- particles of a sample by means of multiple, i.e., more than two, optical traps and comprising a detection system according to the first aspect of the invention.

Fig. 6 shows an enlarged view of the sample plane of the optical ma- nipulation system according to Fig. 5.

Figs. 7-9 illustrate experimental results of experiments performed on an optical manipulation system according to Fig. 5.

More specifically, Fig. 7 shows at the top illustrations A, B and C depicting different experimental conditions used in an optical manipulation sys- tern according to Fig. 5. In condition A only the optical trap of interest con- tains a trapped bead, in condition B all seven optical traps contain trapped beads and in condition C the six optical traps surrounding the optical trap of interest contains beads, while the optical trap of interest is left empty. The central optical trap is selected to be the optical trap of interest, although in principle any of the optical traps could be the optical trap of interest. Below, an overlap of the measured power spectra for conditions A, B and C as a function of frequency is shown. Solid lines are calculated fits to the experimental data, full circles are data points included in the fitting range, open circles are points not included in the fit. The power spectra for conditions A and B clearly overlap within their error margins indicated by dashed lines and the successful fitting of the expected behaviour proves that the traps are fully functional. The signal levels for condition C are orders of magnitude lower, indicating a successful suppression of the signals from the other traps.

Fig. 8 more specifically shows four graphs. The two right graphs show a linear dependency of the difference voltage signal detected on the photodiodes as a function of the lateral (x and y) displacements of the particle in the trap, from this linear region the conversion factor between voltage and meters displacement can be determined. The upper graph shows the linear power dependency of the corner frequency, fc, of the optical trap in the X-direction and the lower graph showing the same but in the Y-direction. Mean and one standard deviation of the measurements (N = 30 per data point) are drawn in black. Full lines are linear ordinary least square fits (R2 > 0.97). The corner frequency f_c is proportional to the spring constant, usually denoted□, characterizing the strength of the optical trap. Hence, the spring constant is proportional to laser power for the multiple traps..

Fig. 9 more specifically shows three graphs illustrating the axial (Z- direction) detection capability. The upper graph shows the fitted power spectrum to the axial position signal data. The inset in the upper graph shows the representative histogram of the axial displacement. The lower left graph shows the linear power dependency of the corner frequency, and hence of the trap stiffness. The lower right graph shows the change of the integrated Quadrant Photodiode signal voltage with Z-displacement where the conversion factor can be determined from the linear region.

Fig. 10 shows a diagram illustrating the steps of an embodiment of a method according to the second aspect of the invention. Fig. 1 shows the setup of a non-limiting embodiment of an optical manipulation system 1 for manipulating micro-particles or nano-particles of a sample by means of two optical traps 61, 62 according to the first aspect of the invention. The optical manipulation system shown in Fig. 1 is thus of the dual-trap type.

The optical manipulation system shown in Fig. 1 comprises a light source 2 emitting at least one beam of light. Seen in the direction of propagation of the light emitted by the light source 2 as illustrated by the arrow 21, the optical manipulation system 1 further comprises a focusing device 5, a collecting device 7 comprising a back focal plane 71 and a detection system comprising a detector 11 and an imaging device 8 imaging the back focal plane 71 of the collecting device 7 onto the detector 11.

Irrespective of the embodiment, the focusing device 5 and the collecting device 7 may be one and the same component or the focusing device 5 and the collecting device 7 may be two different components.

In the following the components of the optical manipulation system 1 and the detection system will be described in general terms with reference to Fig. 1. An example of an optical manipulation system setup with specific types of such components will be described below with reference to Figs. 3 and 4 to illustrate the effects of the invention.

Referring to Fig. 1, the light source 2 emits a Gaussian shaped beam of light and is in the embodiment shown a laser. In principle any type of laser may be used, taking into consideration the type and nature of the sample to be investigated. In principle other types of light sources than lasers may also be used, in which case the light emitted by the light source may be optically manipulated such as to form a beam with a desired intensity profile, such as a Gaussian or a Gaussian-Laguerre shaped beam.

Generally, the focusing device 5 focusses at least one beam obtained from the light source 2 to obtain two or more optical traps. In this case a shaping device adapted for shaping the at least one beam by altering phase and/or intensity may be provided. Such a shaping device may e.g. be one or more spatial light modulators or one or more diffractive optical elements.

In the embodiment shown, however, two beams 3, 4 are obtained from the light source 2.

The two light beams 3, 4 may be obtained from the light emitted by the light source 2 in any way being known as such in the art, any one of which may be employed in an optical manipulating system according to the invention. In one embodiment, an additional optical element, which may comprise one or more components, and which may, e.g., be one or more beamsplitters, may be provided for splitting the beam emitted by the at least one light source into two or more beams for forming the two or more optical traps. In an alternative the two beams of light for forming the two optical traps may be obtained by means of a holographic setup, such setups being known in the art as holographic optical tweezers (HOTs). Yet another alterna- tive is to simply provide two light sources each emitting a beam of light for forming an optical trap.

Furthermore, the two light beams 3, 4 can be provided having orthogonal polarization states.

Referring also to Fig. 2A, the focusing device 5 focusses the two light beams 3, 4 onto a sample plane 6 to obtain two or more optical traps 61, 62 in the sample plane 6 to obtain two or more optical traps 61, 62 in the sample plane 6.

Thus, during investigation of a sample, the sample is placed in or close to the sample plane 6, for instance on a suitable sample stage (not shown). In an embodiment the sample stage is movable in the sample plane for instance by means of an actuator such as a manual actuator, e.g., a micrometer screw, or an automatic actuator, e.g., a piezo-electric actuator.

The two light beams 3, 4 are guided into the focusing device 5 in such a way that the one beam 4 enters the focusing device at an angle of incidence Θ with respect to the direction of propagation of the other beam 3, which is illustrated by means of the arrow 21. Thereby it is obtained that the the optical trap 62 created by means of the beam 4 has a lateral displacement d (cf. Fig. 2A) with respect to the optical trap 61 created by means of the beam 3. The magnitude of the lateral displacement d is dependent on the angle of incidence Θ such that the larger the angle of incidence Θ the larger the lateral displacement d.

The focusing device 5 may in principle be any suitable type of focusing device such as a lens, a system of lenses or an objective. In the embodiment shown the focusing device 5 is a microscope objective.

With reference to Fig. 2A, light originating from the sample plane comprises a signal 3a and 4a, respectively, from each of the two optical traps 61 and 62, respectively.

The light 3a, 4a originating from the sample plane 6 is collected by means of a collecting device 7 comprising a back focal plane 71. The collect- ing device 7 may be any suitable type of collecting device such as a lens, a system of lenses, a condenser or possibly even a collimator. In the embodiment shown the collecting device 7 is a microscope condenser.

The optical manipulation system further comprises a detection system. The detection system comprises in the embodiment shown in Fig. 1 an imaging device 8, a filtering device 9 and a detector 11.

The imaging device 8 is provided for imaging the light 3a, 4a emitted from the sample plane 6 and collected by means of a collecting device 7 onto the detector 11. More precisely, the imaging device images the back focal plane 71 of the collecting device 7 onto the detector 11. Furthermore, the imaging device 8 provides a focal plane 10 which is a conjugate plane of the sample plane 6.

The imaging device 8 may be any suitable type of imaging device capable of providing a focal plane 10. In the embodiment shown the imaging device 8 is a lens. It is noted that the imaging device 8 is, however, an op- tional component.

The detector 11 may be any suitable type of detector. In the embodiment shown the detector 11 is a Quadrant Photodiode (QPD). Alternative detectors are e.g. a camera or a position sensitive detector.

Furthermore, and irrespective of the embodiment, image analysis equipment, such as, e.g., a computer, may be provided for analysing the signals detected by the detector 11.

The detection system further comprises a filtering device 9 spatially filtering the light 3a, 4a emitted from the sample plane 6 such as to separate the signal 3a from the one optical trap 61 from the signal 4a from the other optical trap 62. The filtering device 9 is arranged between the imaging device 8 and the detector 11 in the focal plane 10 created by the imaging device.

Referring also to Fig. 2B the filtering device 9 comprises an opening 91. The filtering device 9 is arranged such that the signal 3a from the optical trap 61 may propagate through the opening 91, while the signal 4a from the optical trap 62 is blocked out. Thus, the filtering device 9 is more specifically arranged such that the beam waist 61a of the signal 3a corresponding to the optical trap 61 is located in the opening 91, while beam waist 62a of the signal 4a corresponding to the optical trap 62 is located next to the opening, such that the beam waist 62a is spatially blocked by the filtering device.

The filtering device 9 may be any type of filtering device enabling spatial filtering of light. In some embodiments the size of the opening 91 of the filtering device 9 is adjustable and/or the filtering device 9 is movable in at least one direction perpendicular to the direction of propagation of the light at the filtering device 9 and/or the filtering device 9 is movable in the focal plane 10. Both an adjustable opening 91 and a movable filtering device 9 may be obtained by means of any suitable type of manual or automatic actuator, examples being a micrometer screw and a piezo-electric actuator.

Furthermore, in some embodiments the opening 91 of the filtering device 9 has a diameter of 150 μηι or less, 100 μηι or less, 50 μηι or less or 20 μηι or less, 10 μηι or less or even 5 μηι or less. The size of the opening 91 of the filtering device 9 may advantageously be chosen in correspondence with the distance d between the two optical traps 61 and 62.

In a specific embodiment the filtering device 9 is a pinhole.

In an alternative embodiment the imaging device 8 of the detection system is omitted. In this embodiment the collecting device 7 provides or creates the focal plane 10 in which the filtering device 9 is arranged.

The detection system of the optical manipulation system 1 shown in Fig. 1 further comprises a further filtering device 12 providing polarization- based filtering of the light 3a, 4a originating from the sample plane 6. It is noted that the further filtering device 12 is an optional element.

The further filtering device 12 is in some embodiments arranged in the back focal plane 71 of the collecting device 7. In other embodiments the further filtering device 12 may be placed in another position between the collecting device 7 and the detector 11.

The further filtering device 12 may be any suitable polarization- based filtering device such as a polarizer. In the embodiment shown the further filtering device 12 is a linear polarizer.

Irrespective of the embodiment the optical manipulation system according to the invention may also optionally comprise a further light source 14 for illumination of the sample plane as well as a dichroic mirror 13 ensur- ing that the light originating from the sample plane is directed towards the detector 11 rather than the further light source 14, while simultaneously letting the light from the further light source 14 pass. Possible alternatives to a dichroic mirror 13 include a beamsplitter.

Irrespective of the embodiment of the optical manipulation system 1,

100, the detection system according to the invention may also optionally comprise at least one duplicating device (not shown) adapted for duplicating light emitted from the sample plane. Such a duplicating the device may for instance be a non-polarizing or a polarizing beamsplitter. The at least one duplicating device can be arranged between the collecting device 7 and the filtering device 9 or the focal plane 10 provided by the collecting device. In such an embodiment the optical manipulation system 1 also comprises at least one further detector and at least one further filtering device adapted for spatially filtering the light originating from the sample plane such as to sepa- rate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

Alternatively, and still irrespective of the embodiment of the optical manipulation system 1, 100, the at least one duplicating device can be arranged between the collecting device 7 and the imaging device 8 where an imaging device 8 is present. The at least one further filtering device may then be arranged between the at least one imaging device and the at least one further detector in the further focal plane created by the at least one imaging device.

In addition to the above, when a duplicating device is provided, the detection system may furthermore comprise at least one further imaging device adapted for imaging the back focal plane of the collecting device onto the detector, the at least one further imaging device providing a further focal plane being a conjugate plane of the sample plane. The at least one further filtering device may then be arranged between the at least one further imag- ing device and the at least one further detector in the further focal plane created by the at least one further imaging device.

EXAMPLE 1

Fig. 3 shows two graphs of which graph A illustrates the amount of crosstalk or spill-over between two optical traps 61, 62 of an optical manipu- lation system 1 according to Fig. 1 as a function of the distance d between the optical traps 61, 62 in the sample plane 6 for a specific setup of the optical manipulation system 1 when employing a filtering device for spatially filtering light originating from the sample plane comprising openings of differ- ent sizes and without polarization-based filtering and graph B illustrates the 3 dB decay distance Γ as a function of the opening diameter p of the filtering device.

The setup of the optical manipulation system 1 is as follows. The two optical traps 61, 62 consist of two expanded Gaussian-shaped beams of a 1064 nm continuous wave Nd :YV0₄-laser having orthogonal polarizations. The focusing device 5 and the collecting device 7 used is a water immersion objective and an oil immersion condenser of a microscope, respectively, and more particularly a water immersion objective (63X, NA= 1.2, Leica HCX PL APO W CORR CS) and an oil immersion condenser (NA= 1.4, Leica SI 551004) of a Leica DM IRBE microscope. The detector 11 used is a Si-PIN Photodiode, and more particularly a Hamamatsu model S5981. The imaging device 8 used is a focusing lens.

Additionally a 1 : 1 telescope was used to enable trap steering of one of the optical traps, namely the optical trap 62 such as to obtain the differ- ence Θ in angle of incidence of the two beams.

The filtering device 9 used is a pinhole, more specifically either a pinhole with an adjustable opening or a number of pinholes with openings of mutually different sizes. Measurements were conducted for pinhole opening diameters p of 10 μηι, 20 μηι, 30 μηι, 50 μηι, 100 μηι and 150 μηι.

In connection with the measurements crosstalk was defined as the quantity Ψ = (S(parasitic) / S(total)). Thus, the crosstalk quantifies the contribution of the parasitic signal, S(parasitic), from the optical trap 62 as measured when the optical trap 61 is turned off in relation to the total measured signal when both optical traps 61 and 62 are turned on and the aim is to measure exclusively the signal originating from the optical trap 61, as this signal contains the desired information.

For both configurations, the transmitted intensities were recorded while varying the distance d between the optical traps 61, 62. This was done by keeping the optical trap 61 at a constant position in the centre of the field of view (FOV), centring the pinhole relative to this trap for maximum trans- mission, and subsequently translating the focus of the optical trap 62 from one edge of the FOV to the opposite one.

The data analysis included subtraction of the photodiode dark current and compensation for a slight power loss (less than 5%) when the optical trap 62 is moved away from the centre of the FOV. This power loss has its cause in the fact that the back focal plane 71 of the collecting device 7 does not coincide with the objective shoulder, but is located slightly inside the collecting device 7 (17.50mm), which leads to a marginal cutting of the impinging beam when being tilted to move the beam laterally in the focal plane. Disregarding this effect would result in an underestimation of the crosstalk when moving towards to the margins of the FOV.

As expected, the maximal crosstalk of 50 % is obtained when the beams overlap perfectly as both beams pass through the pinhole and the signal 4a from the optical trap 62 contributes 50 % to the total signal. With in- creasing distance d the measured crosstalk Ψ decays as shown on graph A in Fig. 3. Notably, the data showed a clear correlation between smaller pinhole opening diameters and more effective crosstalk suppression. To quantify this intensity decay for varying pinhole size a comparison of the distances Γ where the crosstalk Ψ had fallen by 3 dB relative to its maximum value at d = 0 was carried out - cf. graph B in Fig. 3. As may be seen there is a linear proportionality between the distance Γ and the pinhole diameter for diameters larger than 20 μηι, which can be used to estimate the expectable amount of crosstalk and the adequate choice of pinhole size, or in other words the size of the opening 91 of the filtering device 9, when designing experiments.

Fig. 4 shows two graphs of which graph A illustrates the amount of crosstalk or spill-over between two optical traps 61, 62 of an optical manipulation system 1 according to Fig. 1 as a function of the distance d between the optical traps 61, 62 in the sample plane 6 when employing filtering devices for spatially filtering light originating from the sample plane comprising openings of different sizes and for employing polarization-based filtering simultaneously and graph B illustrates the improvement obtained by means of the optical manipulation system and the detection system according to the invention for the curves shown in graph A.

The setup of the optical manipulation system 1 used is identical to that described above in connection with Fig. 3 except for the addition of a further filtering device 12 in the form of a linear polarizer with extinction ratio > 1 : 10⁷, and specifically in the form of a linear polarizer Thorlabs model LPNIR100, arranged in the back focal plane 71 of the collecting device 7 as shown in Fig. 1.

When employing only the linear polarizer and no pinhole for spatial filtering a crosstalk level of 1.1 % was obtained, which is in accordance with comparable prior art results. As expected, this value is not dependent on the distance d separating the traps.

After inserting the pinhole, the crosstalk was measured to further decrease with increasing trap separation d, dropping significantly below the level achievable with the polarization-based filtering alone - cf. graph A in Fig. 4.

It is noted that due to a very low light intensity of the parasitic signal, higher amplification factors provided by means of a signal preamplifier had to be used to perform this measurement. The combination of spatial filtering and polarization-based filtering provided values of crosstalk Ψ below 0.2 % for sufficient trap separation d - cf. graph A in Fig. 4.

To compare the performance of the combination of spatial filtering and polarization-based filtering, Ψ(Ι_Ρ&ΡΗ), with the commonly used polariza- tion-based filtering method, Ψ(Ι-Ρ), one may calculate the improvement Ω as:

Ω = (Ψ(Ι-Ρ) / Ψ(Ι_Ρ&ΡΗ)) - 1.

Thereby the crosstalk levels achieved by the two different methods at a specific trap separation distance d are compared, the result being shown in graph B in Fig. 4). By way of example, cutting the crosstalk by half would correspond to a 100% improvement.

Graph B in Fig. 4 illustrates the fact that the spatial filtering method according to the invention is particularly advantageous for traps separated by a few μηι or more. By combining the signal separation techniques an overall improvement of up to 500% compared to the standard method can be achieved.

Turning now to Fig. 5 a setup of an embodiment of an optical manipulation system 100 for manipulating micro-particles or nano-particles of a sample by means of multiple, i.e. more than two, optical traps according to the first aspect of the invention is shown. The setup shown in Fig. 5 is very similar to that of the optical manipulation system 1 shown in Fig. 1 and described above. Therefore, the optical manipulation system 100 shown in Fig. 5 will be described only with respect to its differences from the optical manipulation system 1 shown in Fig. 1.

First and foremost, the optical manipulation system 100 comprises multiple optical traps, particularly and with reference also to Fig. 6 seven optical traps 61-67.

Generally, the focusing device 5 focusses at least one beam obtained from the light source 2 to obtain three or more optical traps. In this case a shaping device adapted for shaping the at least one beam by altering phase and/or intensity may be provided. Such a shaping device may e.g. be one or more spatial light modulators, one or more elements generalizing phase contrast or one or more diffractive optical elements.

Multiple light beams may be obtained from the light emitted by the light source 2 in several different ways being known as such in the art, any one of which may be employed in an optical manipulating system according to the invention. In one embodiment, an additional optical element, which may comprise one or more components, and which may, e.g., be one or more beamsplitters, may be provided for splitting the beam emitted by the at least one light source into multiple beams for forming the multiple optical traps. In an alternative multiple beams for forming the multiple optical traps may be obtained by means of a holographic setup, such setups being known in the art as holographic optical tweezers (HOTs). Yet another alternative is to simply provide multiple light sources each emitting a beam of light for forming an optical trap.

Furthermore, the multiple light beams may be provided having mutually different polarization or polarization states.

In the optical manipulation system 100 shown in Fig. 5 yet another approach is used to obtain multiple optical traps. Namely, the optical manipu- lation system 100 comprises a shaping device in the form of a diffractive optical element 15, such as a suitable grating, arranged in the light beam 300 of a single light source 2. The light source 2 emits a light beam 300 with a substantially flat wavefront 301, which propagates through the diffractive optical element 15. The diffractive optical element 15 modifies the substan- tially flat wavefront 301 to form a modified wavefront 302 comprising a num- ber of peaks.

Thus, in the embodiment shown the diffractive optical element 15 is adapted for modifying the substantially flat wavefront 301 of the beam 300 to a modified wavefront 302. The modified wavefront 302 then propagates into the focusing element 5 which focusses the beam to form seven optical traps

61- 67. The signal 301a originating from the sample plane 6 therefore comprises seven signal components, one from each of the seven optical traps 61- 67.

The detection system of the optical manipulation system 100 shown in Fig. 5 does not comprise a further filtering device providing polarization- based filtering of the light originating from the sample plane.

In alternative embodiments a further filtering device providing polarization-based filtering of the light originating from the sample plane may, however, be provided. If so, the further filtering device is arranged before the detector 11 which in this embodiment is a photodiode but which may also be a camera.

The detection system of the optical manipulation system 100 further comprises a filtering device 9 comprising an opening 91. The filtering device 9 is arranged such that only one of the seven components, e.g., the compo- nent corresponding to the optical trap 61, of the signal 301a originating from the sample plane 6 may propagate through the opening 91, while the remaining six components, e.g., the components corresponding to the optical traps

62- 67, are blocked out. Thus, the filtering device 9 is more specifically arranged so that the beam waist of the component of the signal 301a corre- sponding to the optical trap 61 is located in the opening 91, while beam waists of the components of the signal 301a corresponding to the optical traps 62-67 are located away from the opening.

The filtering device 9 may be any type of filtering device enabling spatial filtering of light. In some embodiments the size of the opening 91 of the filtering device 9 is adjustable and/or the filtering device 9 is movable in at least one direction perpendicular to the direction of propagation of the light at the filtering device 9 or in other words in the focal plane 10 and/or the filtering device is movable in a direction parallel to the direction of propagation of the light at the filtering device 9. Both an adjustable opening 91 and a movable filtering device 9 may be obtained by means of any suitable type of manual or automatic actuator, examples being a micrometer screw or a piezo-electric actuator.

Furthermore, in one embodiment the opening 91 of the filtering device 9 has a diameter of 150 μηι or less, 100 μηι or less, 50 μηι or less, 20 μηι or less, 10 μηι or less or even 5 μηι or less. The size of the opening 91 of the filtering device 9 may be chosen in correspondence with the distance of separation between each two of the optical traps 61-67.

In a specific embodiment the filtering device 9 is a pinhole.

In an alternative embodiment the imaging device 8 of the detection system is omitted. In this embodiment the collecting device 7 provides or creates the focal plane 10 in which the filtering device 9 is arranged. Also, in this embodiment the collecting device 7 does not necessarily comprise a back focal plane 71. EXAMPLE 2

Fig. 7-9 show different graphs illustrating experimental results of an experiment performed on an optical manipulation system according to Fig. 5.

The optical setup used is essentially the same as described in example 1 above, but used as a multi-trap setup. To this end the multiple optical traps were obtained by phase modulation of the initially flat wavefront of a Gaussian-shaped laser beam from a laser of the type mentioned in example 1 above. Phase modulation was obtained by means of a diffractive optical element 15. The plane of the diffractive optical element 15 was optically conjugated with the back focal plane of the focusing device 5 to ensure an effective conversion of the phase-modulation to the intensity distribution in the focal plane of the focusing device 5.

A filtering device 9 in the form of a pinhole with an opening diameter of 20 μηι was arranged in the plane 10 where an image of the sample plane 6 is formed. Micropositioners, particularly Thorlabs model ST1XY-D, allowed for precise alignment of the pinhole with a specific optical trap of interest. The detector 11 used is a Quadrant Photo Diode (QPD) with four quadrants.

All optical trapping experiments were conducted with the trapping plane far from any surfaces, i.e., 15 μηι above the bottom of the chamber. Optical trap calibration via power spectral analysis was done using a freely available Matlab program . A diffractive optical element, which created seven optical traps arranged as in a hexagonal lattice, i.e., one central optical trap being surrounded by a hexagon of optical traps, was used. The distance between neighbouring optical traps was measured to be 6.7 μηι.

With reference to Fig. 7, the capability to pick out a single optical trap of interest for individual detection was verified. As illustrated in Fig. 7, we compared the optical trap characteristics obtained under three conditions: Condition A where only the optical trap of interest contained a trapped bead, condition B where all seven optical traps contained trapped beads and condi- tion C where the six optical traps surrounding the optical trap of interest contained beads, while the optical trap of interest was left empty.

Comparing of the signal strength in an overlay of individual power spectra revealed that the detection system and method allows for individual detection of the optical trap of interest and efficiently rejects the signal from the remaining optical traps (Fig. 7). The parasitic signal originating from the adjacent optical traps was measured to be orders of magnitude lower than the signal of interest.

With reference to Fig. 8, one important step in a complete optical trap calibration is the determination of the so-called conversion factor, which allows conversion of the measured QPD voltage signals to SI units of distance.

Typically, this conversion factor is assumed to be constant for small displacements of a trapped bead from its equilibrium position, and can be checked by recording the QPD signals while translating a bead, immobilized on the surface of the cover slip, through the focal region of the optical trap. To ensure that this assumption still holds for the detection system and method according to the invention, the relation between the difference pho- todiode voltage signal and lateral displacements was investigated , confirming a linear region which allows for the determination of a simple linear conver- sion factor in the lateral directions - cf. the right graphs in Fig. 8.

A hallmark of the characteristics of an optical trap is the linear relation between the power of the trapping beam and the resulting optical trap stiffness. Figure 8 left shows the result of a total of 3780 recorded and analyzed bead trajectories, confirming the expected linear power dependency between the laser power and the corner frequence, and hence between the laser power and the trap stiffness, for all seven optical traps.

Furthermore, with reference to Fig. 9, the possibility and feasibility to detect the axial Z-displacement of trapped beads without requiring additional modifications of the presented setup was demonstrated. A linear relationship between the integrated photodiode voltage signal and axial displacement was established, thus allowing for the determination of an axial conversion factor - cf. Fig. 9 lower right graph. Hence, the analysis of fluctuations of the summed signal from the photodiode can be used for axial calibration of the optical trap along the optical axis of the focusing device 5 and the collecting device 7. Power spectra (Fig. 9, upper graph) that were fitted perfectly by theoretical expectation were obtained also for the axial direction. Again, the experiments revealed a linear power-dependency of the optical trap corner frequency and hence of stiffness - cf. Fig. 9 lower left graph - and constant conversion factors- cf. Fig. 9 lower right graph.

In the following and with reference to Fig. 10 a method for separating signals from an optical manipulation system 1, 100 for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps obtained in or adjacent to a sample plane 6 according to the second aspect of the invention will be described. It is noted that steps 500, 501, 502, 503 and 504 shown in Fig. 10 are optional steps, as will be apparent from the below.

The method comprises:

a step 505 of providing a filtering device 9,

a step 506 of arranging the filtering device 9 in a focal plane 10 being a conjugate plane of the sample plane 6and,

a step 507 of, by means of the filtering device 9, spatially filtering the light such as to separate the signal 3a from one of the at least two optical traps 61 from the signals 4a from the other one(s) of the at least two optical traps 62.

The method may furthermore comprise:

a step 501 of providing at least one light source 2 adapted for, in operation, emitting light, and

a step 502 of providing a focusing device 5 and, by means of the fo- cusing device 5, focusing at least two light beams 3, 4 obtained from the light emitted by the at least one light source onto a sample plane 6 to obtain two or more optical traps 61, 62 in the sample plane 6.

The method may furthermore comprise a step 503 of providing a collecting device 7, and, by means of the collecting device 7, creating the focal plane 10 and collecting light 3a, 4a comprising a signal 3a and 4a, respectively, from each of at least two optical traps 61, 62,

In embodiments where the collecting device 7 comprises a back focal plane 71, the method may furthermore comprise a step 504 of providing an imaging device 8 and, by means of the imaging device 8, imaging the back focal plane 71 of the collecting device 7 onto the detector 11 and creating a focal plane 10 being a conjugate plane of a sample plane 6 in which the two or more optical traps are obtained. In this embodiment the filtering device 9 may be arranged in the focal plane 10 created by the imaging device 8.

The method may furthermore comprise a step 500 of providing a de- tector 11, and the step of detecting the spatially filtered signal 3a with the detector 11.

The method may furthermore comprise the step of analyzing the detected spatially filtered signal 3a by means of suitable analyzing equipment.

The method may furthermore comprise the step of providing the fil- tering device 9 with an opening 91 and optionally of adjusting the size of the opening 91.

The method may furthermore comprise the step of moving the filtering device 9 in at least one direction perpendicular to the direction of propagation of the light at the filtering device 9, or in other words in at least one direction in the focal plane 10, and/or in a direction parallel to the direction of propagation of the light at the filtering device 9.

The method may furthermore comprise the steps of providing a further filtering device 12 for polarization-based filtering of the light 3a, 4a originating from the sample plane 6 and performing polarization-based filter- ing of the light 3a, 4a originating from the sample plane 6 such as to separate the signal 3a from one of the at least two optical traps 61 from the signals 4a from the other of the at least two optical traps 62.

The method may furthermore comprise the step of arranging the further filtering device 12 in the back focal plane 71 of the collecting device 7.

The method may furthermore comprise the step of providing at least one duplicating device adapted for duplicating light 3a, 4a originating from the sample plane 6.

The method may furthermore comprise the step of arranging the at least one duplicating device between the collecting device 7 and the filtering device 8 and duplicate the light 3a, 4a originating from the sample plane 6.

The method may furthermore comprise the steps of providing a further detector, providing a further imaging device and, by means of the further imaging device, imaging the back focal plane 71 of the collecting device 7 onto the further detector and creating a further focal plane being a conju- gate plane of the sample plane 6, providing a further filtering device, arranging the further filtering device in the further focal plane and, by means of the further filtering device, spatially filtering the light originating from the sample plane 3a, 4a such as to separate the signal 3a from one of the at least two optical traps 61 from the signals 4a from the other one(s) of the at least two optical traps 62.

It should be noted that the above description of preferred embodiments serves only as examples, and that a person skilled in the art will know that numerous variations are possible without deviating from the scope of the claims.

Claims

P A T E N T C L A I M S

1. A detection system for detecting light (3a, 4a) comprising a signal from each of at least two optical traps (61, 62) obtained in a sample plane (6), the detection system comprising :

a detector (11), and

a filtering device (9) adapted for spatially filtering the light (3a, 4a) such as to separate the signal (3a) from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps, and wherein the filtering device is arranged in a focal plane be- ing a conjuagte plane of the sample plane (6).

2. A detection system according to claim 1, wherein the light (3a, 4a) comprising a signal from each of the at least two optical traps (61, 62) is collected by means of a collecting device (7), wherein the filtering device is arranged between the collecting device and the detector and wherein the fo- cal plane (10) is created by the collecting device.

3. A detection system according to claim 1, wherein wherein the light (3a, 4a) comprising a signal from each of at least two optical traps (61, 62) is collected by means of a collecting device (7), wherein the collecting device comprises a back focal plane (71), wherein the detection system further comprises an imaging device (8) adapted for imaging the back focal plane of the collecting device onto the detector, the imaging device providing the focal plane (10), and wherein the filtering device is arranged between the imaging device and the detector in the focal plane created by the imaging device.

4. A detection system according to any one of the above claims, wherein the filtering device (9) comprises an opening (91), the opening having a diameter being 150 μηι or less, a diameter of 100 μηι or less, a diameter of 50 pm or less, a diameter of 20 μηι or less, a diameter of 10 μηι or less or a diameter of 5 μηι or less and/or the size of the opening being adjustable.

5. A detection system according to any one of the above claims, wherein the filtering device is adapted for being movable in at least one direction perpendicular to the direction of propagation of the light at the filtering device and/or in a direction parallel to the direction of propagation of the light at the filtering device.

6. A detection system according to any one of the above claims, and further comprising a further filtering device (12) adapted for polarization- based filtering of the light originating from the sample plane.

7. A detection system according to any one of the above claims, and further comprising at least one duplicating device adapted for duplicating light originating from the sample plane, the at least one duplicating device being arranged between the collecting device and the filtering device, at least one further detector, and at least one further filtering device adapted for spatially filtering the light originating from the sample plane such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

8. An optical manipulation system (1, 100) for manipulating micro- particles or nano-particles of a sample, the optical manipulation system comprising :

at least one light source (2) adapted for, in operation, emitting light, a focusing device (5) adapted for focusing at least one light beam obtained from the light emitted by the at least one light source onto a sample plane (6) to obtain two or more optical traps (61, 62) in or adjacent to the sample plane,

a collecting device (7) adapted for collecting light originating from the sample plane, the light originating from the sample plane comprising a signal from each of the at least two optical traps, wherein

the optical manipulation system further comprises a detection system comprising a detector (11) and a filtering device (9) adapted for spatially filtering the light originating from the sample plane such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps, and wherein the filtering device is arranged between the collecting device and the detector in a focal plane created by the collecting device.

9. A method for separating signals from an optical manipulation system (1, 100) for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps (61, 62) obtained in or adjacent to a sample plane (6), the method comprising the steps of:

providing a filtering device (9),

arranging the filtering device in a focal plane (10) being a conjugate plane of the sample plane, and,

by means of the filtering device, spatially filtering the light such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.

10. A method according to claim 9, and comprising the further steps of providing a collecting device (7) and, by means of the collecting device, creating the focal plane and collecting light comprising a signal from each of at least two optical traps,

11. A method according to claim 10, where the collecting device comprises a back focal plane (71), and the method further comprises the steps of:

providing a detector (11),

providing at least one light (2) source adapted for, in operation, emitting light,

providing a focusing device (5) and, by means of the focusing device, focusing at least one light beam obtained from the light emitted by the at least one light source onto a sample plane to obtain the two or more optical traps in the sample plane,

providing an imaging device (8) adapted for imaging the back focal plane of the collecting device onto the detector, the imaging device providing a focal plane (10) being a conjugate plane of the sample plane, and

arranging the filtering device (9) between the imaging device and the detector in the focal plane created by the imaging device.

12. Use of a filtering device in detection of signals from an optical manipulation system for manipulating micro-particles or nano-particles of a sample, the optical manipulation system comprising at least two optical traps, for signal separation by spatially filtering light comprising a signal from each of the at least two optical traps such as to separate the signal from one of the at least two optical traps at least partially from the signals from the other one(s) of the at least two optical traps.