Abstract
According to one aspect, the present description relates to a device for detecting an SRS resonant non-linear optical signal induced in a sample. The device comprises a light source configured for emitting a first train of pump pulses at a first angular frequency and a second train of Stokes pulses at a Stokes second angular frequency, and first and second amplitude modulators configured to amplitude modulate the train of pump pulses at a first modulation frequency and the train of Stokes pulses at a second modulation frequency different from the first modulation frequency, respectively. The device further comprises optomechanical means for making interact in the sample said amplitude-modulated trains of pump and Stokes pulses, means for optical detection of first and second non-linear optical signals at the first angular frequency and second angular frequency, respectively, and means of synchronous detection of the first and second optical signals at said second modulation frequency and at the first modulation frequency, respectively, allowing an SRL first signal and an SRG second signal that are characteristic of the molecular vibrational resonance of the sample to be extracted.
Claims
1. A device for detecting a resonant non-linear optical signal of stimulated-Raman-scattering type induced in a sample, the device comprising: a light source configured for emitting a first train of pump pulses at a first angular frequency and a second train of Stokes pulses at a second angular frequency, the angular frequencies being such that a difference between the first and second angular frequencies is equal to an angular frequency of molecular vibrational resonance of the sample, the first and second trains of pulses being temporally synchronized; a first amplitude modulator configured to amplitude modulate the first train of pump pulses at a first modulation frequency and a second amplitude modulator configured to amplitude modulate the second train of Stokes pulses at a second modulation frequency different from the first modulation frequency; optomechanical means for making interact in the sample said amplitude-modulated trains of pump and Stokes pulses; first optical detecting means configured for optical detection of a first non-linear optical signal at said first angular frequency, said first non-linear optical signal resulting from the interaction, in the sample, of the amplitude modulated pump and Stokes light pulses in the sample, and means of synchronous detection at said second modulation frequency, allowing a first signal characteristic of the molecular vibrational resonance of the sample to be extracted from the first non-linear optical signal thus detected; second optical detecting means configured for optical detection of a second non-linear optical signal at said second angular frequency, said second optical signal resulting from the interaction, in the sample, of the amplitude modulated pump and Stokes light pulses in the sample, and means of synchronous detection at said first modulation frequency, allowing a second signal characteristic of the molecular vibrational resonance of the sample to be extracted from the second optical signal thus detected; electronic processing means configured to compare said first and second signals characteristic of the molecular vibrational resonance of the sample in order to determine the presence of artefacts.
2. The device according to claim 1, wherein the comparison of said first and second signals characteristic of the molecular vibrational resonance of the sample comprises the computation of a ratio between said signals.
3. The device according to claim 1, wherein said electronic processing means are configured to further compute a sum and/or a difference of said first and second signals characteristic of the molecular vibrational resonance of the sample.
4. The device according to claim 1, wherein said optomechanical means comprise an optical element for focusing the trains of pulses into a common focal volume.
5. The device according to claim 4, wherein said optomechanical means further comprise means for moving relatively the focal volume in the sample.
6. The device according to claim 1, wherein the first and second trains of pulses are trains of frequency-chirped pulses centred on the first and second angular frequencies, respectively.
7. The device according to claim 6, further comprising a delay line configured to generate a time shift between the pulses of the first train of pulses and the pulses of the second train of pulses, such as to make vary the frequency of molecular vibrational resonance of the sample at which the non-linear optical signal is detected.
8. The device according to claim 1, characterized in that it is at least partially fibre-based.
9. A method for detecting a resonant non-linear optical signal of stimulated-Raman-scattering type induced in a sample, comprising: the emission of a first train of pump pulses at a first angular frequency and a second train of Stokes pulses at a second angular frequency, the angular frequencies being such that a difference between the first and second angular frequencies is equal to an angular frequency of molecular vibrational resonance of the sample, the first and second trains of pulses being temporally synchronized; the amplitude modulation of said first train of pump pulses and said second train of Stokes pulses at a first modulation frequency and at a second modulation frequency different from the first modulation frequency, respectively; the interaction in the sample of said amplitude-modulated trains of pump and Stokes pulses; a first optical detection of a first non-linear optical signal at said first angular frequency, said first non-linear optical signal resulting from the interaction of the light pulses in the sample, and a first synchronous detection at said second modulation frequency, allowing a first signal characteristic of the molecular vibrational resonance of the sample to be extracted from the first non-linear optical signal thus detected; a second optical detection of a second non-linear optical signal at said second angular frequency, said second non-linear optical signal resulting from the interaction of the light pulses in the sample, and a second synchronous detection at said first modulation frequency, allowing a second signal characteristic of the molecular vibrational resonance of the sample to be extracted from the second non-linear optical signal thus detected; a comparison of said first and second signals characteristic of the molecular vibrational resonance of the sample in order to determine the presence of artefacts.
10. The method according to claim 9, wherein said comparison of said first and second signals characteristic of the molecular vibrational resonance of the sample comprises the computation of a ratio between said two signals.
11. The method according to claim 9, further comprising the computation of a sum of said two signals characteristic of the molecular vibrational resonance of the sample, the resultant signal being doubled and at least partially freed from said artefacts.
12. The method according to claim 9, further comprising the computation of a difference of said two signals characteristic of the molecular vibrational resonance of the sample, the resultant signal being representative of at least some of said artefacts.
13. The method according to claim 9, wherein the first and second trains of pulses are trains of frequency-chirped pulses centred on the first and second angular frequencies, respectively.
14. The method according to claim 13, further comprising the generation of a time shift between the pulses of the first train of pulses and the pulses of the second train of pulses, such as to make vary the frequency of molecular vibrational resonance of the sample at which the non-linear optical signal is detected.
15. The method according to claim 9 applied to Raman vibrational imaging, wherein the interaction in the sample of said amplitude-modulated trains of pump and Stokes pulses comprises focusing the trains of pulses into a common focal volume and a relative movement of the focal volume in the sample, the first and second signals characteristic of the molecular vibrational resonance of the sample being two-dimensional signals.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0064] Other advantages and features of the invention will become apparent on reading the description, which is illustrated by the following figures:
[0065] FIG. 1A, which has already been described, shows a simplified schematic illustrating the principle of stimulated Raman scattering (SRS);
[0066] FIG. 1B, which has already been described, shows a simplified schematic illustrating the SRL and SRG processes;
[0067] FIG. 1C, which has already been described, shows a schematic of an example of an SRS microscope according to the prior art;
[0068] FIG. 2 shows a schematic of an example of an SRS microscope according to the present description;
[0069] FIG. 3A shows a schematic illustrating the temporal modulation at the first frequency and the frequency spectrum of a train of pump pulses in one example of implementation of a method according to the present description;
[0070] FIG. 3B illustrates the temporal modulation at the second frequency and the frequency spectrum of a train of Stokes pulses in one example of implementation of a method according to the present description;
[0071] FIG. 3C illustrates the temporal modulation and the frequency spectrum of an SRL signal detected at the pump wavelength, in one example of implementation of a method according to the present description;
[0072] FIG. 3D illustrates the temporal modulation and the frequency spectrum of an SRG signal detected at the Stokes wavelength, in one example of implementation of a method according to the present description;
[0073] FIG. 4A illustrates, via schematics, the detection at the second frequency and at the pump wavelength of the optical signal resulting from the non-linear interaction, in steps of an example of a method according to the present description;
[0074] FIG. 4B illustrates, via schematics, the detection at the first frequency and at the Stokes wavelength of the optical signal resulting from the non-linear interaction, in steps of an example of a method according to the present description;
[0075] FIG. 5A shows experimental SRL, SRG and SRL/SRG images of the surface of an oyster shell, which images were obtained with an SRS microscope according to the present description, at the resonant frequency of phosphate (1090 cm.sup.−1);
[0076] FIG. 5B shows experimental SRL, SRG and SRL/SRG images of mouse nerve cells, which images were obtained with an SRS microscope according to the present description, at the resonant frequency of the CH.sub.2 bond (2850 cm.sup.−1);
[0077] FIG. 6 shows experimental SRL, SRG and SRL+SRG images and experimental images of the signal-to-noise ratios of SRL, SRG and SRL+SRG of the surface of an onion cell, which images were obtained with an SRS microscope according to the present description, off resonance, at the resonant frequency of the CH.sub.2 bond (2850 cm.sup.−1);
[0078] FIG. 7 shows a schematic of another example of an SRS microscope according to the present description, in an endoscopic mode using an optical fibre;
[0079] FIG. 8 shows schematics illustrating pump and Stokes pulses, after they have been frequency chirped, for two time-delay values separated by Δt.
DETAILED DESCRIPTION OF THE INVENTION
[0080] In the figures, the elements have not been shown to scale in order to be more easily seen. FIG. 2 shows a simplified schematic illustrating an example of a device 200 for detecting a resonant non-linear optical signal of stimulated-Raman-scattering (SRS) type induced in a sample S, and more precisely a schematic illustrating an example of an SRS microscope according to the present description. FIGS. 3A-3D show schematics illustrating the optical intensity as a function of time of trains of pulses at the pump and Stokes angular frequencies during the method and schematics showing the frequency spectrum of said trains of pulses.
[0081] The device 200 comprises a light source 210 that is configured to emit a first beam 202, or “pump beam”, formed from a first train of pump pulses at a first angular frequency ω.sub.p and that is configured to emit a second beam 203, or “Stokes beam” formed from a second train of Stokes pulses at a second angular frequency ω.sub.s, these angular frequencies being such that a difference ω.sub.p−ω.sub.s between the first and second angular frequencies is equal to an angular frequency Ω.sub.R of molecular vibrational resonance of the sample S that it is sought to observe.
[0082] The first train of pulses and the second train of pulses are synchronized temporally in order to allow the interaction of the pump and Stokes pulses in the sample.
[0083] The pulses are for example picosecond pulses, of durations comprised between about 1 ps and about 10 ps and for example between about 1 ps and about 3 ps, and of spectral width comprised between about 15 cm.sup.−1 and about 5 cm.sup.−1, or may be frequency-chirped pulses as will be described in more detail below. Typically, the pulses are emitted at rates of a few tens of MHz and comprised for example between about 10 MHz and about 100 MHz and for example about 80 MHz, for a duration of about 1 μs.
[0084] The light source 210 may comprise synchronized independent lasers 211, 212, as shown in FIG. 4.
[0085] In other exemplary embodiments, the light source 210 may comprise a laser system with a master laser that emits trains of pulses at the pump angular frequency and a laser OPO (or optical parametric oscillator) that receives from the master laser the pump pulses and that is configured to emit pulses at the Stokes angular frequency. The light source 210 may also comprise a laser system with a master laser and two OPOs configured to generate the trains of pump and Stokes pulses from pulses emitted by the master laser. In both cases, the trains of pulses are automatically synchronized. It is moreover possible to modify the angular frequency of the Stokes pulses, and optionally of the pump pulses in the case of two OPOs since OPOs are wavelength tunable.
[0086] For example, in the case of a laser system consisting of a master laser and of an OPO, the master laser may emit pump pulses with a pump angular frequency corresponding to a wavelength comprised between about 1000 nm and about 1100 nm and for example between about 1030 nm and about 1065 nm, this wavelength range covering the wavelengths of emission of an ytterbium laser and of a YAG laser. The OPO may emit Stokes pulses with a Stokes angular frequency corresponding to a wavelength comprised between about 600 nm and about 1000 nm and for example between about 640 nm and about 960 nm.
[0087] In the case of a laser system comprising a master laser and two OPOs, the master laser may emit pulses with an angular frequency corresponding to a wavelength comprised, as above, between about 1000 nm and about 1100 nm and for example between about 1030 nm and about 1065 nm, and the OPOs may each emit pump and Stokes pulses with angular frequencies corresponding to wavelengths comprised between about 600 nm and about 1000 nm and for example between about 640 nm and about 960 nm. In the example of FIG. 2, a delay line 221 allows the trains of pump and Stokes pulses to be synchronized temporally in order to ensure a temporal overlap of the pulses in the sample.
[0088] As will be described in more detail below, the delay line may be configured to introduce a variable time delay. It may be positioned on either or both of the pump and Stokes channels.
[0089] Moreover, the device 200 comprises a first amplitude modulator 231 configured to amplitude modulate the first train of pump pulses at a first modulation frequency f.sub.1 and a second amplitude modulator 232 configured to amplitude modulate the second train of Stokes pulses at a second modulation frequency f.sub.2 different from the first modulation frequency. As a result, pump and Stokes beams formed from pulse trains that are amplitude modulated at the frequencies f.sub.1 and f.sub.2, and that are denoted 204 and 205 in FIG. 2, respectively, are produced.
[0090] FIG. 3A thus illustrates, according to one example, the intensity I.sub.p of a train of pulses 204 at the pump angular frequency ω.sub.p, which train is amplitude modulated at the frequency f.sub.1 (curve 32). Curve 31 in FIG. 3A illustrates the power spectral density (PSD) as a function of frequency for the train of pulses 204. As may be seen in FIG. 3A, the frequency spectrum comprises, at low frequencies, a 1/f contribution related to noise, and the contribution at f.sub.1. It will be noted that the frequency peak at the repetition frequency of the pulses has not been shown in the figure.
[0091] In the same way, FIG. 3B illustrates, according to one example, the intensity I.sub.S of a train of pulses 205 at the pump angular frequency ω.sub.p, which train is amplitude modulated at the frequency f.sub.2 (curve 34). Curve 33 in FIG. 3A illustrates, as above, the power spectral density as a function of frequency for the train of pulses 205 with, in particular, the peak at the frequency f.sub.2 corresponding to the modulation of the train of pulses. The amplitude modulators 231, 232 are for example acousto- or electro-optical modulators that receive modulation signals from a radiofrequency (RF) generator 230. The modulation frequencies f.sub.1 and f.sub.2 are for example comprised between 1 MHz and 40 MHz.
[0092] Advantageously, they are selected so as not to be multiples of one another in order to avoid, when the amplitude modulators are not perfect and generate harmonics, generating pump and Stokes beams at the same frequencies.
[0093] In the example of FIG. 2, the device 200 further comprises optomechanical means for making interact in the sample S said amplitude-modulated trains of pump and Stokes pulses 204, 205. The optomechanical means comprise, in this example, reflective elements 261, 262, 263, which for example include a dichroic mirror 262, and a microscope objective 252 that is configured to focus the pump and Stokes pulses into a common focal volume in the sample S. The microscope objective 252 and the steering mirror 263 are, in this example, arranged in the body of a microscope 250. The microscope objective is for example a microscope objective of numerical aperture comprised between about 0.3 and about 1.3 and for example of a numerical aperture NA=0.5. The microscope objective is moreover advantageously achromatic in the wavelength domain of interest, for example in the near infrared (600-1100 nm).
[0094] According to exemplary embodiments, the optomechanical means may also comprise, on each of the pump and Stokes channels, a telescope (not shown) of given magnification, allowing the divergence of the pump and Stokes beams to be adjusted in order to optimize their spatial overlap at the focal point of the microscope objective. For example, the pump and Stokes beams are excited in their fundamental TEMOO mode so that the electric and magnetic fields are both perpendicular to the direction of propagation of these signals; the pump and Stokes beams are for example linearly polarized with the same polarization direction, allowing the signal to be optimized in a homogenous medium.
[0095] The optomechanical means may also comprise a motorized stage 256 allowing the sample S to be moved relative to the common focal volume of the trains of pulses, in order to form an image of the sample for an application of the device to SRS imaging. Alternatively, or in addition, a system 244 scanning the pump and Stokes beams, for example comprising two galvanometric mirrors 241, 242, may also be used to move the focal volume in the sample.
[0096] The device 200 moreover comprises first optical detecting means configured for optical detection, at the pump angular frequency ω.sub.p, of a first non-linear optical signal 206 resulting from the interaction of the light pulses in the sample.
[0097] FIG. 3C illustrates, according to one example, the intensity I.sub.p of the first non-linear optical signal 206 at the pump angular frequency W.sub.p (curve 36), and curve 35 in FIG. 3C illustrates the power spectral density as a function of frequency for the optical signal 206. As may be seen in FIG. 3C, in addition to the modulation at f.sub.1 (351) provided by the modulator 231 (FIG. 2) an SRL signal appears at the frequency f.sub.2 (352).
[0098] The device 200 also comprises second optical means configured for optical detection, at the Stokes angular frequency ω.sub.S, of a second non-linear optical signal 207, the second optical signal resulting from the interaction of the light pulses in the sample.
[0099] In the same way, FIG. 3D illustrates, according to one example, the intensity I.sub.S of the second non-linear optical signal 207 at the pump angular frequency ω.sub.s (curve 37), and curve 38 in FIG. 3D illustrates the power spectral density as a function of frequency for the optical signal 207. As may be seen in FIG. 3D, in addition to the modulation at f2 (362) provided by the modulator 232 (FIG. 2) an SRG signal appears at the frequency f.sub.1 (361).
[0100] In the example of FIG. 2, the first and second optical detecting means share a collecting objective 254, which is for example arranged in the microscope body 250, and a set of reflective elements 264, 265, which includes a dichroic mirror 265 allowing the two detection channels to be split. The collecting objective 254 is for example a microscope objective of higher numerical aperture than the microscope objective 252, and for example of a numerical aperture NA=0.60, in order to collect the trains of pulses returned from the sample without the need for a diaphragm.
[0101] Moreover, the first optical detecting means comprise a first optical detector 271 and the second optical detecting means comprise a second optical detector 272. The first and second detectors 271, 272 are for example photodiodes that are sensitive at the pump and Stokes angular frequencies, respectively. Each detection channel may moreover comprise one or more optical conjugating elements and optical filtering means (not shown), an interference optical filter for example, allowing the radiation at the angular frequency of interest to be selected for each detector.
[0102] In the example of FIG. 2, the first and second optical detecting means are configured for a detection in forward configuration. Of course, it is entirely possible to envisage first and second optical detecting means configured for a detection in epi configuration, which is especially advantageous for thick/not very transparent samples. In this case, the collected signals depend on the back-scattering nature of the sample.
[0103] As illustrated in FIG. 2, the device 200 moreover comprises first means 281 of synchronous detection, allowing, at the second modulation frequency f.sub.2, and from the first non-linear optical signal 206 detected by the first optical detector 271, a first signal 208 characteristic of the molecular vibrational resonance of the sample to be extracted. As will be explained in more detail below, the signal 208 is none other than the SRL signal. The device 200 also comprises second means 282 of synchronous detection, allowing, at the first modulation frequency f.sub.1, and from the second non-linear optical signal 207 detected by the second detector 272, a second signal 209 characteristic of the molecular vibrational resonance of the sample to be extracted. As will be explained in more detail below, the signal 209 is the SRG signal. The device 200 lastly comprises electronic processing means 290 that are configured to compare the first signal characteristic of the molecular vibrational resonance of the sample S (SRL signal) and the second signal characteristic of the molecular vibrational resonance of the sample S (SRG signal) in order to determine whether artefacts are present, as will be illustrated by means of FIGS. 5A, 5B and 6.
[0104] The first and second means 281, 282 of synchronous detection may for example comprise an analogue synchronous detection at the modulation frequencies f.sub.1, f.sub.2, respectively.
[0105] Alternatively, the synchronous detection of the signal on each of the channels may be achieved digitally, via digital processing of the signals output directly from the detecting optics.
[0106] FIGS. 4A and 4B illustrate the synchronous detection of the first non-linear optical signal 206 and the synchronous detection of the second non-linear optical signal 207, respectively, at the frequency f.sub.2 and at the frequency f.sub.1, respectively.
[0107] In FIG. 4A, the schematic 41 illustrates the intensity as a function of time of the train of pulses 204 (FIG. 2) at the pump angular frequency ω.sub.p, this train being modulated at the first frequency f.sub.1. The schematic 42 illustrates the intensity as a function of time of the first non-linear optical signal 206 detected by the first optical detector 271. As may be seen in the schematic 42, the intensity of the first non-linear optical signal 206 is decreased with respect to the optical signal 204. This decrease in intensity results from the SRL process on the train of pulses at the pump angular frequency, the process being induced by the interaction with the train of pulses at the Stokes angular frequency, which train is modulated at the frequency f.sub.2. The synchronous detection at the second frequency f.sub.2 thus allows the signal 208 (schematic 43), which is the SRL signal corresponding to the decrease in the intensity of the pump beam, to be extracted.
[0108] In the same way, in FIG. 4B, the schematic 44 illustrates the intensity as a function of time of the train of pulses 205 (FIG. 2) at the Stokes angular frequency ω.sub.S, this train being modulated at the second frequency f.sub.2. The schematic 45 illustrates the intensity as a function of time of the second non-linear optical signal 207 detected by the second non-linear optical detector 272. As may be seen in the schematic 45, the intensity of the second optical signal 207 is increased with respect to that of the optical signal 205. This increase in intensity results from the SRG process on the train of pulses at the Stokes angular frequency, the process being induced by the interaction with the train of pulses at the pump angular frequency, which train is modulated at the frequency f.sub.1. The synchronous detection at the frequency f, thus allows the signal 209 (schematic 46), which is the SRG signal corresponding to the gain of Stokes-beam intensity, to be extracted.
[0109] The applicant has shown that comparison of these signals allows information to be obtained on artefacts.
[0110] Specifically, the amplitude of these SRL and SRG signals after calibration should be equal. A difference in amplitude is therefore indicative of an artefact in the measurement of the SRS signal.
[0111] FIGS. 5A, 5B and 6 illustrate the exploitation of the SRL and SRG signals determined by means of a method according to the present description, in the case of application to imaging.
[0112] The images of FIGS. 5A, 5B and 6 use a calibration comprising the determination of a spatial mean over all of the pixels of the image and the normalization of each image with the computed mean. Alternatively, it is possible to calibrate the device such that the gains of the SRL and SRG electronics give equal SRL and SRG signals for a test sample having a high SRS signal (for example oil at 2850 cm.sup.−1).
[0113] More precisely, FIG. 5A shows experimental images representing a two-dimensional SRL signal (image 511), a two-dimensional SRG signal (image 512) and the ratio of the SRL/SRG signals (image 513) of the surface of an oyster shell. The SRL and SRG signals are obtained with an SRS microscope according to the present description, at the resonant frequency of phosphate (1090 cm.sup.−1). It may be seen that the SRL/SRG ratio (513) has a value strictly higher than 1 on elliptical structures that are attributed to parasites and that exhibit 2-photon absorption (increase of the SRL signal in the image 511 and decrease of the SRG signal in the image 512). These elliptical structures are thus interpreted as being artefacts in the SRL (511) and SRG (512) images.
[0114] FIG. 5B shows experimental images representing a two-dimensional SRL signal (image 521), a two-dimensional SRG signal (image 522) and the ratio of the SRL/SRG signals (image 523) of mouse nerve cells. The SRL and SRG signals are obtained with an SRS microscope according to the present description, at the resonant frequency of the CH.sub.2 bond (2850 cm.sup.−1). It may be seen that the SRL/SRG ratio (523) has a value strictly higher than 1 on discrete structures that are interpreted as being local accumulations of myelin that produce cross phase modulation. These discrete structures are thus interpreted as being artefacts in the SRL (521) and SRG (522) images.
[0115] FIG. 6 shows, in the schematic 61, experimental images showing, in a coordinate system XY, a two-dimensional SRL signal (image 611) and a two-dimensional SRG signal (image 612) of the surface of an onion cell, and the sum SRL+SRG of the SRL and SRG signals (image 613), which is called the SRGAL signal in the present description. The SRL and SRG signals are obtained with an SRS microscope according to the present description, at the resonant frequency of the CH.sub.2 bond (2850 cm.sup.−1).
[0116] Moreover, FIG. 6 shows, in the schematic 62, curves representing signal-to-noise ratios (or SNRs) of the SRL (621), SRG (622) and SRL+SRG (624) images illustrated in the schematic 61, as a function of the direction X. These signal-to-noise ratios are the ratio between the squared mean of the signal and its variance. They are established in regions of interest of each image, which are indicated by the dashed rectangles 631, 632, 633 in the images 611, 612 and 613, respectively. They are integrated in the direction Y of the regions of interest and represented along X in the graph 62. It may be seen that the SNRs of the SRL (621) and SRG (622) images (denoted SNR.sub.SRL, and SNR.sub.SRG, respectively) are similar. In contrast, the SNR of the sum SRL+SRG (624) (denoted SNR.sub.SRGAL) is higher by a factor 2. This is graphically illustrated by curve 623, which shows the ratio 2SNR.sub.SRGAL/(SNR.sub.SRL+SNR.sub.SRG). This demonstrates that the image SRL+SRG (613) has an SNR two times higher than the SRL (611) and SRG (612) images.
[0117] FIG. 7 shows a schematic of a device 700 for detecting a resonant non-linear optical signal of stimulated-Raman-scattering (SRS) type induced in a sample S, and more precisely a schematic illustrating an example of an SRS microscope according to the present description in an endoscopic mode using an optical fibre.
[0118] As described with reference to FIG. 2, the device 700 comprises a light source 710 that is configured to emit a first beam 702, or “pump beam”, formed from a first train of pump pulses at a first angular frequency ω.sub.p and that is configured to emit a second beam 703, or “Stokes beam”, formed from a second train of Stokes pulses at a second angular frequency ω.sub.s, these angular frequencies being such that a difference ω.sub.p−ω.sub.s between the first and second angular frequencies is equal to an angular frequency ΩR of molecular vibrational resonance of the sample S that it is sought to observe. The first train of pulses and the second train of pulses are temporally synchronized in order to allow the pump and Stokes pulses to interact in the sample and the light source 710 may be a source such as those described with reference to FIG. 2. A delay line (not shown in FIG. 7) may allow the trains of pump and Stokes pulses to be synchronized temporally in order to ensure a temporal overlap of the pulses in the sample. The delay line may also be configured to introduce a variable time delay. It may be positioned on either or both of the pump and Stokes channels. The device 700 comprises a first amplitude modulator 731 configured to amplitude modulate the first train of pump pulses at a first modulation frequency f.sub.1 and a second amplitude modulator 732 configured to amplitude modulate the second train of Stokes pulses at a second modulation frequency f.sub.2 different from the first modulation frequency. The amplitude modulators 731, 732 for example receive modulation signals from a radiofrequency (RF) generator 730. As a result, pump and Stokes beams formed from pulse trains that are amplitude modulated at the frequencies f.sub.1 and f.sub.2, and that are denoted 704 and 705 in FIG. 2, respectively, are produced. The frequencies f.sub.1 and f.sub.2 may be similar to those described with reference to FIG. 2.
[0119] In the example of FIG. 7, the device 700 further comprises optomechanical means for making interact in the sample S the amplitude-modulated trains of pump and Stokes pulses 704, 705.
[0120] The optomechanical means are configured in this example for an application to endoscopy. The optomechanical means thus comprise, in this example, a transporting optical fibre 753, a single-mode optical fibre for example, and optical elements for injecting trains of pump and Stokes pulses into the fibre 753. The optical elements for injecting into the fibre comprise, in this example, reflective elements 761, 762, 763, which for example include dichroic mirrors 761, 762 and a semi-reflective element 763, and a microscope objective 752. On exiting the fibre, the trains of pulses are collected by a microscope objective 754 and transmitted to a microscope objective 755 that is configured to focus the pump and Stokes pulses into a common focal volume in the sample S. The sample S may be scanned either by moving the latter using an XY translational stage (referenced 756) or, especially when the sample S is not accessible, by inserting a scanning device (not shown in FIG. 7) configured to scan the trains of pump and Stokes pulses over the sample. Such a scanning device may be similar to that shown in FIG. 2 or more compact, by virtue of the use for example of piezoelectric tubes arranged in the distal portion of the endoscope.
[0121] The example of FIG. 7 illustrates a device in “epi” configuration, in which:
[0122] The first and second optical detecting means are configured for the optical detection, at the pump angular frequency ω.sub.p, of a first non-linear optical signal (signal referenced 706 in FIG. 7) backscattered by the sample, and for the optical detection, at the Stokes angular frequency ω.sub.S, of a second non-linear optical signal (signal referenced 707 in FIG. 7) backscattered by the sample, respectively, the two signals resulting from the interaction of the light pulses in the sample. Thus, the first and second optical detecting means comprise optical elements common to the optomechanical means in order to make interact in the sample S the pump and Stokes pulse trains, namely the microscope objectives 755, 754, 752 and the optical fibre 753. The backscattered pump and Stokes signals are thus collected by the same optical fibre 753 and redirected to a first optical detector 771 and to a second optical detector 772, respectively, by means of the semi-reflective plate 763 and a dichroic plate 764 allowing the signals 706 and 707 to be split. As with reference to FIG. 2, synchronous detections 781, 782 allow the SRL (708) and SRG (709) signals, respectively, to be extracted, which signals are processed by the processing means 790 with a view to implementing a method according to the present description, as for example described with reference to FIGS. 5A, 5B and 6.
[0123] Irrespectively of whether it is a question of the example of FIG. 2 or the example of FIG. 7, for an application of the device to spectroscopy or to hyperspectral imaging, it is also possible to make the angular frequencies ω.sub.p and/or ω.sub.S of the trains of pump and Stokes pulses vary in order to probe the SRS signal as a function of molecular vibrational frequency. The processing means 290 (FIG. 2) or 790 (FIG. 7) thus allow the sought-after characteristic signal to be extracted as a function of molecular vibrational frequency in order to form a spectrum.
[0124] FIG. 8 illustrates a particular example in which the pump and Stokes pulses are ultrashort pulses that are temporally stretched by means of a stretcher in order to form trains of frequency-chirped pulses that are centred on the angular frequencies ω.sub.p and ω.sub.S, respectively. A modulation of the time delay between the trains of pulses at the angular frequencies ω.sub.p and ω.sub.S is then possible by means for example of a delay line, as was described with reference to FIG. 2 for example. The same synchronous-detection method may be implemented for the detection of the SRG and SRL signals. As illustrated in FIG. 8, a variation in the time delay Δt then allows the vibrational resonance of interest Ω to be probed.
[0125] Although described through a certain number of exemplary embodiments, the methods and devices according to the present description comprise various variants, modifications and improvements that will appear obvious to anyone skilled in the art, and it will be understood that these various variants, modifications and improvements form part of the scope of the invention such as defined by the following claims.
REFERENCES
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