Patent Description:
For imaging some biological phenomena such as the synaptic transmission, the scanning speed of a system used should be at least of the order of <NUM> meters per second. It is thus desirable to be able to scan a sample with a fast laser scanning system.

For this, it is known to use acousto-optic deflectors (often named after their acronym AOD) which are fast pointing devices based on the interaction between an acoustic compression wave propagating in an acousto-optical crystal and an electromagnetic wave. In most cases, the electromagnetic wave has a planar or a spherical wavefront. The resulting diffractive process deflects a fraction of the electromagnetic wave at an angle proportional to the acoustic frequency of the acoustic compression wave.

An acousto-optic deflector can be used as a scanning device by changing the acoustic frequency by elementary steps. However, each new acoustic frequency must propagate in the acousto-optical crystal throughout the region of interaction with the electromagnetic wave in order to fully redirect the electromagnetic power to the new angle. The characteristic time of switching associated is in the range of a few microseconds to several tens of microseconds. Thus, to acquire an image of a sample, a scanning device with an acousto-optic deflector would usually take several seconds. This time is prohibitive to efficiently acquire high resolution images.

It is known from <CIT> to drive a single acousto-optic deflector by a linear frequency chirp of slope s. The linear chirp produces a linear drift of the central frequency of the acousto-optic deflector and, thus, an angular scan of constant speed, proportional to s. The linear chirp also induces a lensing effect by introducing a cylindrical curvature of the wavefront of the electromagnetic wave. This lensing effect corresponds to a cylindrical lens of optical power proportional to s (according to the paraxial approximation). This lensing effect can be compensated by a cylindrical lens with a fixed focal lens.

However, the use of a fixed focal lens implies that the slope s is constant, which means that the scanning speed is fixed.

An acousto-optic scanning system known from document <CIT> which relies on two linear chirps of identical slopes propagated in opposite directions in a single acousto-optic crystal or in two separate acousto-optic crystal conjugated optically. The spatial gradients of frequencies are thus opposite, generating a lensing effect corresponding to two cylindrical lenses of opposite power (diverging and converging). The lensing effect is therefore cancelled, as long as the acousto-optic deflectors are perfectly conjugated optically, while the scanning speeds of each acousto-optic deflector are added.

But, this technique requires a tedious alignment of the various optical elements and induces a loss of power of the diffracted electromagnetic wave, resulting from the use of pairs of two acousto-optic successive interactions. Indeed, the deflection of the electromagnetic wave by the first acousto-optic interaction changes the incidence angle for the second acousto-optic interaction. This second interaction does no longer occur at the optimal angle (Bragg's angle), hence a loss in diffraction efficiency and transmitted power in the first diffraction order.

It is known from the article by <NPL>) that using acousto-optical deflectors at high deflection speeds via acoustical frequency chirping induces astigmatism, deforming the laser beam in an unfavorable way. Within this paper, a method to prevent this effect for an ultrashort pulsed laser beam is presented via acoustical frequency jumps synchronized to the pulse-to-pulse pause. A method to calculate beam shaping capability of acousto-optical deflectors via arbitrary spatial frequency developments during ultrashort laser pulse transit through the deflector is also given. Cylinder-lens-free redirection at more than <NUM> rad/s (if jumping from one end to the other end of the deflection range in one switch time) is demonstrated experimentally. However, continuous scan of the laser beam is not achievable in this way. In the experiments, the switching time between two beam shapes is equal to <NUM> microsecond (µs).

It is also known from document <CIT> a scanning system characterized by including acousto-optic means for deflecting a beam of electromagnetic radiation in first and second directions, wherein the system is configured for scanning the beam in third and fourth directions, which are inclined angularly relative to the first and second directions, by simultaneous operation of the first and second acousto-optic means.

The invention aims at solving the problems of the known laser scanning systems with a new laser scanning system which is tunable in scanning speed and in field of scan.

A laser scanning system according to the claimed invention is defined in appended claim <NUM>.

To this end, the invention concerns a laser scanning system comprising a first acousto-optical deflector adapted to deflect an input beam in a first direction to obtain a first deflected beam, the first acousto-optical deflector comprising a first acousto-optical crystal and a first transducer adapted to command the first acousto-optical crystal by applying a first acoustic wave having a variation in frequency over time according to a first law of command. The laser scanning system also comprises a second acousto-optical deflector adapted to deflect the first deflected beam in a second direction to obtain a second deflected beam, the second acousto-optical deflector comprising a second acousto-optical crystal and a second transducer adapted to command the second acousto-optical crystal by applying a second acoustic wave having a variation in frequency over time according to a second law of command. The first direction and the second direction define at least an angle comprised between <NUM>° and <NUM>°. The first law of command and the second law of command are chosen so that the speed of the laser scanning system is superior to <NUM> radians per second and the two acousto-optic deflectors introduce a defocusing in the input beam and the laser scanning system comprises a compensating unit for compensating the defocusing introduced by the two acousto-optic deflectors.

Thanks to the invention, the laser scanning system provides a way of carrying out a simple, precise and repeatable scanning of a sample with a tunable scanning speed. The values of speed which can be reached are, in addition, faster than the values reached by classical mechanical scanning devices such as resonant scanners or galvanometric mirrors.

According to further aspects of the invention which are advantageous but not compulsory, the laser scanning system might incorporate one or several of the following features, taken in any technically admissible combination:.

The invention also concerns a two-photon microscope comprising a laser scanning system as previously described.

The invention will be better understood on the basis of the following description which is given in correspondence with the annexed figures and as an illustrative example, without restricting the object of the invention. In the annexed figures:.

For the remainder of the description, a longitudinal direction is defined: the longitudinal direction corresponds to the general direction of the propagation of light. Two transversal directions perpendicular to the longitudinal direction are also defined, the first transversal direction being further perpendicular to the second transversal direction. The longitudinal and transversal directions are respectively symbolized by an axis Z and axes X and Y on <FIG>.

A two-photon microscope <NUM> adapted to achieve a two-photon microscopy on a sample <NUM> is represented on <FIG>.

The sample <NUM> is, for instance, a soft tissue. In the meaning of the present invention, a soft tissue is an organic tissue which can have an animal or vegetal origin. For instance, such a soft tissue can be a muscle or any portion of a human body, of an animal body or of a vegetable. A soft tissue can also be a non-metallic part of a prosthesis.

The microscope <NUM> comprises a laser unit <NUM>, a first optical system <NUM>, a laser scanning system <NUM>, a radiofrequency power supply <NUM> of the laser scanning system <NUM>, a second optical system <NUM>, a beam splitter <NUM>, an objective <NUM> and a detecting unit <NUM>.

The laser unit <NUM> is adapted to emit a laser beam. The laser unit <NUM> comprises a laser source. The laser source is adapted to emit a coherent light whose wavelength is comprised between <NUM> nanometers (nm) and <NUM> micrometers (µm). The laser source is preferably a femtosecond laser adapted to emit laser pulses with a duration comprised between <NUM> fs and <NUM> picoseconds (ps).

Preferably, the femtosecond laser is adapted to emit laser with a full width at half maximum strictly inferior to <NUM> picoseconds.

Alternatively, the laser unit <NUM> also comprises an element of spatial and/or temporal precompensation of the pulses emitted by the laser source. Such element is, for example, a prism or a grating or another acousto-optical deflector.

The first optical system <NUM> is adapted to make the laser beam emitted by the laser unit <NUM> propagate towards the laser scanning system <NUM>.

The laser scanning system <NUM> is adapted to carry out a scan of the sample <NUM> in two dimensions.

The laser scanning system <NUM> comprises a first acousto-optical deflector <NUM>, a second acousto-optical deflector <NUM> and a compensating unit <NUM>.

The first acousto-optical deflector <NUM> is adapted to deflect an input beam in the first transverse direction X to obtain a first deflected beam. In other words, the first acousto-optical deflector <NUM> is adapted to carry out a scan of the sample <NUM> in the first transverse direction X.

The first acousto-optical deflector <NUM> comprises a first acousto-optical crystal <NUM>, a first transducer <NUM> and a first casing <NUM> protecting the first acousto-optical crystal <NUM>.

The first acousto-optical crystal <NUM> has a parallelepipedic shape, the light entering by a first input face <NUM> and leaving by a first output face <NUM>.

The distance between the first input face <NUM> and the first output face <NUM> along the longitudinal direction Z is the thickness of the crystal.

The first acousto-optical crystal <NUM> is a crystal made in TeO<NUM>. Any other material exhibiting acousto-optical properties may be considered.

The first transducer <NUM> is adapted to command the first acousto-optical crystal <NUM> by applying a first acoustic wave having a variation in frequency over time according to a first law of command L1.

The second acousto-optical deflector <NUM> is adapted to deflect the first deflected beam in the second transverse direction Y to obtain a second deflected beam. In other words, the second acousto-optical deflector <NUM> is adapted to carry out a scan of the sample <NUM> in the second transverse direction Y.

The first transverse direction X and the second transverse direction Y define at least an angle α comprised between <NUM>° and <NUM>°.

Preferably, as can be seen on <FIG> and <FIG>, the angle α is equal to <NUM>°. Such configuration for the two acousto-optical deflectors <NUM>, <NUM> is named a "crossed" configuration.

The second acousto-optical deflector <NUM> comprises a second acousto-optical crystal <NUM>, a second transducer <NUM> and a second casing <NUM> protecting the second acousto-optical crystal <NUM>.

The second acousto-optical crystal <NUM> has a parallelepipedic shape, the light entering by a second input face <NUM> and leaving by a second output face <NUM>.

The distance between the second input face <NUM> and the second output face <NUM> along the longitudinal direction Z is the thickness of the crystal.

In the specific case illustrated, the second acousto-optical crystal <NUM> is identical to the first acousto-optical crystal <NUM>.

The optical distance d between the first acousto-optical crystal <NUM> and the second acousto-optical crystal <NUM> along the longitudinal direction Z is as small as possible and in all cases such that the difference in position along the longitudinal direction Z of the theoretical focal points generated in the sample <NUM> by the lensing effects of the twoacousto-optical deflectors <NUM>, <NUM> be less than half of the diffraction-limited optical resolution along the longitudinal direction Z of the objective <NUM> under all conditions of scanning.

To fulfill this requirement, it is proposed that the optical distance d between the first acousto-optical crystal <NUM> and the second acousto-optical crystal <NUM> along the longitudinal direction Z be inferior to <NUM> millimeters (mm). This enables to limit the astigmatism of the lensing effect generated by the use of both acousto-optical deflectors <NUM>, <NUM>. By definition, the optical distance d is the distance between the first output face <NUM> and the second input face <NUM> in the absence of optical relay system. In such case, the optical distance d is the geometrical distance between the first output face <NUM> and the second input face <NUM>.

In case an optical relay system is present, this optical relay system images the first output face <NUM> into a conjugate plane and the optical distance d12 is the distance from this image plane to the second input face <NUM>.

According to a preferred embodiment of the invention, the optical distance d is inferior to <NUM> millimeters (mm).

The second transducer <NUM> is adapted to command the second acousto-optical crystal <NUM> by applying a second acoustic wave having a variation in frequency over time according to a second law of command L2.

Both acousto-optic deflectors <NUM>, <NUM> generate a lensing effect which mainly results in a defocusing in the input beam. The compensating unit <NUM> is adapted to compensate the defocusing introduced by the two acousto-optic deflectors <NUM>, <NUM>.

According to the example of <FIG>, the compensating unit <NUM> comprises a spherical lens and a translation device for modifying the position of the spherical lens with respect to the two acousto-optic deflectors <NUM>, <NUM>.

In an alternative example, both acousto-optical crystals <NUM>, <NUM> are in the same casing.

The radiofrequency power supply <NUM> or radiofrequency generator <NUM> of the laser scanning system <NUM> is adapted to provide, to each transducer <NUM>, <NUM>, radiofrequency waves. Each transducer <NUM>, <NUM> is adapted to convert radiofrequency waves in an acoustic wave. Thus, the radiofrequency generator <NUM> is able to provide a first sequence of radiofrequency waves to the first transducer <NUM> corresponding to the first law of command L1. The radiofrequency generator <NUM> is also able to provide a second time-dependent radiofrequency wave to the second transducer <NUM> corresponding to the second law of command L2.

The radiofrequency generator <NUM> is a direct digital synthesizer. Such device (whose usual acronym is DDS) is a type of frequency synthesizer used for creating arbitrary waveforms from a single, fixed-frequency reference clock.

Alternatively, the radiofrequency generator <NUM> is an analog device.

The second optical system <NUM> is adapted to serve as an optical relay between the output of the laser scanning system <NUM> and the microscope <NUM>. The second optical system <NUM> is, for instance, a 4f relay.

The beam splitter <NUM> is adapted to reflect the light issued from the laser scanning system <NUM> towards the objective <NUM> and to transmit the light collected by the objective <NUM> to the detecting unit <NUM>. For instance, the beam splitter <NUM> is a dichroïc mirror.

Alternately, instead of the objective <NUM>, any combination of lenses may be used.

The objective <NUM> is adapted to make the light received from the beam splitter <NUM> converge on a focal point situated in a focal plane located in the sample <NUM>, to gather light emitted by the sample <NUM> and to send it to the beam splitter <NUM>.

The objective <NUM> is an oil-immersion objective or water-immersion objective comprising a combination of several optical elements. The objective <NUM> provides a magnification usually ranging from <NUM> to <NUM> and a numerical aperture comprised between <NUM> and <NUM>.

The detecting unit <NUM> comprises a third optical system <NUM> and a detector <NUM>. The third optical system <NUM> is adapted to collect the light transmitted by the beam splitter <NUM> and to focus it on the detector <NUM>. The detector <NUM> is adapted to convert the light received in an electrical signal. The detector <NUM> is, for instance, a photomultiplier.

Operation of the microscope <NUM> for two-photon imaging is now described when the first law of command L1 and the second law of command L2 applied are linear function of time, as in the first example of <FIG>.

The laser unit <NUM> emits a first laser beam F1 towards the first optical system <NUM> which converts the first laser beam F1 into a second laser beam F2 whose waist is located in the laser scanning system <NUM>. The second laser beam F2 can thus be considered as the input beam for the first acousto-optical deflector <NUM>.

Then, the radiofrequency generator <NUM> applies a first sequence of radiofrequency waves to the first transducer <NUM> such that the first law of command L1 be a linear function of time. Simultaneously, the radiofrequency generator <NUM> applies a second sequence of radiofrequency waves to the second transducer <NUM> such that the second law of command L2 be a linear function of time. In this example, the two linear functions of time S share the same slope named and are represented on <FIG>. This slope S is the temporal gradient of frequency or the value of the local averaged slope of frequency over time. In case the function is continuous over time, the local averaged slope of the function at an instant is equal to the value of the derivative of the function at the same instant.

The first acousto-optical deflector <NUM> deflects the second laser beam F2 by a first angle of deviation in the first transverse direction X. This first deflected beam is labeled F3. With time, the first angle of deviation gets more and more important.

It can be shown that when the first law of command L1 is a linear function of time, the lensing effect generated by the first acousto-optical deflector <NUM> corresponds to a first cylindrical lens with a first power.

The first deflected beam F3 propagates towards the second acousto-optical deflector <NUM>.

The second acousto-optical deflector <NUM> deflects the first deflected beam F3 by a second angle of deviation in the second transverse direction Y. This second deflected beam is labeled F4. With time, the second angle deviation gets more and more important.

It can be shown that when the second law of command L2 is a linear function of time, the lensing effect generated by the second acousto-optical deflector <NUM> corresponds to a second cylindrical lens with a second power.

The second power is identical to the first power since the slope of the first law of command L1 and the slope of second law of command L2 are the same.

Thus, the lensing effect generated by both acousto-optical deflectors <NUM>, <NUM> is equivalent to the optical system illustrated on <FIG>. This optical system comprises a first cylindrical lens adapted to focus light along the first transverse direction X and a second cylindrical lens adapted to focus light along the second transverse direction Y. This equivalent optical system therefore behaves like a spherical lens having the power of the two cylindrical lenses. This power causes an axial displacement of the focal plane in the sample or defocusing to be compensated.

The second deflected beam F4 then propagates towards the compensating unit <NUM>, which is a spherical lens, so as to obtain a fifth laser beam F5 in which the defocusing generated by both acousto-optical deflector <NUM>, <NUM> is compensated.

The fifth laser beam F5 propagates towards the objective <NUM> via the second optical system <NUM> which serves as a relay and reflects on the beam splitter <NUM>.

The objective <NUM> makes the fifth laser beam F5 convergent on the focal point of the focal plane in the sample <NUM>.

Because the slope S is identical for both acousto-optical deflectors <NUM>, <NUM>, the focal point is scanned along the direction of the diagonal of the field of view of the objective <NUM>. The initial frequencies applied to the two acousto-optical crystals <NUM>, <NUM> set the point of origin of the diagonal scanning line, which can thus be displaced at will over the desired scanning area in the sample <NUM>.

As two-photon microscopy relies on the two-photon absorption phenomena predicted by Göpert-Mayer in <NUM>, at the focal point, an interaction between the sample <NUM> and light occurs by simultaneous absorption of pairs of photons of the fifth laser beam F5 by the sample <NUM>. Following most of these biphotonic absorption events, the sample <NUM> emits one photon by fluorescence. The wavelength of the photon emitted is larger than half the wavelength of the photons of one pair.

The objective <NUM> then gathers the fluorescence light emitted by the sample <NUM> so as to form a sixth laser beam F6.

The beam splitter <NUM> transmits the sixth laser beam F6 towards the detecting unit <NUM>.

The third optical system <NUM> makes the sixth laser beam F6 converge on the detector <NUM> which converts the sixth laser beam F6 in an electrical signal. This electrical signal contains information relative to the arrangement of sample <NUM> in space. Based on the electrical signal, an image of the sample <NUM> in the focal point can thus be reconstituted point by point according to the diagonal scanning pattern. The reconstitution has been shown by the applicant for a scanning speed up to <NUM> radians per second.

The laser scanning system <NUM> enables to scan the sample <NUM> with a speed commanded by the first law of command L1 and the second law of command L2. As the slope S can be freely chosen, the scanning speed can be set to any arbitrary value, as long as the compensating unit <NUM> can compensate for the defocusing created by the equivalent spherical lens <NUM>. Thus, the scanning speed of the laser scanning system <NUM> is tunable.

Furthermore, the laser scanning system <NUM> is easy to implement since only two acousto-optical deflectors <NUM>, <NUM> are involved.

According to a second example, the first law of command L1 and the second law of command L2 are step functions of time, as represented on <FIG>. A step function comprises several intervals of constant frequency separated by a step. Each acousto-optical deflector <NUM>, <NUM> has an optical resolution. To minimize the degradation of the spatial resolution, the largest frequency step between two intervals of constant frequency should remain inferior to the highest one of the two optical resolutions of the acousto-optic deflectors <NUM>, <NUM>. By this expression, it is meant that the largest frequency step between two intervals of constant frequency should remain inferior to the frequency value corresponding to the highest one of the two optical resolutions of the acousto-optic deflectors <NUM>, <NUM>.

Operation of the microscope <NUM> for two-photon imaging when the first applied law of command L1 and the second applied law of command L2 are step functions of time as in the second example of <FIG> is similar to the operating of the microscope <NUM> previously presented because the local averaged slope of each law of command L1, L2 over time is similar. Therefore, the operating of the microscope <NUM> is mainly determined by the behavior of the local averaged slope of each law of command L1, L2 over time.

Thus, by extension, many laws of command L1 and L2 enables to obtain an operating of the microscope <NUM> which is adapted for two-photon imaging provided the behavior of the local averaged slope of each law of command L1, L2 be adapted.

Preferably, the local averaged slope of the first law of command L1 over time is comprised between <NUM>% and <NUM>% of a first constant value. This enables that the lensing effect, generated by the first acousto-optical deflector <NUM> alone, substantially corresponds to the effect of a first cylindrical lens with a constant power linked to the first constant value.

Preferably, the local averaged slope of the second law of command L2 over time is comprised between <NUM>% and <NUM>% of a second constant value. This enables that the lensing effect, generated by the second acousto-optical deflector <NUM> alone, substantially corresponds to the effect of a second cylindrical lens with a constant power linked to the second constant value.

Preferably, the first constant value and the second value are the same so as to ensure that the lensing effect generated by both acousto-optical deflectors <NUM>, <NUM> be close to the behavior of a spherical lens.

Advantageously, the first law of command L1 and the second law of command L2 are chosen so that the speed of the laser scanning system <NUM> is superior to <NUM> radians per second, preferably <NUM> radians per second, more preferably <NUM> radians per second.

The speed of the laser scanning system <NUM> is the scanning speed. A scanning speed implies that a surface is scanned by a laser in a plurality of contiguous points with time. The angular speed for following the path linking said points on the surface is the scanning speed.

Such scanning speed differs from the deflecting speed of an acousto-optical deflector. Indeed, such deflecting speed is equal to the ratio between the deflecting angle imposed by an acousto-optical deflector and the duration between the time when the generator starts to apply an acoustic wave at an extremity of the crystal and the time when the beam is actually deflected.

It should be understood that various law of commands L1 and L2 fulfill this criteria Linear functions of time with the same slope constitute a specific example. However, in some circumstance, it may be desirable that the first law of command L1 and the second law of command L2 be different. It is notably the case if the optical distance d is not small.

According to the claimed invention, the compensating unit <NUM> is a translation device for modifying the position of the objective <NUM> with respect to the two acousto-optic deflectors <NUM>, <NUM>.

The defocusing introduced by the two acousto-optic deflectors <NUM>, <NUM> is given by the following formula: <MAT> wherein:.

This formula accounts for the large convergence angles under the objective <NUM> and is therefore more precise than the paraxial approximation obtained by standard lens conjugation formulas.

Thus, by translating the translation device for modifying the position of the objective <NUM> along the optical axis by a quantity of - Δz, the defocusing introduced by the two acousto-optic deflectors <NUM>, <NUM> is compensated.

Alternatively, not according to the claimed invention, the compensating unit <NUM> comprises adaptive optics such as a deformable mirror. Adaptive optics is notably interesting so as to compensate any optical aberration the laser scanning system <NUM> can generate in the focal plane.

Notably, such optical aberration to compensate can be astigmatism. Indeed, in reality, the equivalent optical system represented on <FIG> is a spherical lens exhibiting an astigmatism defect since the distance d between the two acousto-optic crystals <NUM>, <NUM> is not zero.

According to another example of the laser scanning system <NUM>, not according to the claimed invention, the laser scanning system <NUM> is deprived of compensating unit <NUM>. Indeed, if it is desired to image any focal plane of the sample <NUM>, there is no need to compensate for the defocusing generated by both acousto-optical deflector <NUM>, <NUM>.

Claim 1:
Laser scanning system (<NUM>) comprising:
- a first acousto-optical deflector (<NUM>) adapted to deflect an input beam in a first direction (X) to obtain a first deflected beam, the first acousto-optical deflector (<NUM>) comprising a first acousto-optical crystal (<NUM>) and a first transducer (<NUM>) adapted to command the first acousto-optical crystal (<NUM>) by applying a first acoustic wave having a variation in frequency over time according to a first law of command (L1), and
- a second acousto-optical deflector (<NUM>) adapted to deflect the first deflected beam in a second direction (Y) to obtain a second deflected beam, the first direction and the second direction define at least an angle (α) comprised between <NUM>° and <NUM>° and the second acousto-optical deflector (<NUM>) comprising a second acousto-optical crystal (<NUM>) and a second transducer (<NUM>) adapted to command the second acousto-optical crystal (<NUM>) by applying a second acoustic wave having a variation in frequency over time according to a second law of command (L2),
wherein the two acousto-optic deflectors (<NUM>, <NUM>) introduce a defocusing in the input beam and the laser scanning system (<NUM>) comprises a compensating unit (<NUM>) for compensating the defocusing introduced by the two acousto-optic deflectors (<NUM>, <NUM>),
characterized in that the first law of command (L1) and the second law of command (L2) are chosen so that the speed of the laser scanning system (<NUM>) is superior to <NUM> radians per second, and in that
the compensating unit (<NUM>) comprises an objective (<NUM>) and a translation device configured to modify the position of the objective (<NUM>) with respect to the two acousto-optic deflectors (<NUM>, <NUM>) by a quantity of - Δz, Δz being the defocusing introduced by the two acousto-optic deflectors (<NUM>, <NUM>) and being such that: <MAT> wherein:
• F is the focal distance of the objective (<NUM>);
• NA is the numerical aperture of the objective (<NUM>);
• n is the refractive index of the medium of immersion of the objective (<NUM>), and
• Δθ is the angular variation between the ray entering at the center of the objective (<NUM>) aperture and the ray passing at the edge of its effective back-aperture.