The present invention is related to a device for controlling the axial position of a laser focal point produced by a microscope obj ective . In particular, the device according to the invention can be used for different applications where high-speed 3D focal spot scanning is necessary such as optogenics , optical manipulation, confocal microscopy, two-photon microscopy or two-photon polymerisation .

It is usual to use an obj ective microscope to strongly focali ze a laser beam . The location of the laser focal point is determined by the angle of incidence ( in the planar axis X, Y) and the degree of collimation ( in the axial axis Z ) of the laser beam that reaches the entrance of the microscope obj ective . The quality of the focused laser spots ( for instance in optical manipulation the stability of the traps ) is very dependent on the qual ity of the beam that reaches the entrance of the microscope obj ective . However, given the correlation between the beam that reaches the obj ective and the focal point , it is difficult to change the three- dimensional position of the focal point without distorting its shape and introducing aberrations that will degrade its performance .

To overcome these constraints in the axial direction of the laser beam, it is known to use a deformable mirror which can focus or defocus the laser beam on the axial axis .

Deformable mirrors are mirrors whose curvature can be dynamically changed . They contain an array of actuators allowing a parabolic deflection of their surface , changing the mirror focal length . However, deformable mirrors have a small surface deflection, with a maximum stroke of for instance of 5 pm, and a high settling times of for instance 5 ms ( 10 - 90 % ) .

Consequently, the known devices are limited to a narrow working space of the laser focal point and a full scale bandwidth of the deformable mirror of for instance 200 Hz . These two limitations restrain the working space in which the laser focal point is produced and the scanning frequency at which the laser focal point can be moved between a set of arbitrary positions . This brings restrictions to the different applications , for instance in the imaging systems ( such as confocal microscopy, multi-photon microscopy) the 3D scanning of planes at arbitrary orientation, curved surfaces , and targeted scanning are still challenges . In optical manipulation, this limits the number of traps that can be created and the si ze of the obj ects that can be out- of-plane rotated, for instance .

The present invention proposes to remedy these drawbacks .

To this end, a device for controlling the axial position of a laser focal point produced by a microscope obj ective comprises :

- a laser source for emitting a laser beam,

- a deformable mirror for focusing or defocusing the laser beam axially,

- a microscope obj ective for focusing the laser beam coming from the deformable mirror on a laser focal point ,

The device according to the invention further comprises 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 concave configuration of the deformable mirror) or the def ocalisation ( in the case of a convex configuration of the deformable mirror) is amplif ied . Therefore , the convergence of the laser beam at the entrance of the microscope obj ective is increased ( in the case of a concave configuration of the deformable mirror) or the divergence of the laser beam at the entrance of the microscope obj ective is increased ( in the case of a convex configuration of the deformable mirror) . Thus , the axial position of the laser focal point can be increased ( in the case of a concave configuration of the deformable mirror) or decreased ( in the case of a converging configuration of the deformable mirror) . The invention allows the high-speed motion control of the laser focal point in a large working space . As for a given axial position, the deformable mirror has less amplitude of deformation ( compared to the case in which the laser beam passes only once through the deformed mirror) the settling times of the system is reduced, increasing the scanning frequency . As the device is sti ll solely based on mirrors , the light path is bidirectional , i . e . the path is independent from the propagation direction, and the optical efficiency is maximi zed .

The invention enlarges the actuation axis workspace 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 , whi le ensuring that the si ze 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, and the movement of the laser focal point in the axial direction . 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 optical relay system for conj ugating the deformable mirror plane with the next deformable mirror plane between said two consecutive passages .

The optical relay system can comprise an afocal system with two positive lenses ( or one negative and one positive lens ) .

The device can comprise a 2D planar scanning system for controlling the planar position of the laser focal point .

The planar scanning system is typically a galvanometer mirror .

The device can comprise a first optical relay system placed between the deformable mirror and the planar scanning mirror and a second optical relay 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 ob j ective .

The device can be an optical manipulation device , a two-photon polymeri zation device , a confocal microscopy device , a multi-photon microscopy device or an optogenetics device , or any other device that needs 3D fast scanning laser .

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 is a schematic view of a a device for controlling the axial position of a laser focal point produced by a microscope obj ective according to the prior art ,

Figure 2 is a schematic view of a a device for controlling the axial position of a laser focal point produced by a microscope obj ective according to the invention,

- Figure 3 is a partial view of the device of figure 2 ,

- Figure 4 is a schematic view of a device with an actuation system according to the prior art , in a first embodiment ,

- Figure 5 is a schematic view of a device with an actuation system according to the invention, in a first embodiment ,

- Figure 6 is a schematic view of a device with an actuation system according to the prior art , in a second embodiment ,

- Figure 7 is a schematic view of a device with an actuation system according to the invention, in a second embodiment ,

- Figure 8 is a schematic view of a device with an actuation system according to the invention, in a second embodiment ,

- Figure 9 is a schematic view of a device with an actuation system according to the prior art , in a third embodiment ,

- Figure 10 is a schematic view of a device with an actuation system according to the invention, in a third embodiment , and Figures 11 to 15 represent schematically various embodiments of a device according to the invention .

As shown in figure 1 , a device 1 for controlling the axial position of a laser focal point produced by a microscope obj ective according to the prior art comprises a laser source 2 which emits a laser beam 3 .

The laser beam 3 passes through an actuation system for controlling the three-dimensional position of the laser focal point , i . e . in the axial Z direction as well as in the planar X, Y directions . It includes a deformable mirror 4 for controlling the axial Z position of the laser focal point and a galvanometer (not shown) for controlling the planar X, Y position of the laser focal point .

A microscope obj ective 5 is used to focus the laser beam 3 coming from the actuation system on the laser focal point 6 and to image the laser focal point 6 .

The deformable mirror 4 which can focus or defocus the laser beam 3 is used on the Z axis . The deformable mirror 4 and the galvanometer are preferably positioned in a conj ugate plane on the entrance aperture of the microscope obj ective 5 . Hence , the laser beam 3 will pivot around the entrance aperture of the microscope obj ective 6 and retain the same degree of overfilling, independently of the angle or the degree of collimation of the incident beam 2 , producing equally and efficient laser focal points 6 . The conj ugate planes are pictured by the symbols

When the deformable mirror 4 is in a flat configuration (position 4a in figure 1 ) , the microscope obj ective 5 focuses the laser beam 3 on the laser focal point 6a . When the deformable mirror 4 is not in a flat configuration, for instance when it is in a convex configuration, as shown in figure 1 , the microscope obj ective 5 focuses the laser beam 3 on the laser focal point 6 . The laser focal point 6 is located a distance 1 from the laser focal point 6a .

According to the invention, and as shown in figure 2 , the device 10 further comprises a system for passing the laser beam 3 coming from the laser source 2 several times through the deformable mirror 4 .

The system can include a plurality of mirrors 7 . For instance , two reflecting mirrors 7 can be used between two successive passages of the laser beam 3 on the deformable mirror 4 .

The laser beam 3 coming from the laser source 2 i s directed to the deformable mirror (passage Pl of the laser beam 3 ) . Then a second passage P2 of the laser beam 3 is performed using two mirrors 7 . In the same manner, succes sive passages P3 and P4 are then carried out . Of course the number of passages is not limited and thus Pn passages can be carried out in the same manner .

Thus , by passing the laser beam 3 coming from the laser source consecutively several times through the deformable mirror 4 , the def ocalisation is amplified and therefore the angle of the laser beam at the entrance of the microscope obj ective is increased . Thus , the axial position of the laser focal point 6 is increased . 1 being the axial position of the laser focal point 6 from the axial position of the laser focal point 6a in the case of a flat configuration of the deformable mirror 4 ( figure 1 ) , the axial position of the laser focal point 6 from the axial position of the laser focal point 6a when the laser beam passes four times through the deformable mirror 4 i s around 4 * 1 . More generally, the axial position of the laser focal point 6 from the axial position of the laser focal point 6a after n passages of the laser beam through the deformable mirror 4 is approximately l *n . Deformation of the deformable mirror is used to changing their focal length as well as compensating for optical aberrations .

The workspace and the bandwidth are dependent of the used deformable mirror . In current system, the full working range of the axial displacement is estimated for instance as 10 pm between the maximal and minimal defocusing position of the deformable mirror and can be performed at 200Hz for instance ( limited by the time of travel of the mirror between his maximal position to his minimal position) . For small relative displacements in the z axis (below 2 pm, for instance ) , the sampling rate can be increased ( typically to 2 kHz ) as the deformable mirror maximal and minimal deformation is smaller .

With the invention, using the same deformable mirror, if the laser beam is deflected six times through the deformable mirror, the axial workspace will be approximatively of 60 pm at 200 Hz or around 12pm at 2 KHz . Therefore , this new solution will enlarge the working space and also the bandwidth .

At each passage of the laser beam 3 through the deformable mirror 4 , the deformable mirror 4 is advantageously conj ugated with the entrance aperture of the microscope obj ective 5 . To this end, the system for passing the laser beam 3 several times through the deformable mirror 4 can comprise between two consecutive passages of the laser beam 3 through the deformable mirror an optical relay for conj ugating the deformable mirror 4 with the deformable mirror 4 itself . The optical relay system can be an afocal system with a set of two positive lens , where the distance between the two lens is equal to the sum of each element' s focal length . For the passage P2, the beam coming from the deformable mirror 4 passes successively through a first lens fl, a first mirror 7, a second mirror 7, a second lens f2 and the deformable mirror 4 again. For the passage P3, the beam coming from the deformable mirror 4 passes successively through a first lens f3, a first mirror 7, a second mirror 7, a second lens f4 and the deformable mirror 4 again. For the passage P4, the beam coming from the deformable mirror 4 passes successively through a first lens f5, a first mirror 7, a second mirror 7, a second lens f6 and the deformable mirror 4 again.

As shown in figure 3, for instance for a passage P4, dl is the length of the laser beam 3 path between the deformable mirror 4 and a first lens fl, d2 is the length of the laser beam 3 path between the first lens fl and the second lens f2, d3 is the length of the laser beam 3 path between the second lens f2 and the deformable mirror 4, fl being the focal length of the first lens fl and f2 being the focal length of the second lens f2.

In the afocal system (d2=fl+f2) , two successive passages of the laser beam 3 on the deformable mirror 4 are conjugated if dl, d2, d3, fl and f2 satisfy the following relation (with the thin-lens formalism) : dl = fl/f2* (f l+f2-f l/f2*d3)

To image the same surface diameter of the deformable mirror in each passage, the magnification of the afocal telescope can be set to 1 : 1. In this case fl=f2, then dl=2fl- d3. If d3=fl, then dl=fl, making the optical relay system a 4f system.

As illustrated on figure 4, an actuation system controls the three-dimensional position of the laser focal point 6, i.e in the axial Z direction as well as in the planar X, Y directions. It includes the deformable mirror 4 and a galvanometer 8 which are used to control the axial Z and the planar X, Y positions of the laser focal point 6 respectively .

The deformable mirror 4 which can focus or defocus the laser beam 2 is used on the Z axis . The deformable mirror 4 and the galvanometer 8 are preferably positioned in a conj ugate plane on the entrance aperture of the microscope obj ective 5 . Hence , the laser beam 3 will pivot around the entrance aperture of the microscope obj ective 5 and retain the same degree of overfilling, independently of the angle and the degree of collimation of the incident beam 3 .

The deformable mirror 4 can be a microelectromechanical component with 111 actuators and 37 piston-tip-tilt segments with an update rate of 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 pm . Electrostatic actuation allows precise positioning of each segment with nanometer and microradian resolution (wavefront resolution < 15 nm rms ) .

The laser beam 3 is guided into the microscope obj ective 5 through the galvanometer 8 , the deformable mirror 4 , and standard optical elements . Two afocal systems with lens f7 , f 8 and f 9 , f l O are preferably used to conj ugate the two actuators 4 , 8 with the entrance aperture of the microscope obj ective 5 and to expand the laser beam 3 .

Figures 4 and 5 show the deformable mirror 4 is in a flat configuration . Figure 4 illustrates a laser beam 3 passing one time through the deformable mirror 4 whereas figure 5 illustrates a laser beam 3 passing three times through the deformable mirror 4 . The laser focal point 6 has the same axial position in figure 4 and in figure 5 .

In a second embodiment , as illustrated in figures 6 , 7 and 8 , the deformable mirror 4 is in a focalisation configuration ( i . e . the deformable mirror 4 is in a concave configuration) . When the laser beam 3 passes one time through the deformable mirror 4 ( figure 6 ) , the axial position of the laser focal point 6 is decreased from 1 compared to the laser focal point reference when the deformable mirror is in a flat configuration ( figure 4 ) . When the laser beam 3 passes two times through the deformable mirror 4 ( figure 7 ) , the axial position of the laser focal point 6 i s decreased from around 21 compared to the laser focal point reference when the deformable mirror 4 is in a flat configuration . When the laser beam 3 passes three times through the deformable mirror 4 ( figure 8 ) , the axial position of the laser focal point 6 is decreased from around 31 compared to the laser focal point reference when the deformable mirror 4 is in a flat configuration .

In a third embodiment , as illustrated in figure 9 and 10 , the deformable mirror 4 is in a def ocalisation configuration ( i . e . the deformable mirror 4 is in a convex configuration) . When the laser beam 3 passes one time through the deformable mirror 4 ( figure 9 ) , the axial position of the laser focal point 6 is increased from 1 compared to the laser focal point reference when the deformable mirror is in a flat configuration ( figure 4 ) . When the laser beam 3 passes three times through the deformable mirror 4 ( figure 10 ) , the axial position of the laser focal point 6 i s increased from around 31 compared to the laser focal point reference when the deformable mirror 4 is in a flat configuration .

The actuation system as described above can be used in several devices according to the invention .

The device 10 according to the invention can be used for optical manipulation, typically for trapping a plurality of obj ects ( figure 11 ) . In this case , the laser beam 3 is deflected by the actuation system 4 , 8 between several trapping points 6 . A camera 9 determines the position of each of the trapped obj ects .

As illustrated in figure 12 , the device 10 according to the invention can be used for multi-photon polymeri zation .

Two photons or more can be absorbed simultaneously by a photo-sensitive polymer 11 in a very small volume called "voxel" at the laser focal point 6 . A chemical reaction starts , and the liquid monomer becomes a solid polymer inside the voxel and a structure 12 is formed .

In another embodiment , the device 10 is a confocal microscopy device ( figure 13 ) . Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole 13 to block out-of-focus light in image formation .

As the system advantageously only uses mirrors , and the light path is bidirectional , the detector can be used in a "descanned" configuration, where the emitted light returns along the same path as the excitation laser beam . Capturing multiple two-dimensional images at different depths in a sample with a high-sensitivity detector 14 , such as a photomultiplier tube enables the reconstruction of three- dimensional structures within an obj ect .

In another embodiment , the device 10 is a multi-photon microscopy device ( figure 14 ) . Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness . Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light . Two-photon excitation microscopy typically uses near-infrared excitation light which can also excite fluorescent dyes . The fluorescence from the sample is then collected by a light detector 14 , such as a photomultiplier tube .

In a last embodiment , the device 10 is an optogenetics device using a camera 9 ( figure 15 ) . Optogenetics most commonly refers to a biological technique that involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels . Thus , the laser beam 3 is used for activating photosensitive cells 15 .