Abstract:
A method and a device for detecting a resonant non-linear optical signal induced in a sample including a resonant medium and a non-resonant medium forming an interface is disclosed. The device includes an excitation light beam that intercepts the sample along an optical axis. The device further includes a first optical detection module for detecting a nonlinear optical signal resulting from the interaction of the beam with the sample, and a mirror that reflects the excitation beam. The device further includes a second optical detection module for detecting a nonlinear optical signal resulting from the interaction of the reflected excitation beam with the sample, and a processing module for processing the optical signals, detected by the first and second detection modules. Processing the optical signals includes calculating a difference in the detected signals, the difference being characteristic of a vibrational or electronic resonance of the resonant medium.

Description:
PRIOR ART 
     1. Technical Field of the Invention 
     The present invention relates to a method for detecting a resonant nonlinear optical signal and a device for implementing said method. It is particularly applicable to the detection of CARS scattered signals. 
     2. Prior Art 
     All chemical bonds have their own characteristic vibration frequency. Methods aimed at using the light/matter interaction to obtain information on these molecular vibrations are called vibrationally-sensitive optical techniques. The most well-known of these techniques is infrared (IR) spectroscopy, which observes the absorption lines specific to chemical bonds present in a sample. Discovered in 1928, Raman scattering (from the name of a physicist, Chandrasekhara Venkata Raman, who discovered the effect) allows visible light to be used to access the vibrational spectrum of molecules which interact with a light beam. In Raman scattering, a pump wave of angular frequency ω P  incident on a molecule is scattered inelastically into a wave called a Stokes wave, of angular frequency ω S  ( FIG. 1A ) and a wave called an anti-Stokes wave, of angular frequency ω AS  ( FIG. 1B ). The difference in frequency between the generated waves and the pump wave depends on the molecular Raman transition (of angular frequency Ω R ) such that ω p −ω s =ω as −ω p· =Ω R . In a photonic view of the process, the Stokes and anti-Stokes waves correspond to absorption from the fundamental or excited vibrational level respectively. The process generating the anti-Stokes wave, from the excited vibrational level (B), is much less probable than the process creating the Stokes wave, which is the only one observed in practice in spontaneous Raman spectroscopy. Detailed study of the spectral distribution of Stokes waves yields information about the densities of chemical bonds present in the sample. This spontaneous process of inelastic scattering is very inefficient compared with fluorescence (Raman cross-sections are of the order of 10 −+ cm 2 /molecule, compared with the absorption cross-section of 1 photon of a fluorophore, which reaches 10 −16  cm 2 /molecule). 
     Stimulated CARS (Coherent Anti-Stokes Raman Scattering) Raman spectroscopy is a four-wave mixing process that allows the vibrational bonds present in a sample to be addressed. This process is described, for example, in R. W. Boyd,  Nonlinear Optics  (Academic Press, Boston, 1992). It involves sending two laser pulses of angular frequencies ω p  and ω s  (or of frequencies ν p  and ν s ), the angular frequency difference of which is equal to the angular frequency Ω at the vibrational level under investigation. In this resonance configuration ω p −ω s =Ω, the vibrational level of angular frequency Ω is populated in a stimulated manner and will be able to scatter inelastically the beam of angular frequency ω p  into a beam of angular frequency ω as =2 ω p −Ω s  ( FIG. 2A ). The presence of this new radiation ω as  is the signature of the presence of the bond vibrating at the angular frequency Ω in the sample. A first implementation of CARS consists directing at the sample two pulses which are spectrally picosecond narrow, the angular frequency difference of which addresses only one specific vibrational bond. For optimum identification, all the vibrational bonds present in the sample are tested. This is done by operating in a mode called “Multiplex CARS” (see, for example, M. Muller and J. Schins, “Imaging the thermodynamic state of lipidic membranes with multiplex CARS spectroscopy”, Physical Chemistry B 106, 3715-3723 (2002)) where a spectrally narrow pulse ω p  and a spectrally wide pulse ω s  are directed at the sample ( FIG. 2B ). Thus all the vibrational levels Ω i  present in the sample can be addressed, and a spectrum of the generated signal ω as  can be obtained. From a technical point of view, the narrow spectrum originates, for example, from a picosecond laser and the wide spectrum, for example, from a femtosecond laser, or a photonic crystal fibre generating a supercontinuum (SC). 
     In  FIG. 3A  the process of resonant CARS scattering is described, which is used to access the signature of the molecular to be identified. However, a non-resonant CARS contribution exists, represented in  FIG. 3B , which arises from an electronic contribution of the sample. This non-resonant contribution may be important when CARS spectroscopy is performed on a sample comprising a wide diversity of chemical bonds. 
     In the article “Focused field symmetries for background-free coherent anti-Stokes Raman spectroscopy”, Physical Review A 77 (2008), in the name of D. Gachet et al., an original method is presented which allows the non-resonant contribution to be eliminated.  FIGS. 4 to 6  illustrate the method. This consists in producing a differential CARS image between an object and its mirror image about a transverse interface  43  between a resonant medium (reference numeral  41  in  FIGS. 4A and 4B ) and a non-resonant medium (reference numeral  42  in  FIGS. 4A and 4B ). The 3rd order nonlinear susceptibility is defined in the resonant medium  41  by a resonant term χ (3)   1R  and a non-resonant term χ (3)   1NR . In the non-resonant medium  42 , it is defined by the non-resonant term χ (3)   2NR .  FIGS. 4A and 4B  depict an active CARS volume  45  (focal point of pump and Stokes beams of frequencies ω p  and ω s  respectively), located on the transverse interface  43  between the resonant medium and the non-resonant medium. Two situations are envisaged: case α wherein the pump and Stokes beams are incident on the non-resonant medium side, and case β wherein the pump and Stokes beams are incident on the resonant medium side. It is demonstrated in this article that the difference between the CARS signals obtained in cases α and β comprises only the resonant contribution of the resonant medium.  FIG. 5  illustrates the results of a numerical calculation taking into account the vectorial nature of the pump and Stokes beams focused on a transverse interface as illustrated in  FIGS. 4A and 4B . The analysis consists in studying the difference ΔI Fwd  of the CARS signals emitted in problems α (I α (Fwd)) and β (I β (Fwd)) as a function of the normalised Raman shift ζ=(ω p −ω s −Ω R )Γ (where Γ is the spectral width of the vibrational line studied). It is demonstrated that the difference ΔI Fwd  exactly follows the imaginary part of χ (3)   1R  which is known to be the Raman spectrum of medium  1 . An experimental implementation of the method is illustrated in  FIG. 6B  and the experimental results are presented in  FIG. 6A .  FIG. 6B  represents a sample composed of a layer  61  of DMF (N,N-dimethylformamide) between two glass slides  62 ,  63 . Case α corresponds to the case wherein the pump and Stokes beams are focused on the glass-DMF interface (interface between  62  and  61 ), while case β corresponds to the case wherein the excitation beams are focused on the DMF-glass interface (interface between  61  and  63 ). In  FIG. 6A , as a function of the Raman shift respectively, curve C 1  illustrates the CAR intensity of the DMF alone (when the excitation beams are focused in the resonant medium); curve C 2 , the intensity I α (Fwd); curve C 3 , the intensity I β (Fwd); curve C 4 , the differential ΔI Fwd , and curve C 5  the Raman spectrum. It appears, as demonstrated theoretically, that the method enables elimination of the non-resonant component which soils the CARS scattered signal represented by curve C 1 . 
     However, this method has a number of drawbacks. Notably, it is limited to symmetric samples, such as shown in  FIG. 6B , or reversible ones, in order to have access to resonant/non-resonant interfaces on the one hand and non-resonant/resonant interfaces on the other. This has a limitation in the cases of biological samples which rarely have these properties. Furthermore, although it allows spectroscopy applications, this method is limited for microscopy applications. 
     The present invention proposes a novel device for detecting a resonant nonlinear optical signal, based on the principle of transverse interfaces detection as described in the prior art, but which may be applied to any sample having an interface between a resonant medium and a non-resonant medium, both for spectroscopy and microscopy applications. 
     SUMMARY OF THE INVENTION 
     According to a first aspect, the invention relates to a device for detecting a resonant nonlinear optical signal induced in a sample of the type comprising a resonant medium and a non-resonant medium forming an interface, the device comprising: an emission source of at least one first excitation light beam, called a pump beam, at a first given angular frequency ωp for the excitation of the resonant medium of a sample of the given type, a first optical module for detecting the nonlinear optical signal resulting from interaction of said incident pump beam the sample when said pump beam is incident on the sample along an optical axis and intercepts the sample at a given position of a transverse interface between the resonant and non-resonant media of the sample, means of reflection of said pump beam, arranged in such a way that said reflected pump beam intercepts said transverse interface substantially at the same position as said incident pump beam, a second optical module for detecting the nonlinear optical signal resulting from interaction of said reflected pump beam with the sample, an optical signal processing module detected by said first and second detection modules, comprising the calculation of a difference in the detected signals, the difference being characteristic of a vibrational or electronic resonance of the resonant medium. 
     According to a variant embodiment, the emission source allows the emission of a pump beam of angular frequency op and a Stokes beam of angular frequency ωs, the nonlinear optical signal resulting from the interaction of said pump and Stokes beams is a signal called a CARS scattered signal, of angular frequency ωas=2ωp−ωs and the difference in signals detected by the first and second detection module is characteristic of a Raman resonance of the resonant medium. 
     According to another variant embodiment, the device according to the invention comprises a lens for focusing incident excitation beams in a common focal volume, allowing said interface between the resonant medium and the non-resonant medium to be intercepted and a lens for collecting the nonlinear signal resulting from interaction of the incident excitation beams with the sample, said collecting lens being identical to the focusing lens for focusing the incident beams and the collecting lens forming a lens for focusing the reflected excitation beams and the lens for focusing the incident beams forming a lens for collecting the nonlinear signal resulting from interaction of the reflected excitation beams with the sample. 
     According to another variant embodiment, each of the optical detection modules comprises an image recording device, the nonlinear optical signal being collected in each of the optical detection modules respectively in the symmetrical directions about the optical axis, the difference being effected for each signal couple thus detected. 
     According to another variant embodiment, a device for angular scanning of the excitation beams allows the excitation beams to intercept the sample at different positions of the interface between the resonant and non-resonant medium. 
     According to another variant embodiment, the emission source emits at least one variable wavelength excitation beam, allowing a spectrum of vibrational or electronic resonances of the resonant medium to be obtained. 
     According to a second aspect, the invention relates to a method for detecting a resonant nonlinear optical signal induced in a sample, the sample comprising a resonant medium and a non-resonant medium forming an interface, the method comprising: the emission of at least one first light beam for the excitation of the resonant medium, called a pump beam, at a first given angular frequency wp, said pump beam being incident on the sample along an optical axis, and intercepting the sample at a given position of a transverse interface between the resonant and non-resonant medium, the detection of a first nonlinear optical signal resulting from interaction of said beam or beams with the sample, the reflection of said excitation beam or beams, the reflected excitation beam or beams intercepting said transverse interface substantially at the same position as the incident excitation beam or beams, the detection of a second nonlinear optical signal resulting from interaction of said reflected excitation beam or beams with the sample, the processing of the first and second detected optical signals, comprising the calculation of a difference in the detected signals, the difference being characteristic of a vibrational or electronic resonance of the resonant medium. 
     According to a variant embodiment, the method comprises the emission of a pump beam of angular frequency ωp and of a Stokes beam of angular frequency ωs, the nonlinear optical signal resulting from the interaction of said pump and Stokes beams being a signal called a CARS scattered signal, of angular frequency ωas=2ωp−ωs and the difference in the first and second detected signals being characteristic of a Raman resonance of the resonant medium. 
     According to another variant embodiment, the first and second nonlinear optical signals are detected respectively in symmetrical directions about the optical axis of the incident excitation beams, the difference being effected for each signal couple thus detected. 
     According to another embodiment, the excitation beam or beams are subject to an angular scan to intercept the sample at various positions of the interface between the resonant and non-resonant medium. 
     According to another variant embodiment, at least one of the excitation beams has a variable emission wavelength, allowing a spectrum of vibrational or electronic resonances of the resonant medium to be obtained. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Other advantages and characteristics of the invention will become apparent from reading the description, illustrated by the following FIGS.: 
         FIGS. 1A and 1B  (previously described), principle of Stokes and anti-Stokes emission in a Raman scattering process; 
         FIGS. 2A and 2B  (previously described), principle of CARS emission in two different modes; 
         FIGS. 3A and 3B  (previously described), illustrations of the resonant and non-resonant CARS process; 
         FIGS. 4A and 4B  (previously described), illustrations of cases α and β for implementation of the method according to the prior art; 
         FIG. 5  (previously described), numerical simulations of the results obtained with the method according to the prior art; 
         FIGS. 6A and 6B  (previously described), experimental results obtained with a symmetrical sample, by the method according to the prior art; 
         FIGS. 7A ,  7 B, illustrations of cases α and β for implementation of the method according to the invention; 
         FIGS. 8A ,  8 B, example of the experimental setup for implementation of the method according to the invention; 
         FIG. 9 , experimental results obtained with a sample of the type of that in  FIG. 8B ; 
         FIGS. 10A to 10D , images obtained by numerical simulation with a polystyrene bead of 3-μm diameter, immersed in an aqueous liquid with a refraction index n=1.33, by the method according to the invention; 
         FIG. 11 , example of the experimental setup for implementation of the method according to the invention according to a variant embodiment; 
         FIGS. 12A ,  12 B, illustrations of cases α and β for implementation of the method in the example of  FIG. 11 ; 
         FIG. 13 , diagram of the geometric conditions for implementing the CARS scattering at an axial interface between resonant and non-resonant media; 
         FIG. 14A to 14E , illustration of the deviation of the CARS scattered signal as a function of the relative position of the focal point of the excitation beams with an axial interface between resonant and non-resonant media; 
         FIGS. 15A to 15C , curves obtained by numerical simulation, illustrating the shift in the CARS scattered signal as function of the parameter. ζ=(ω p −ω s −Ω R )/Γ (normalised Raman shift) ( FIG. 15A ), the light intensities calculated respectively in the space of (k x &gt;0) and (k x &lt;0) and the difference in intensities, as a function of the parameter ζ ( FIG. 15B ) and the x position of the focal point of the excitation beams relative to an axial interface ( FIG. 15C ); 
         FIGS. 16A to 16C , diagrams illustrating 3 possible modalities for implementation of the CARS detection according to the invention. 
     
    
    
     DETAILED DESCRIPTION 
       FIGS. 7A and 7B  illustrate in two diagrams the principle of the detection method according to the invention in the case of CARS scattering. According to the method, a pump beam of angular frequency ω p  and a Stokes beam of angular frequency ω s , which are collinear, intercept a transverse interface  70 , that is, an interface having a non-zero component along a plane perpendicular to the axes of the incident beams (optical axis), between a non-resonant medium and a resonant medium. In general, the two beams are focused,  71  designating the common focal volume which intercepts the transverse interface  70 . According to the invention, the excitation beams cross the interface in a first direction, called case α, and are then reflected towards the sample in such a way as to intercept the same interface at substantially the same position, but in the opposite direction (case β). In the example of  FIG. 7 , the excitation beams first cross the interface in the direction non-resonant medium/resonant medium ( FIG. 7A , case α), then in the direction resonant medium/non-resonant medium ( FIG. 7B , case β). The CARS scattered signal intensities I α (Fwd) and I β (Fwd) are measured respectively in the two cases α and β, and their difference ΔI Fwd  is calculated, after calibration, to give a signal, which the Applicant has demonstrated is proportional to the imaginary part Im[χ (3)   1R ] of the 3rd order nonlinear susceptibility of the resonant medium. According to the invention, a single pulse of the pump beam and Stokes beam is used to excite the sample in cases α and β, allowing the signal-to-noise ratio to be increased as compared with the method according to prior art described in  FIGS. 4 to 6 . 
     Taking the difference of the CARS signals generated by an object and its mirror image about a plane perpendicular to the optical axis, the method according to the invention is called Dz-CARS in the following description (Dz standing for Differential imaging in Z symmetry). 
       FIG. 8A  illustrates an example device for implementing the detection method according to the invention. The detection device  800  generally comprises a laser system  801  permitting emission of a first excitation beam of angular frequency ω p  (pump beam) and of a second excitation beam of angular frequency ω s  (Stokes beam), which are collinear, the two excitation beams being symbolised by the arrow  802 . The device  800  also comprises an optical element, for example reflective sheeting  804 , allowing the two excitation beams to be directed into a first optical detection module of the device, generally assigned the reference numeral  803 , according to a main direction Z. 
     The laser system  801  comprises, for example, in a so-called bi-colour application, two spectrally narrow, tunable laser sources  808 , for example of the Ti:Sapphire type, emitting at wavelengths between 690 and 1000 nm, pumped by a pump laser  809 , Nd:YVO4 type emitting at 532 nm. The tuneable lasers emit, for example, picosecond pulses, typically of the order of 3 ps, to form a pump excitation beam of angular frequency ω p  (of typical wavelength 730 nm) and Stokes excitation beam of angular frequency ω s . A pulse picker  810  may be used to reduce the pulse repetition frequency of the pump and probe excitation lasers without reducing the peak pulse power. Using a tuneable Stokes beam or pump beam enables, in particular, the anti-Stokes emission spectrum to be scanned for applications in spectroscopy aimed at determining the Raman spectrum of the resonant medium. Other tuneable laser sources may be used, for example, optical parametric oscillators (OPO), optical parametric amplifiers (OPA), picosecond Nd:glass oscillators, ytterbium or erbium-doped optical fibres, etc. The sources may also be nanosecond or femtosecond laser sources, depending on the spectral width of Raman lines to be observed. However, nanosecond pulses, although very good spectrally, have a lower peak power than ps pulses. Moreover, the thermal effects associated with ns pulses are more capable of damaging biological samples. Raw femtosecond pulses are generally too wide spectrally. In condensed phase (solid or liquid), the line widths are around 10-20 cm −1 , corresponding to the use of ps pulses. 
     In the example in  FIG. 8A , the first optical detection module  803  comprises a focusing lens  807  intended for focusing the pump and Stokes beams in a common focal volume for analysis of the sample  805  represented in  FIG. 8B . Using a focusing lens is particularly appropriate in microscopy applications. However, it is not essential for the emission of the CARS signal to work using focused beams, in particular when studying thin samples. In this example, the sample is formed, as in the example of  FIG. 6B , of a layer  61  of DMF (N,N-dimethylformamide) between two glass slides  62 ,  63 . The first optical detection module  803  similarly comprises a collecting lens  811  allowing the emitted nonlinear optical signal to be collected, in this example the CARS scattered signal, and a detector  816 , for example a point detector of the avalanche photodiode (APD), rapid photodiode (PIN), or photomultiplier (PMT) type, preceded by a collecting lens  818  and a filter  812  to cut the residual excitation beams. 
     In this example, the transition from problem a to problem R for a given sample is made by returning the pump and Stokes beams as indicated in  FIG. 8A  by means of a mirror  813 , the coefficient of reflection of which is appropriate for the wavelengths of the excitation beams on the one hand and of the CARS scattered signal on the other, in such a way as to reflect the pump and Stokes beams and transmit the CARS scattered signal resulting from the interaction of the excitation beams with the sample. In this situation, during the first passage of the incident pulses, a first CARS signal is projected and collected by the detector  816 ; this is then it therefore concerns case α and the collected signal is I α (Fwd). The pump and Stokes pulses are then reflected by the mirror to be returned on to the sample, which is then seen to be in case β, in a second optical detection module overall assigned the reference numeral  806  in  FIG. 8A . The second optical detection module  806  comprises in common with the first optical detection module, lenses  811  and  807 , but lens  811  acts as a focusing lens for the excitation beams returned by the mirror  813  and lens  807  acts as a collecting lens for the nonlinear optical signal resulting from interaction of the reflected excitation beams with the sample  805 . The second optical module  806  furthermore comprises a detector  817 , for example a point detector of the same type as detector  816 , preceded by a collecting lens  819  and a filter  820  to cut the residual excitation beams. The signal collected behind by detector  817  is then I β (Fwd). The difference of signals I α (Fwd)−I β (Fwd) is operated in real time, which the Applicant has shown to be proportional to the Raman spectrum of the resonant medium, by means of a processing unit labelled  830  in  FIG. 8A . The reflective sheeting  804  is advantageously dichroic sheeting, allowing the excitation beams emitted by the laser source  801  to be reflected towards the sample  805  (case α) while transmitting the CARS scattered signal in case β. The focusing  807  and collecting  811  lenses are advantageously identical, enabling a symmetrical set-up in cases α and β. In practice, detectors  816 ,  817  are calibrated prior to measurement. For example, this calibration is performed on a sample comprising only solvent. 
     As can be seen in  FIG. 8B , the device according to the invention allows the same pump and Stokes pulses to intercept the sample at the same position of the transverse interface, in cases α and β respectively. The method can thus be used with any type of sample presenting an interface between a resonant medium and a non-resonant medium, and not just a symmetrical or reversible sample. 
     According to one example, the device  800  also comprises an excitation beam scanning device in plane XY of the sample (not shown). This scanning device may be useful at one and the same time in spectroscopy applications, for adjusting the focal point of the excitation beams over a transverse interface of the resonant and non-resonant media forming the sample, that is, in imaging applications. It may act as a device allowing displacement of the sample, or preferably, as an excitation beam scanning device. A spherical excitation beam-reflecting mirror  813  can advantageously be used in such a way as to reflect the excitation beams in an antiparallel direction, which will allow the reflected excitation beams (case β to intercept the sample at the same position as that of the incident beams (case α). 
       FIG. 9  illustrates the experimental results obtained with the device of  FIG. 8A  and a sample of the type in  FIG. 8B , in which a fine layer of DMF (N,N-dimethylformamide) serves as the resonant medium between two glass slides (here serving as the non-resonant medium). The wavelength of the pump beam is 730 nm, that of the Stokes excitation beam around 814 nm. The numerical aperture in air of lenses  807 ,  811  is 0.6. In  FIG. 9 , as a function of the Raman shift respectively, curve D 1  illustrates the CARS scattered signal measured in the DMF; curve D 2 , the intensity I α (Fwd) measured in case α ( FIG. 6B ); curve D 3 , the intensity I β (Fwd) measured in case β, curve D 4 , the difference ΔI Fwd , curve D 5  (dotted line) the Raman spectrum, and curve D 6  the CARS scattered signal measured on the glass. Curve D 1  reveals clearly the distortion effect due to the non-resonant contribution of the resonant medium, while the difference ΔI Fwd  is superimposed exactly on the Raman spectrum of DMF (dotted line). Thus one can appreciate the capacity of Dz-CARS to extract the Raman spectrum of the resonant medium without any distortion due to the non-resonant part of the resonant medium. 
     The experimental results demonstrate the relevance of the Dz-CARS approach for noiseless, non-resonant CARS spectroscopy, and with a precision distinctly improved by comparison with the prior art, as illustrated in  FIG. 6A . Furthermore, the method according to the invention allows perfect identification of the position on the interface on which one is working, and focusing of identical excitation pulses at the same position of the interface as in cases α and β, notably enabling microscopy applications. 
       FIGS. 10A to 10D  present numerical simulations obtained with the method according to the invention on another type of sample. The images are calculated by taking as a sample a bead of 3-μm diameter in an aqueous solvent (pump wavelength 730 nm, Stokes wavelength 814 nm, numerical aperture in water of the excitation lens 1.2, numerical aperture in water of the collecting lens 1.2). The image is calculated in each case in a plane XZ of the bead corresponding to a longitudinal plane comprising the direction of incidence of the excitation beams.  FIGS. 10A and 10B  represent an image of the bead in conventional detection, in other words only the CARS scattered signal in case α is represented. On-resonance ( FIG. 10A ), the signal is more intense than off-resonance ( FIG. 10B ), but the contrast difference is weak due to the non-resonant contributions of the bead and of its environment.  FIGS. 10C and 10D  represent images of the bead on-resonance and off-resonance, but calculated with the Dz-CARS method according to the invention, in other words by subtracting the CARS scattered signals in cases α and β, with a setup of the type of  FIG. 8A . Off-resonance ( FIG. 10D ), the contrast is zero, because the difference in the signals which contain only a non-resonant contribution is cancelled out. In contrast, in  FIG. 10C , calculated on-resonance, the contrast at the transverse interfaces is maximal. These results establish the feasibility of Dz-CARS in a microscopy configuration. 
       FIG. 11  illustrates an example experimental setup for implementation of the detection according to the invention according to a variant embodiment; The setup is substantially identical to that of  FIG. 8A , but the point detectors  816 ,  817  are replaced by matrix detectors  901 ,  902 , for example of CCD or CMOS type. According to this variant, the difference in CARS scattered signals integrated respectively for cases α and β in all the space of wave vectors             contained in the numerical aperture of detection lenses is no longer detected as previously; instead the difference in CARS scattered signals in symmetrical directions about the optical axis of the excitation beams incident on the sample is measured, the signals being detected for the first in case α, for the second in case β.
     Thus, as is apparent in  FIG. 12A , in case α, the CARS scattered signal is measured in a direction represented by the wave vector            , of coordinates k x , k y  in the XY projection plane perpendicular to the main axis z, and in case β, the CARS scattered signal is measured in a direction represented by the wave vector           of coordinates −k x , −k y  in the XY projection plane. Here, as previously, case α corresponds to the generation of a CARS scattered signal resulting from the interaction of incident excitation beams with a sample, while case β corresponds to the generation of a CARS scattered signal resulting from the interaction of reflected excitation beams with the sample.
     The Applicant has in fact demonstrated, both theoretically and experimentally, that besides allowing detection at the transverse interfaces of the sample, this method would allow detection at the axial interfaces of the sample, in other words having a non-zero component along the optical axis of the incident excitation beams. Hereinafter in the application, the method is called D-CARS. 
     For an improved understanding of D-CARS,  FIGS. 13 to 15  illustrate, the following approach called Dk-CARS for detection at axial interfaces (Dk standing for Differential imaging in K-space). 
       FIG. 13  represents a sample comprising the resonant medium  131 , for example a medium containing the medium to be analysed, in other words the medium of biological interest, and the non-resonant medium  132 , typically a medium containing the solvent. The 3rd order nonlinear susceptibility is defined in the resonant medium  131  by a resonant term χ (3)   1R  and a non-resonant term χ (3)   1NR . In the non-resonant medium  132 , it is defined by the non-resonant term χ (3)   2NR . According to this aspect of the method according to the invention, the pump excitation beam of angular frequency ω p  and probe excitation beam of angular frequency ω s , which are collinear, are incident on the sample in a focal volume  135 , intercepting an axial interface  133  of the sample. According to this aspect of the method, as is explained in detail in what follows, analysis of the light intensity of the nonlinear optical beam in the space of wave vectors            , that is, in the space of the emission directions of the signal emitted by the CARS process, on both sides of the interface, this intensity being indicated in  FIG. 13  I Fwd (         ) and I Fwd (         ) on both sides of the interface respectively, the abbreviation “Fwd” representing the CARS forward scattered signal, as opposed to the signal called “Epi”, scattered in a backward direction.
     Indeed, the Applicant has demonstrated experimentally and theoretically that at an axial interface, the signal emitted by the CARS process is deviated at the resonance. 
       FIGS. 14A to 14E  represent, by a series of diagrams, the deviation of the CARS scattered signal as a function of the relative position of the pump and Stokes beams incident with the interface.  FIGS. 14A to 14E  represent the active CARS volume  135  (focal point of the pump and Stokes beams) which is displaced through a CARS object  140  (each illustration corresponds to a different position of the active volume in the object). The CARS object is considered as resonant when the medium surrounding the object is considered as non-resonant (in the rest of the description it will be called “the solvent”). It appears that, at the interfaces between the CARS object and the solvent, the CARS scattered signal is affected by a deviation (or tilt). The Applicant has demonstrated that this deviation arises from a purely interferential process between the CARS object and the solvent and is in no way due to refractive effects. In the two illustrations  1  ( FIGS. 14A and 14E ), the CARS volume is focused in the solvent and the CARS scattered signal is emitted in the normal direction (parallel to the axis of incidence of the pump and Stokes beams, symbolised by the arrow  141 ); in illustration  2  ( FIG. 14B ), the CARS volume is focused on the interface between the CARS object and the solvent, the CARS scattered signal is then emitted at a positive angle α (relative to the axis of incidence of the pump and Stokes beams), thus deviating the beam in a direction defined by (k x &gt;0) in the space of wave vectors            . In illustration  3  ( FIG. 14C ), the CARS volume is centred in the CARS object, the CARS signal is then intense and is directed in the normal direction (parallel to the axis of incidence of the pump and Stokes beams). A similar situation is then found in the following illustrations (illustration  4 ,  FIG. 14D  and illustration  1 ,  FIG. 14E ); however, it is important to note that in illustration  4 , α is negative and corresponds to a deviation in a direction defined by (k x &lt;0). The Applicant has demonstrated both theoretically and experimentally that the change in angle α as a function of the normalised parameter ζ=(ω p −ω s −Ω R )Γ (where Γ is the spectral width of the vibrational line studied), follows the phase of the tensor χ (3)   1 =χ (3)   1R +χ (3)   1NR  describing medium  1 . The Applicant has also demonstrated that by analysing the CARS signal in the symmetrical scattering directions, it is possible to determine the Raman spectrum.
       FIG. 15A  shows the results of a rigorous numerical calculation considering the vectorial nature of the pump and Stokes beams focused on an axial interface between a resonant medium  1  and a non-resonant medium  2  ( FIG. 13 ). The analysis consists in studying in the space of wave vectors             the deviation of the CARS scattered signal emitted as a function of the normalised Raman shift ζ=(ω p −ω s −Ω R )/Γ. Off-resonance (ζ=−10), the beam is centred, while on-resonance (ζ=0), an angular displacement clearly appears.
       FIGS. 15B and 15C  represent numerical simulations in which the CARS scattered signal is integrated into the half-spaces (k x &gt;0) and (k x &lt;0) respectively, then the difference in the signals thus integrated is determined.  FIG. 15B  shows the CARS spectra integrated on the half-spaces (k x &gt;0) and (k x &lt;0) when the pump and Stokes beams are focused on the interface (x=0), as well as their difference ΔI. This difference exactly follows the Raman spectrum given by Im[χ (3)   1R ]. This demonstrates the pertinence of the Dk-CARS approach for a CARS spectroscopy without non-resonant noise. It is thus for example possible, by varying the frequency of the Stokes beam, to determine the Raman spectrum of the resonant medium. FIG.  15 CB represents the CARS signals integrated on half-spaces (k x &gt;0) and (k x &lt;0) as a function of the focal point of the pump and Stokes beams relative to the interface. Their difference is non-zero uniquely in the vicinity of the interface (x=0). A non-resonant CARS image without background noise can thus be obtained in the vicinity of the interface. 
       FIGS. 16A to 16C  thus present  3  possible detection modalities for the D-CARS microscopy combining the Dz-CARS and Dk-CARS approaches. For each detection modality, a numerical simulation represents the image obtained for a bead of 3-μm diameter in an aqueous solvent (pump wavelength 730 nm, Stokes wavelength 814 nm, numerical aperture in water of the excitation lens 1.2, numerical aperture in water of the collecting lens 1.2).  FIG. 16A  represents the XZ detection modality enabling detection at the interfaces perpendicular to the X axis and at the interfaces perpendicular to the Z axis. For this, the difference of the CARS scattered signals in cases α and β respectively is calculated by integrating the CARS signal in space (k x &gt;0) (case α,  FIG. 7A ) and in space (k x &lt;0) (case β,  FIG. 7B ), the referential system selected being that of the direction of the excitation beams. Thus, for example, by changing the relative position of the focal point of the pump and Stokes beams, the image of  FIG. 16A  is obtained in an equatorial plane of the bead.  FIG. 16B  represents the YZ detection modality allowing detection at the interfaces perpendicular to the Y axis and at the interfaces perpendicular to the Z axis. In this example, for different positions, the difference in light intensities integrated in space (k y &gt;0) (case α,  FIG. 7A ) and in space (k y &lt;0) (case β,  FIG. 7B ) is calculated.  FIG. 16C  shows detection modality XYZ. The image is calculated by taking the two by two difference in light intensities I α (k x , k y ) and I β (−k x , −k y ) measured in two opposing directions             (k x , k y , k 2 ) and          ′ (−k x , −k y , k z ), in cases α and β respectively, the directions being contained in the angular cone, the aperture angle of which is defined by the numerical aperture for collection of the CARS scattered signal (for example 1.2 in water). Again, the coordinates of wave vectors           and          ″ are expressed in the reference of the excitation beams specific to cases α and β respectively.
     In the example of  FIG. 11 , it will advantageously be possible to provide means for angular scanning of the excitation beams, in particular for microscopy applications. As in the example of the device in  FIG. 8A , it will be possible to select a spherical mirror  813  for reflecting the excitation beams  813 . Furthermore, it is advantageous to position cameras  901 ,  902  in the exit pupils of lenses  811 ,  807  respectively, so as to keep the direction of incidence of the excitation beams centred on the camera in each of cases α and β. A calibration of cameras in solution is also possible to identify, in each case α and β, and for each scanning angle, the direction of the excitation beams relative to which the deviation of the CARS scattered signal will be measured. 
     Dz-CARS or D-CARS detection has been described by means of the implementation examples of  FIGS. 8A and 11  in a bi-colour application, using two spectrally narrow laser sources. In the application called multiplex, a spectrally wide emission source of the Stokes beam can be chosen, generated, for example, by a femtosecond pulse or by a supercontinuum generated by an optical fibre or another dispersive medium. The pump signal remains spectrally narrow. In this application, it will be possible to acquire a Raman spectrum in a single pulse, for example, by using two slit spectrometers or a single spectrometer equipped with a CCD camera into which the two signals detected in the two cases α and β are injected. In this application, it is a matter of acquiring the spectrums in each of the cases (α or β) and making their difference. 
     In an application called a tricolour application, three wavelengths of associated frequencies ω 1 , ω 2  and ω 3  are used to generate a CARS signal at angular frequency ω 1 −ω 2 +ω 3 . The CARS signal may be rendered non-resonant without noise by detecting the signals at the angular frequency ω 1 −ω 2 +ω 3  in the cases (α or β) and taking their difference. 
     Although the detection method has been described in the case of CARS scattering, it applies just as well to other nonlinear, 2nd or 3rd order processes, both for spectroscopy applications and for microscopy applications by detection at axial interfaces, thus enabling the interfaces between the resonant and non-resonant media to be revealed. In each case, an analysis of the nonlinear optical signal resulting from the interaction of one or more excitation beams is performed with a sample presenting an interface between a resonant medium and a non-resonant medium. This spatial analysis allows either the interface between the resonant medium and the non-resonant medium to be revealed, or a spectrum of the resonant medium to be characterised. 
     According to one example, a process for generating the third resonant harmonic can be used wherein the resonance is an electronic resonance, by exciting a sample comprising an interface between a resonant medium and a non-resonant medium with a single pump excitation beam, of angular frequency ω p . For example a picosecond or femtosecond laser source of the oscillator type Ti: Sapphire, Nd: glass, or ytterbium or erbium-doped optical fibres. 
     According to another example, a four-wave mixing process can be used wherein the resonance is an electronic resonance, by exciting a sample comprising an interface between a resonant medium and a non-resonant medium with a single pump excitation beam, of angular frequency ω p . For example a picosecond or femtosecond laser source of the oscillator type Ti: Sapphire, Nd: glass, or ytterbium or erbium-doped optical fibres. 
     The two examples described above deal with electronic resonances. They are found in atoms, molecules, semi-conductor crystals, etc. 
     According to another embodiment, the second resonant harmonic can be excited with a single pump beam, or the sum of the frequency can be made with a pump beam and probe beam (nonlinear effect of the 2nd order). 
     Although described using a certain number of detailed example embodiments, the detection device and method according to the invention comprise different variants, modifications and developments which will be obvious to the person skilled in the art, it being understood that these different variants, modifications and developments fall within the scope of the invention, as defined by the claims below.