The present invention is related to a device for determining the position of a plurality of obj ects that are simultaneously trapped by a single laser beam . In particular , the device according to the invention makes it possible to determine the external forces applied to the obj ects .

Optical tweezers (originally called single-beam gradient force trap) are scientif ic instruments that use a highly focused laser beam to provide an attractive or repulsive force ( typically on the order of piconewtons ) , depending on the relative refractive index between particle and surrounding medium; these forces can be used to physically hold and move microscopic obj ects , in a manner similar to tweezers . They are able to trap and manipulate small particles , whose size is typically in microns , including dielectric and absorbing particles . Optical tweezers have been particularly successful in studying a variety of biological systems in recent years .

As shown in f igure 1 and f igure 2 , optical tweezers are capable of manipulating nanometer and micron- sized dielectric particles 1 by exerting extremely small forces via a highly focused laser beam 2 extending along an optical axis Z . The beam 2 is typically focused by sending it through a microscope obj ective . The narrowest point of the focused beam, known as the beam waist , contains a very strong electric f ield gradient 3 . Dielectric particles are attracted along the gradient to the region of strongest electric f ield, which is the center of the beam . The laser light also tends to apply a force on particles in the beam along the direction of beam propagation . This is due to conservation of momentum : photons that are absorbed or scattered by the tiny dielectric particle impart momentum to the dielectric particle . This is known as the scattering force 4 and results in the particle being displaced slightly downstream from the exact position of the beam waist , as seen in f igure 1 .

In an optical trap , the tightly focused laser beam provides equilibrium position near the beam waist . When the trapped obj ects 1 are laterally or axially displaced away from the equilibrium position, the optical forces act to pull it back towards the equilibrium position . For small displacements , the force applied to the particle is a linear function, and the optical trap can be compared to a simple spring system, which follows Hooke ' s law . In this model the restoring optical force F is proportional to the distance from its equilibrium position, and the trap stif fness is proportional to the light intensity . Considering the motion range and the trap stif fness , three-dimensional pico-Newton resolution is reachable .

This particularity has led to the use of optical trapping for quantitative force measurements , such as the strength of inter-molecular bonds , the stif fness of a cell membrane or intracellular measurements for micro- rheology .

Force calibration of arbitrary obj ects with reasonable accuracy is still a challenge as the optical stif fness depends on several factors such as the material properties , the refractive index of both obj ect and environment , the sizes and shapes of trapped obj ects etc . For these reasons , silicon or polystyrene microspheres are often used as a force probe to indirectly manipulate and sense the force during micromanipulation tasks . By using artif icial microbeads which are uniform in material and shape , the optical force applied on the microbead can be obtained . For small displacements , the optical force can be described by a spring model :

F = K* ( Pi aser Pprobe )

Where Piaser and Pprobe represent the laser and the particle 1 position respectively . F is the three-dimensional optical force . K [K _x , K _y , K _z ] is the trap stif fness where K _x , K _y , K _z represent the stif fness in x, y, and z direction respectively . Therefore , the optical force F is obtained by measuring the particle ' s displacement from the equilibrium trap position .

Well -established methods exist for precise stif fness calibration, as equipartition method and Stokes ' drag method . Hence , force sensing directly stems from the capability to detect the position of the laser and the one of the trapped obj ects .

The determination of this position can be done by imagebased position detection where the images of video cameras are used to extract the information, or by interferometric methods where beam displacements after the laser passes through the sample are measured via quadrant photodiode QDP .

The displacement in optical tweezers systems can be achieved by moving the platform or moving the laser beam, referred as passive actuation or active actuation .

In passive-actuation the incorporation of a motorized stage allows dynamic control of the sample chamber , while the manipulated obj ect remains f ixed on the trap .

In active-actuation, the laser direction is changed through active optical components . In addition, it is possible to multiplex the trapping laser beam in order to simultaneously trap several obj ects using a unique laser source . By rapidly def lecting the laser beam between several trap positions , it is possible to trap several obj ects , in such a way that the obj ects do not have time to spread between two laser visits . These methods are referred as time- shared methods . Or by using active dif fractive optical elements such as Spatial Light modulators (SLM) , several traps can be dynamically controlled creating a number of dif fraction spots at dif ferent positions , referred as spatial - shared methods .

In quantitative force measurements with optical tweezers , the passive methods are usually used as the optical trap is maintained f ixed, and only the trapped obj ect position information is needed .

The invention is to perform the position and thus the force measurement for all traps created by a three- dimensional multi - trap actuation system expanding the force measurements capabilities to several traps .

To this end, the invention aims to provide a device for determining the position of a plurality of obj ects that are simultaneously trapped by a single laser beam .

The device according to the invention comprises :

- a laser source for emitting a laser beam, an actuation system for a three-dimensional def lecting of the laser beam between several trapping points ,

- a microscope obj ective for focusing the laser beam coming from the actuation system on the trapping points ,

- a sample chamber including the obj ects to be trapped on the trapping points ,

- a light source for lighten the sample chamber , and an image sensor ( for instance a camera) for determining the position of each of the trapped obj ects , said image sensor being located between the laser source and the actuation system, so that the light emitted by the light source passes successively through the sample chamber , the microscope obj ective , the actuation system and the image sensor . The actuation system controls the three-dimensional position of the obj ects . As the image sensor is placed between the laser source and the actuation system and the actuation system is reversible , the image obtained by the image sensor is always aligned with the laser spot . Then by tracking the three-dimensional position of each obj ect it is possible to obtain the three-dimensional force information of several traps similar as was performed in passiveactuation methods .

The actuation system can include a deformable mirror for controlling the axial positions of the trapping points . The wording "axial" refers to the axis of the laser beam . The planar scanning mirror can be a mirror galvanometer .

The deformable mirror can typically focus or defocus the laser beam axially .

The device can comprise a system for passing the laser beam emitted by the laser source several times through the deformable mirror .

Thus , by passing the laser beam several times through the same deformable mirror , the focalisation ( in the case of a converging conf iguration of the deformable mirror) or the def ocalisation ( in the case of a diverging conf iguration of the deformable mirror) is amplif ied and therefore the angle of the laser beam at the entrance of the microscope obj ective is increased ( in the case of a diverging conf iguration of the deformable mirror) or decreased ( in the case of a converging conf iguration of the deformable mirror) . Thus , the axial position of the trapping points can be increased ( in the case of a diverging conf iguration of the deformable mirror) or decreased ( in the case of a converging conf iguration of the deformable mirror) . The invention allows the high- speed motion control of the trapping points in a large working space . As the device is still solely based on mirrors the light path is bidirectional , i . e . the path is independent from the propagation direction, and the optical ef f iciency is maximized . Thi s property allows the simultaneous trapping of obj ects in the axial direction and the visualization of trapped obj ects in the axial plane , as the illumination light returns along the same path as the manipulation laser .

Thus , the actuation axis workspace can be enlarged by using several "virtual" deformable mirrors in series . The idea is to pass the laser beam several times through the same deformable mirror using for instance a set of mirrors . By ensuring that virtual deformable mirrors are placed on conj ugate planes of the entrance aperture of the obj ective , it is possible to increase considerably the workspace , while ensuring that the size of the laser beam diameter at the entrance aperture of the obj ective remain the same , regardless the degree of convergence or divergence of the laser beam .

The working space , the number of traps that can be created and the maximum applicable force are increased . It could be used to simultaneously manipulated or stimulated several biological and synthetic obj ects in a large 3D working space . This actuation technique is also useful in dif ferent applications where the 3D orientation of microscopic obj ects (biological or synthetic ) is needed, such as cell surgery applications (e . g . nuclear transplantation, embryo micro- inj ections , polar-body biopsy) , 3D tomographic imaging of living samples , or microassembling in microf luidic devices .

The system for passing the laser beam several times through the deformable mirror can comprise at least one set of two mirrors for guiding the laser beam between two successive passages of the laser beam on the deformable mirror .

The system for passing the laser beam several times through the deformable mirror advantageously comprises between two consecutive passages of the laser beam through the deformable mirror an afocal system for conj ugating said two consecutive passages of the laser beam through the deformable mirror .

The afocal system can comprise two converging lenses .

The actuation system can include a planar scanning mirror for controlling the planar positions of the trapping points . The wording "planar" refers to a plan perpendicular to the axis of the laser beam . The planar scanning mirror can be a mirror galvanometer .

The device advantageously comprises a f irst afocal system placed between the deformable mirror and the planar scanning mirror and a second afocal system placed between the planar scanning mirror and the microscope obj ective , for conj ugating the deformable mirror and the planar scanning mirror with the entrance aperture of the microscope obj ective .

The device can comprise a system for calculating the force on each trapped obj ect as a function of the position of each of the trapped obj ect determined by the camera .

The force F on each trapped obj ect can be calculated by the system using the optical force model :

F = K* ( Pi aser Pprobe ) wherein Piaser - Pprobe represents the displacement between the laser position and the position of the trapped obj ect , K being the stif fness of the trap .

The force on the trapped obj ect is the sum of the external forces that move the obj ect from its equilibrium position, i . e the sum of the external forces that move the obj ect with the exception of the force applied by the laser beam .

The force and torque applied to a synthetic obj ect with several obj ects attached to its body, can be calculated as a function of the force F on each trapped obj ect .

The obj ects to be trapped can be biological obj ects , such as red blood cells and bacteria cells or synthetic obj ects such as optical robots (especially printed-design micro- structures with dif ferent types of end-ef fectors ) which can be used to indirectly manipulate and measure forces . Thus , the obj ects to be trapped can be biological obj ects , microspheres or synthetic obj ects .

Other aims , features and advantages of the invention will emerge from reading the following description, given purely by way of non- limiting example , and with reference to the accompanying drawings in which :

- Figure 1 , previously described, is a schematic view of an optical tweezer ,

Figure 2 , previously described, illustrates the determination of the external force applied to the obj ect trapped in the optical tweezer of f igure 1 ,

Figure 3 schematically represents a device for determining the position of trapped obj ects in optical tweezers according to the invention,

- Figure 4 is a diagram illustrating a haptic interface associated with a device according to the invention ,

Figure 5 schematically illustrates a mirror galvanometer used in the device according to the invention, Figure 6 and Figure 7 schematically illustrate a deformable mirror used in the device according to the invention, and

- Figure 8 is a diagram illustrating an optical robot associated with a device according to the invention . Figure 3 illustrates a device 10 for determining the position of a plurality of obj ects 1 that are simultaneously trapped by a single laser beam 2 according to the invention .

The device 10 comprises a laser source 5 which emits a laser beam 2 . The laser source 5 can be a 1070 nm laser . The stif fness of the trap depends on the laser power , and the output laser power usually used can be from lOOmW to 2W .

The laser beam 2 passes through an actuation system for def lecting the laser beam 2 between several trapping positions . The generation of several simultaneously trapping points using a single laser source can be achieved using spatial or temporal sharing methods . Multiple stable trapping points can be created by rapidly def lecting a laser beam among a set of positions . The displacement in optical traps systems can be achieved by moving the platform or moving the laser beam, referred to as passive actuation or active actuation .

In passive actuation, the incorporation of a motorized stage allows dynamic control of the sample chamber , while the manipulated obj ect remains f ixed on the trap .

In active actuation, the laser direction is changed through active optical components . In addition, it is possible to multiplex the trapping laser beam in order to simultaneously trap several obj ects using a unique laser source . By rapidly def lecting the laser beam between several trap positions , it is possible to trap several obj ects , in such a way that the obj ects do not have time to spread between two laser visits . This method is referred to as time- shared method . Or by using active dif fractive optical elements such as Spatial Light Modulators (SLM) , several traps can be dynamically controlled creating a number of dif fraction spots at dif ferent positions . This method is referred to as spatial - shared method . A microscope obj ective 6 is used to focus the laser beam 2 coming from the actuation system on the trapping points and to image the trapped obj ects 1 . The obj ects 1 of a sample 7 are disposed in a sample chamber 8 placed on a platform 9 (can be a three-dimensional nanostage) .

A light source 11 is used to lighten the sample . The light source can be a LED ( light -emitting diode) .

The light 20 is focused on the sample 8 in the axial direction Z via a lens 12 .

The actuation system controls the three-dimensional position of the obj ects 1 , i . e in the axial Z direction as well as in the planar X , Y directions . It includes a deformable mirror 13 and a galvanometer 14 which are used to control the axial Z and the planar X , Y positions of the trapping points respectively .

The deformable mirror 13 which can focus or defocus the laser beam 2 is used on the Z axi s . The deformable mirror 13 and the galvanometer 14 are positioned in a conj ugate plane on the entrance aperture of the microscope obj ective 6 . Hence , the laser beam 2 will pivot around the entrance aperture of the microscope obj ective 6 and retain the same degree of overf illing , independently of the angle or the degree of collimation of the incident beam 2 , producing equally and stable traps .

The deformable mirror 13 can be a microelectromechanical component with 111 actuators and 37 piston- tip- tilt segments with an update rate of more than 2 kHz . Each segment has 700 pm diameter while the array has an aperture of 3 . 5 mm and with a maximum dynamic range (Stroke) of 5 . 8 pm . Electrostatic actuation allows precise positioning of each segment with nanometer and microradian resolution (wavefront resolution < 15 nm rms) . The mechanical step-response speed is less than 200 ps (10-90%) and has high reflectance (>99.9%) .

The segmented deformable mirror 13 can effectively create smooth shapes, and it is used to control the degree of collimation of the laser beam 2. By synchronizing the orientation of the mirror galvanometer 14 and the motion of the deformable mirror 13, it is possible to displace the focal spot laterally and axially while maintaining the diffraction- limited performance. This axial scan implies a very low mechanical inertia, hence the Z bandwidth is compatible with the galvanometer 1 . The overall switching speed between three-dimensional trap position is the same order of magnitude of equivalent two-dimensional systems in the prior art. Thus, it is possible to obtain very stable traps without introducing any significant aberrations.

The Gaussian laser beam (1070 nm) is guided into the inverted microscope 6 through the galvanometer 14, the deformable mirror 13, and standard optical elements. Two afocal systems fi, f ₂ and f ₃ , f4 are preferably used to conjugate the two actuators 13, 14 with the entrance aperture of the microscope objective 6 and to expand the laser beam 2. It is expanded in order to overfill (20%) the objective entrance to improve the trapping efficiency.

Figure 5 shows how the laser beam 2 will pivot around the microscope objective's entrance aperture for small movements of the mirror galvanometer 14 creating the motion of the optical trap in the optical plane and thus creating several trapping points 15.

It is shown the effect of focusing (Figure 6) or defocusing (Figure 7) the deformable mirror 13 to change the Z position of the trapping points 15. The size of the beam 2 at the objective's entrance aperture remains the same, regardless of the degree of collimation of the incident beam. According to the invention, an image sensor 16 for determining the position of each of the trapped objects 1 is located between the laser source 5 and the actuation system 13, 14, more precisely between the laser source 5 and the deformable mirror 13, so that the light emitted by the light source 11 passes successively through the sample 7, the microscope objective 6, the actuation system 13, 14 and is then guided via a dichroic mirror 21 to the camera 16. The image sensor can be an event -based camera.

Due to the fact that the actuation system 13, 14 only uses mirrors, the light pass is bidirectional, i.e. the path is independent from the propagation direction. Using this property and thanks to the specific location of the camera 16, it is possible to track the position of the trapped objects 1 by looking at one trapping point 15 at a time, where it three-dimensional position is, and as the objects 1 are always maintained in the center of the field of view of the camera 16.

Placing the camera 16 between the laser source 5 and the actuation system 13, 14, the white light 20 reaches and goes through the actuation system 13, 14 in the exact opposite direction from the laser beam 2. In this way the image on the sensor 16 is always cantered on the laser spot. In this configuration, only one sensor receives the measurement of all the scanned points. By synchronizing the multi-trap actuation technique and the event-based camera 16 (or other cameras) , it will be possible to differentiate events (or frames) coming from different traps 15. This solution bypasses the problem of determining the center of the mobile laser in a multi-trap force measurement. A tradeoff between spatial and temporal resolution may remain, as the totality of pixels are used to determine the position of each trapped object, but the time to take the measurements depends on the number of created traps and the scanning frequency . In this way, it is for instance possible to arbitrarily position in a space three dif ferent traps , while the sensor receives the information from all the scanned points , thus measuring the position with respect to the laser of each trapped obj ect , and f inally extracting the information of external forces for each trap .

This ability to measure force at multiple three- dimensional locations simultaneously is important in many biomechanical studies , such as characterizing the stif fness of the membrane of a cell at multiple points during a fusion phase or measure the stif fness at dif ferent cel ls simultaneously . Multi - trap force measurement wil l be also useful in the investigation of mechanotransduction, for example the cell adaptation to repetitive stimulations and clarify how cells change shape and control their migratory behavior .

Optical robots 22 are especially printed-design microstructures with dif ferent types of end-ef fectors , actuated in 6DoF ( six degrees of freedom) by optical tweezers through spherical handles attached to its bodies ( f igure 8 ) .

By measuring the three-dimensional force for each handle of the robots , a six-axis force/torque measurement can be performed by combining the information of each trap . These 6 -DoF Optobots with a six-axis force sensor will allow the study of new biological interactions , such as the mechanism of Toxoplsama gondii . This latter requires mechanical work of translation and rotational twisting motion to complete cellular invasion, and therefore only characterizable with a six-axis force/torque sensor .

As illustrated in f igure 4 , a haptic interface 17 can be used . The user controls the three-dimensional position of the microrobot and feels the three-dimensional external forces F applied to it via a force sensor 18 that determines the force applied as explained above . A computer 19 makes it possible to have a visual perception and environment information to operators .