Method and system for obtaining CARS spectra

Abstract

The system (1) includes an optical path (20) configured to irradiate a portion (5a) of a target (5) with a Stokes optical pulse (11), a pump optical pulse (12), and a hybrid probe optical pulse (13) including a first probe optical pulse (13a) and a second optical pulse (13b), where the first probe optical pulse and the second probe optical pulse overlap in time, the first and second probe optical pulses overlap with the Stokes and pump pulses, and at least one of the first probe optical pulse and the second probe optical pulse changes phase. The system further includes a detector (50) configured to detect CARS spectra (15x) and (15y) generated by the Stokes optical pulse, the pump optical pulse, and the hybrid probe optical pulse to obtain a set of CARS spectra (15) in relation to the phase of the hybrid probe optical pulse.

Description

[Technical field]

[0001] The present invention relates generally to systems and methods for acquiring CARS (Coherent Anti-Stokes Raman Scattering (Spectroscopy)) spectra and / or multiple spectra. [Background technology]

[0002] US Patent No. 7,388,668 discloses a system for detecting a nonlinear coherent field induced in a sample volume. The system includes a first source for generating a first electromagnetic field at a first frequency, a second source for generating a second electromagnetic field at a second frequency, a first optical system for directing the first and second electromagnetic fields into the sample volume, a second optical system for directing the first and second electromagnetic fields into a local oscillator volume, and an interferometer. The interferometer is for interfering a first scattered field generated by the interaction of the first and second electromagnetic fields in the sample volume with a second scattered field generated by the interaction of the first and second electromagnetic fields in the local oscillator volume.

[0003] Methods and systems involving detection of coherent anti-Stokes Raman scattering (CARS) spectra or scattered light find wide application in a variety of fields including, for example, the fields of biochemical and structural characterization of objects of interest in living subjects, and in particular, invasive and non-invasive assessment of the biochemical composition of objects of interest in living subjects and their applications. Summary of the Invention

[0004] However, the CARS spectrum contains resonant and non-resonant components. Heterodyne detection using a local oscillator (LO) was one of the solutions to obtain the resonant components from the CARS spectrum. This method includes (a) overlapping the signal with a coherent and phase-stable LO that has a constant phase relationship with the signal, and (b) scanning the phase difference between the LO and the signal. The phase difference can be controlled by a delay stage, a single moving mirror, moving the entire object (epi, backscattering), a pulse shaper, two glass wedges, etc. Possible local oscillator sources include an oscillator that generates an LO signal from a second focus of a non-resonant sample, as disclosed in U.S. Pat. No. 7,388,668, an external laser, a laser generated by a coherent process (NOPA / OPA), a broadband laser blue wing (SB-CARS), etc. However, such a method of obtaining LO from outside, i.e., from a source other than the target, has problems such as instability due to the use of different optical systems, as well as the time required to obtain and switch LO.

[0005] One aspect of the present invention is a method including: (i) irradiating a part of a target with a Stokes light pulse, a pump light pulse, and a hybrid probe light pulse including a first probe light pulse and a second probe light pulse while changing the phase of at least one of the first probe light pulse and the second probe light pulse to obtain a set of CARS spectra; and (ii) comparing the obtained sets of CARS spectra to extract a resonant component. The first probe light pulse and the second probe light pulse of the hybrid probe light pulse partially overlap in time, and the first probe light pulse and the second probe light pulse partially overlap with the Stokes light pulse and the pump light pulse.

[0006] According to the results of the inventor's simulation, by using a negatively delayed probe light pulse, the resonant component is erased or nearly erased from the spectrum, so that the spectrum can be used as an LO signal (local oscillator signal) or a signal. In this method, one of the hybrid probe light pulses (the second probe light pulse) having a negative delay is irradiated from a target, and an LO signal can be obtained simultaneously with a signal containing a resonant component generated by the other hybrid probe light pulse (the first probe light pulse). Therefore, in this method, the second probe light pulse can be selected to generate a local oscillator (LO) signal by the Stokes light pulse and the pump light pulse, and the first probe light pulse can be selected to generate a signal containing a resonant component by the Stokes light pulse and the pump light. Therefore, in this method, the interference can be essentially stabilized, a reference measurement is not required, switching of the optical system is not required, and the scanning speed can be improved. When switching the sample to obtain the LO and a signal having a resonant component, an unstable factor such as laser drift may occur. However, with this method, the LO and the signal with the resonant component can be acquired from the target simultaneously using the same optical system, so the above-mentioned changes and drift do not occur between the measurement of the LO and the measurement of the signal with the resonant component.

[0007] The pulse widths of the first and second probe light pulses may be greater (wider) than the pulse widths of the Stokes light pulse and the pump light pulse. Acquiring the set of CARS spectra may include emitting a hybrid probe light pulse such that the first probe light pulse has a first relative temporal relationship with the Stokes light pulse and the pump light pulse such that the first probe light pulse overlaps with the Stokes light pulse and the pump light pulse within the pulse width of the first probe light pulse, and the second probe light pulse has a second relative temporal relationship with the Stokes light pulse and the pump light pulse within the pulse width of the second probe light pulse and includes a negative delay with respect to the first relative temporal relationship. Acquiring the set of CARS spectra of this method may include emitting a hybrid probe light pulse such that the second probe light pulse is emitted earlier than the Stokes light pulse and the pump light pulse. Obtaining the set of CARS spectra of this method may include launching a hybrid probe light pulse such that the Stokes light pulse, the pump light pulse and the first probe light pulse are launched substantially simultaneously, and the Stokes light pulse and the pump light pulse are launched substantially at the end (terminal portion) of the pulse width of the second probe light pulse.

[0008] The method may further include scanning the target with a plurality of Stokes light pulses, a plurality of pump light pulses, and a plurality of hybrid probe light pulses to perform 2D (two-dimensional) CARS microscopic imaging and acquiring a set of CARS spectra at each pixel. The method may further include scanning the target with a plurality of Stokes light pulses, a plurality of pump light pulses, and a plurality of hybrid probe light pulses to perform 3D (three-dimensional) CARS microscopic imaging and acquiring a set of CARS spectra at each voxel.

[0009] Another aspect of the invention is a system having (i) an optical path configured to irradiate a portion of a target with a Stokes light pulse, a pump light pulse, and a hybrid probe light pulse, where a first probe light pulse and a second probe light pulse in the hybrid probe light pulse partially overlap in time and further where the first probe light pulse and the second probe light pulse partially overlap with the Stokes pulse and the pump pulse, while varying the phase of at least one of the first probe light pulse and the second probe light pulse, and (ii) a detector configured to detect CARS spectra produced by the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to obtain a set of CARS spectra associated with the phases of the hybrid probe light pulses.

[0010] Yet another aspect of the invention is a computer program or computer program product stored on a non-transitory medium for causing a computer to operate as the system described above, the computer program (program product) including instructions for controlling the system to acquire a set of CARS spectra and extract resonant components by comparing the sets of acquired CARS spectra. [Brief description of the drawings]

[0011] The embodiments herein will be better understood from the following detailed description taken in conjunction with the drawings, in which: [Figure 1] FIG. 1 illustrates one embodiment of the system of the present invention. [Diagram 2] Figure 2 shows a typical CARS process and the signal dependencies using different charts. [Diagram 3] Figure 3 shows typical pulses of the Stokes, pump and probe beams, as well as the generated CARS spectrum. [Figure 4] Figure 4 shows examples of CARS spectra when the delay of the probe beam is changed. [Diagram 5]Figure 5 shows an example of a CARS measurement and an internal reference measurement. [Figure 6] Figure 6 shows an example of CARS measurement using a hybrid probe light pulse and an example of an internal reference measurement. [Figure 7] Figure 7 shows an example of a CARS spectrum produced by out-of-phase hybrid probe light pulses. [Figure 8] FIG. 8 shows an example of the CARS spectrum when the delay of the probe beam is varied using a time-frequency map. [Figure 9] FIG. 9 is a block diagram showing an example of a probe pulse generator. [Figure 10] FIG. 10 shows the signal in the internal heterodyne method using a time-frequency map. [Figure 11] FIG. 11 shows an example of a process by which a first probe light pulse is generated in a probe pulse generator. [Figure 12] FIG. 12 shows the signal when CARS is generated by the first probe light pulse using a time-frequency map. [Figure 13] FIG. 13 shows an example of a process for generating a second probe light pulse with a probe pulse generator. [Figure 14] FIG. 14 shows the signal when CARS is generated by the second probe light pulse using the time-frequency map. [Figure 15] FIG. 15 shows an example of a process for extracting resonance components from a set of CARS spectra. [Figure 16] FIG. 16 shows examples of waveforms and resonance components of two types of CARS spectra obtained by simulation. [Figure 17] FIG. 17 shows the theoretical background of the internal heterodyne method. [Figure 18] FIG. 18 shows an example set of CARS spectra and resonance components. [Figure 19] FIG. 19 shows another example of a probe pulse generator. [Figure 20] FIG. 20 shows a flow diagram of the interheterodyne detection method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The embodiments herein and their various features and advantageous details will be more fully described with reference to the non-limiting embodiments illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as not to unnecessarily obscure the embodiments herein. The examples used herein are merely intended to facilitate understanding of how the embodiments herein can be implemented and further enable those skilled in the art to implement the embodiments herein. Thus, the examples should not be interpreted as limiting the scope of the embodiments herein.

[0013] FIG. 1 shows a system 1 according to an embodiment of the present invention. The system 1 includes an optical module 10 configured to provide (emit) a Stokes light pulse 11, a pump light pulse 12, and a hybrid probe light pulse 13 to irradiate a part 5a of the target 5 to generate (create) a CARS (Coherent Anti-Stokes Raman Scattering or Coherent Anti-Stokes Raman Spectroscopy) signal (CARS spectrum, CARS light) 15 on the part 5a of the target 5. The system 1 can be used as a measuring device, an analyzing device, a monitoring device, a monitor, etc., depending on the application. The optical system 10 uses CARS to acquire data indicative of the condition and components on the surface and inside of the target 5, such as a sample in a cuvette or a human body.

[0014] The system 1 further includes a scanner (scanning interface) 60 configured to scan the target 5 with the Stokes light pulse 11, the pump light pulse 12, and the probe pulse 13 and acquire the CARS light 15 from the target 5 through the lens 28 and other optical elements, a probe pulse generator 70 configured to generate and control the phase of the hybrid probe light pulse 13, a detector 50 configured to detect the CARS light 15 for analysis, and a controller 55 configured to control the system 1 and modules such as the scanner 60, the probe pulse generator 70, and the laser source 30. The scanning module 60 may be configured to scan a cuvette or may function as a non-invasive sampler, an invasive sampler, a flow path, or a wearable scanning interface such as a fingertip scanning interface module. The controller 55 includes a laser controller 58 that controls the laser source 30, and an analyzer 56 that analyzes the internal composition by CARS (CARS spectrum). The analyzer 56 may include a number of modules for identifying a portion (site) 5a of the target 5 from which the CARS light 15 is generated. A program (program product, software, application) 59 stored in a memory of the controller 55 is provided for executing processes on the controller 55 having computer resources such as memory, a CPU, etc. The program (software) 59 may be stored in a processor or other storage medium (non-transitory medium) readable by a computer. The program may include instructions for controlling the system 1 to perform the processes described herein.

[0015] The optical system 10 includes a laser light source 30 for generating a plurality of first laser pulses 30a having a first wavelength of 1040 nm for a plurality of Stokes light pulses (Stokes beam pulses) 11 and a plurality of pump light pulses (pump beam pulses) 12. One of the preferable laser light sources 30 is a fiber laser. Each of the plurality of first laser pulses 30a has a pulse width of 1 to several hundred fs (femtoseconds) order of several tens to several hundreds mW, and generates (generates) pulses of the Stokes light 11 and the pump light 12 having a pulse width of the femtosecond order. The pulse width PW1 of the pulses of the Stokes light 11 and the pump light 12 may be 1 to several hundred fs, for example, 1 to 900 fs, 10 to 600 fs, or 50 to 400 fs. The optical system 10 includes a number of optical elements 29, such as lenses, filters, mirrors, dichroic mirrors and prisms, for positioning the optical paths for splitting and combining the laser light pulses.

[0016] The optical system 10 includes an optical path 20 configured to irradiate a portion 5a of the target 5 with a plurality of Stokes light pulses 11, a plurality of pump light pulses 12, and a plurality of hybrid probe light pulses 13. The optical path 20 includes a Stokes light path (first optical path, Stokes unit) 21 configured to supply a plurality of broadband Stokes light pulses 11 having a first range R1 of wavelengths 1080 to 1300 nm from a first laser pulse 30a common to the pump light pulse 12 via a PCF (Photonic Crystal Fiber, photonic crystal fiber, fiber) 21a. The optical path 20 includes a pump light path (second optical path, pump unit) 22 configured to supply a plurality of pump light pulses (second light pulses) 12 having a second wavelength range R2 of wavelengths 1070 nm that is shorter than the first wavelength range (first range) R1 from the first laser pulse 30a common to the Stokes light pulse 11. The optical path 20 includes a common optical path that supplies the Stokes optical pulse 11 supplied by a path 21 and the pump optical pulse 12 supplied by a path 22 to an optical input / output unit (lens system) 28 together with the hybrid probe optical pulse 13. The optical paths include optical elements necessary to configure each optical path, such as filters, fibers, dichroic mirrors, and prisms. The same applies to the optical paths described below.

[0017] The laser light source 30 generates a plurality of first laser pulses 30a with a first wavelength of 1040 nm for the plurality of Stokes light pulses 11 and the plurality of pump light pulses 12, as well as a plurality of second laser pulses 30b with a second wavelength of 780 nm for the plurality of probe light pulses (probe beam pulses) 13. The second laser pulses 30b may include pulses on the order of 1 to several tens of ps (picoseconds) of several tens to several hundreds of mW in order to generate pulses of probe light 13 having a pulse width on the order of picoseconds. The pulse width PW2 of the pulses of the probe light 13 may be 1 to several tens of ps, for example, 1 to 90 ps, ​​1 to 50 ps, ​​or 2 to 10 ps. The second laser pulses 30b with a wavelength of 780 nm may be generated from a light source oscillator with a wavelength of 1560 nm. In addition to the Stokes optical path 21 and the pump optical path 22, the optical path 20 includes a probe optical path (third optical path) 23 configured to supply a plurality of probe light pulses (probe beam pulses) 13 having a third wavelength range R3 of 780 nm wavelength, which is shorter than the second wavelength range R2.

[0018] The optical path 20 further includes an optical I / O unit (optical unit) 28 configured to coaxially output the plurality of Stokes light pulses 11, the plurality of pump light pulses 12, and the plurality of probe light pulses 13 to the target 5 and acquire the CARS light 15 from the target 5 via a common optical path. A typical optical input / output unit 28 is an objective lens or lens system facing the target 5 and acquiring the backward scattered CARS light pulse (backward CARS light pulse, Epi-CARS) 15. The optical system 10 may include an optical path configured to acquire the forward scattered CARS light (forward CARS light). In this system 1, the plurality of CARS light pulses 15 in the wavelength range of 680 to 760 nm, which is shorter than the wavelength range R3, are generated (produced) by the probe light pulse 13 and acquired to be detected by the detector 50.

[0019] The probe light path 23 includes a probe pulse generator (probe pulse generator, probe pulse generator, probe pulse conditioner) 70 configured to generate a hybrid probe light pulse (paired probe light pulse, composite probe light pulse) 13 consisting of a first probe light pulse 13a and a second probe light pulse 13b. The first probe light pulse 13a and the second probe light pulse 13b overlap in time, and the first probe light pulse 13a and the second probe light pulse 13b overlap in time with the Stokes pulse 11 and the pump pulse 12, respectively. Furthermore, the probe pulse generator 70 is configured to change the phase of at least one of the first probe light pulse 13a and the second probe light pulse 13b. The probe pulse generator 70 is configured to control the relative temporal relationship between each pulse of the probe light 13a and 13b and each pulse of the Stokes light 11 and the pump light 12 within the pulse width PW2 of the probe light pulses 13a and 13b. Typically, the probe pulse generator 70 controls (changes, sets, or modulates) the time difference Δt between the emission (release) of the probe light pulses 13a and 13b and the emission (release) of the Stokes light pulse 11 and the pump light pulse 12. The probe pulse generator 70 may include a time delay stage (time delay unit) having a collimator and an actuator such as a motor or a piezoelectric actuator that can modulate the optical path (length of the optical path) of each of the probe light pulses 13a and 13b, as described below. The probe pulse generator 70 may include an LC-SLM (Liquid Crystal Spatial Light Modulator), an AWG (Arrayed Wave-guide Grating), or the like that controls the distance between the collimators.

[0020] In this system 1, a second probe light pulse 13b having a negative delay (reverse time delay) Δtn, which is one of a pair of hybrid probe light pulses 13, is selected to generate a local oscillator (LO) signal with the Stokes light pulse 11 and the pump light pulse 12. Thus, the probe pulse generator 70 is configured to generate a hybrid probe light pulse 13 with the first probe light pulse 13a and the second probe light pulse 13b having a negative delay Δtn with respect to the first probe light pulse 13a.

[0021] In the present system 1, the probe pulse generator 70 includes a first probe pulse conditioner (first probe pulse unit, first probe pulse generator) 71, a second probe pulse conditioner (second probe pulse unit, second probe pulse generator) 72, an input interface 73, an output interface 74, and a TD probe pulse conditioner (TD probe pulse unit, TD probe pulse generator) 75. The first probe pulse unit 71 is configured to generate a plurality of first probe light pulses 13a having a pulse width PW2 larger than the pulse width PW1 of the Stokes light pulse 11 and the pump light pulse 12 and having a first relative time relationship (relative time relationship, relative time relationship) Δt1 that partially overlaps with the Stokes light pulse 11 and the pump light pulse 12 within the pulse width PW2 of the first probe light pulse 13a. The second probe pulse unit 72 is configured to generate a plurality of second probe light pulses 13b having a pulse width PW2 larger than the pulse width PW1 of the Stokes light pulse 11 and the pump light pulse 12, and having a negative delay time Δtn with respect to the first relative time relationship Δt1 and a second relative time relationship Δt2 that partially overlaps the Stokes light pulse 11 and the pump light pulse 12 within the pulse width PW2 of the second probe pulse 13b. The TD probe pulse unit 75 is configured to generate a plurality of TD probe light pulses 13c having a positive delay Δtd to generate a TD-CARS spectrum.

[0022] In this system 1, the probe pulse generator 70 is configured to generate (generate) the hybrid probe light pulse 13 such that the second probe light pulse 13b having a negative delay Δtn is emitted earlier than the pulses of the Stokes light 11 and the pump light 12. Typically, the probe pulse generator 70 emits the second probe light pulse 13b such that the first probe light pulse 13a is emitted substantially simultaneously with the Stokes light pulse 11 and the pump light pulse 12 (first relative time relationship Δt1=0), and the Stokes light pulse 11 and the pump light pulse 12 are emitted substantially at the end (end, the second relative time relationship Δt2 and the negative delay time Δtn are approximately equal to or coincident with PW2) of the pulse width PW2 of the second probe light pulse 13b. The negative time delay may be several thousand fs (several picoseconds) or more relative to the excitation by the pulses of the Stokes light 11 and the pump light 12.

[0023] The first probe unit 71 and / or the second probe unit 72 may include a function to vary the phase difference Δφ between the phase φ1 of the first probe light pulse 13a and the phase φ2 of the second probe light pulse by around plus or minus Pi / 2 ((+ / -)π / 2). In this system 1, the second probe pulse unit 72 modulates the second probe light pulse 13b to vary the phase φ2 so that the hybrid probe light pulse 13 includes a phase difference Δφ by around plus or minus Pi / 2 ((+ / -)π / 2). The relative phase difference Δφ provided by the probe pulse generator 70 may be varied or set under the control of a phase controller (timing controller, modulation controller) 56t in the controller 55. Using the generator 70, the probe light path 23 supplies two kinds (two types) of hybrid probe light pulses 13x and 13y having different phase differences Δφ between the first probe light pulse 13a and the second probe light pulse 13b of plus or minus Pi / 2 ((+ / -)π / 2), and irradiates the hybrid probe light pulse 13 to the point 5a of the target 5 via the optical input / output unit 28, thereby obtaining two kinds (two types) of CARS pulses 15x and 15y, which contain resonant components (resonant components) having different phases in the LO signal, generated by the pulses of the Stokes light 11, the pump light 12, and the hybrid probe light 13.

[0024] The controller 55 further includes a CARS spectrum acquisition module (CARS acquisition module, CARS acquisition device) 56a. The CARS acquisition module 56a controls a probe pulse generator 70 via a phase controller 56t to synchronously irradiate a part 5a of the target 5 with a plurality of Stokes light pulses 11, a pump light pulse 12, and a hybrid probe light pulse 13 while changing the phase of the hybrid probe light pulse 13, thereby acquiring a set (a plurality of sets) of CARS spectra 15 including CARS spectra 15x and 15y associated with the phase of the hybrid probe light pulse 13. The controller 55 may further include a TD-CARS acquisition module 56b that controls the probe pulse generator 70 via a phase controller 56t to acquire a TD-CARS spectrum using a TD (time delay) probe light pulse 13c having a positive delay Δtd with respect to the Stokes light pulse 11 and the pump light pulse 12.

[0025] The detector 50 is configured to detect CARS spectra 15x and 15y generated by pulses of the Stokes light 11, the pump light 12, and the hybrid probe lights 13x and 13y to obtain a set of CARS spectra 15 including spectra 15x and 15y related to the phase difference of the hybrid probe lights 13x and 13y.

[0026] The controller 55 may further include, as one of the functions of the analyzer 55, an extraction module (extractor) 56d configured to extract a resonance component (resonance feature) Rf from the set of CARS spectra 15 by comparing the set of CARS spectra acquired to analyze the characteristics or composition of the portion 5a of the target 5. The extraction module 56d may include a function of scanning the target 5 using a scanner 60. The scanner 60 is configured to scan the target 5 with a plurality of Stokes light pulses 11, a plurality of pump light pulses 12, and a plurality of hybrid probe light pulses 13x and 13y to acquire a set of CARS spectra 15 at each pixel. The analyzer 56 may include an image generation module (image generator) 56e that generates an image (two-dimensional image) of the target 5 by a plurality of pixels having the resonance feature Rf. Thus, the system 1 may have the functions of CARS spectroscopy and CARS microscopy.

[0027] In CARS spectroscopy, the system 1 can generate a depth profile of the target 5 by moving the focal points or spots of the Stokes light 11, the pump light 12, and the probe light 13. Thus, the scanner 60 may be configured to three-dimensionally scan the target 5 with multiple Stokes light pulses 11, multiple pump light pulses 12, and multiple hybrid probe light pulses 13x and 13y to obtain a set of CARS spectra 15 at each voxel. The analyzer 56 may include a 3D image generation module (3D image generator) 56f that generates a 3D image of the target 5 with voxels having a resonant component Rf. Thus, the system 1 may have the functionality of a CARS 3D microscope.

[0028] By using a broadband Stokes pulse (Stokes beam) 11, many resonances can be excited at once and a full spectrum can be recorded in one shot. Therefore, by scanning the target (sample) 5, full CARS spectra (full-range CARS spectra) 15x and 15y can be acquired at each pixel or voxel at each shot, and 2D (two-dimensional) or 3D (three-dimensional) CARS imaging can be performed in a short time. In addition, by using this system 1, CARS spectra 15x and 15y including both LO signals and resonance features can be obtained at each pixel or voxel at once from the actual measurement location in the target (subject) 5 including tissues (cells) and the like. Since differences in the generation environment, light path, scattering, sample inhomogeneity, and other artifacts are canceled out between the LO signal and the resonance features, the system 1 generates a CARS spectrum with improved sensitivity.

[0029] FIG. 2 shows a typical CARS process and the relationship between signals. FIG. 2(a) shows the energy maps of the Stokes light pulse 11, the pump light pulse 12, the probe light pulse 13, and the generated CARS signal 16. FIG. 2(b) shows these signals in the frequency domain, and FIG. 2(c) shows these signals in the time domain. Each domain alone is insufficient to explain the signal behavior in the present invention based on the relationship of the time overlap of the signals and the relationship of the signals generated by the frequencies. In the explanation of the present invention, a time-frequency map is used for better understanding. FIG. 2(d) is an example of explaining the Stokes light pulse 11, the pump light pulse 12, the probe light pulse 13, and the generated CARS signal 16 using a time-frequency map.

[0030] FIG. 3 shows a typical CARS spectrum (light, signal, spectrum, spectroscopy) (FIG. 3(b)) including a broad non-resonant background (NRB) and a resonant component (Rf) generated by a pulse (FIG. 3(a)) of the Stokes light 11, the pump light 12, and the probe light (first probe light pulse) 13a with a time delay Δt1 (0 fs), in which the Stokes pulse 11, the pump pulse 12, and the probe pulse 13a are simultaneously emitted, and the time-delay characteristics (composition, components) of the CARS signal 16a are acquired within the pulse width PW2 of the probe light pulse 13a. The Stokes pulse 11 and the pump pulse 11 overlap in time, and the position of the probe light pulse 13a can be controlled. The time difference (time relationship, time delay) Δt between the pulses of the Stokes light 11 and the pump light 12 and the probe pulse 13a is arbitrarily selected. In FIG. 3(a), the time delay Δt is Δt1 (t=0). In other words, the Stokes pulse 11 and the pump pulse 12 overlap with the start (the first to arrive, the part that arrives earlier) 13s of the probe pulse 13a, and then the remainder of the probe pulse 13a, including the final (the last to arrive, the part that arrives later) 13e, arrives with a delay.

[0031] FIG. 4 shows an example of CARS light (signal, spectrum, spectroscopy) 16 as a simulation result when the delay (time relationship) Δt of the probe lights 13a, 13b, and 13c is changed. FIG. 4(c) shows an example of a CARS spectrum 16a generated when the Stokes pulse 11, the pump pulse 12, and the first probe pulse 13a are simultaneously emitted (when the time delay Δt is set to Δt1 (Δt=0)). FIG. 4(a) and FIG. 4(b) show an example of an internal reference (signal for internal reference, LO signal) 16b generated by the Stokes pulse 11, the pump pulse 12, and the second probe pulse 13b having a negative delay Δt2 (Δtn). That is, the Stokes pulse 11 and the pump pulse 12 are emitted later than the emission of the second probe pulse 13b, but overlapping with the second probe pulse 13b. A typical internal reference (LO signal) 16b is obtained when a Stokes pulse 11 and a pump pulse 12 are emitted so as to overlap with the end of a second probe pulse 13b, as shown in FIG. 4(a).

[0032] 4(d) and 4(e) show examples of TD-CARS spectra 16c generated by the Stokes pulse 11, the pump pulse 12, and the probe pulse 13c with a positive delay Δt3 (Δtd). That is, the probe pulse 13c is emitted later than the Stokes pulse 11 and the pump pulse 12, and further, does not overlap with the Stokes pulse 11 and the pump pulse 12. A typical TD-CARS spectrum 16c is formed almost exclusively of the resonance component (resonance component, resonance feature) Rf, as shown in FIG. 4(e), but the intensity is very small compared to the target CARS spectrum 16a and the internal reference 16b.

[0033] As shown in Figures 4(a) to (e), phenomena such as molecular vibration changes with long decay times, which correspond to the resonant component (Rf), take a long time to build up, while phenomena such as molecular vibration changes with short decay times, which correspond to the NRB, take a short time to build up. In other words, the NRB is an instantaneous electronic response. The resonant component (Rf) takes time to build up, and the TD-CARS simulation results show a similar trend. In addition, when the decay time is long, the build-up also takes longer (the line width becomes narrower), and when the decay time is short, the time required for the response to build up takes less time (the line width becomes wider). Therefore, by emitting the first probe pulse 13a simultaneously with the Stokes pulse 11 and the pump pulse 12, a CARS spectrum 16a containing a large resonant component with NRB is obtained, and by emitting the Stokes pulse 11 and the pump pulse 12 in time with the end of the pulse width PW2 of the second probe pulse 13b, an internal reference (internal reference signal, internal reference signal) 16b, which is a CARS spectrum that is almost NRB (in this specification, a spectrum that is almost NRB or a spectrum that is entirely NRB refers to a spectrum that does not contain a resonant component to a sufficient extent and can be treated (referenced, used as a standard) as NRB), is obtained.

[0034] 5 to 7 show the basic concept of the present invention (heterodyne internal reference, internal heterodyning, internal heterodyne method). As shown in FIG. 5, by changing the time relationship Δt of the probe pulses 13a and 13b with respect to the Stokes pulse 11 and pump pulse 12, it is possible to obtain a CARS spectrum 16a (FIG. 5(b)) having NRB and resonance features and a CARS spectrum (internal reference, INR) 16b (FIG. 5(a)) having only NRB under exactly the same experimental conditions (focus, scattering, absorption, light path, etc.) without changing the sample and container (cuvette). Therefore, by using the INR 16b as a local oscillator (LO), it is possible to extract the resonance component by the heterodyne method. That is, by simultaneously generating the INR 16b and the CARS signal 16a having the resonance component at the same position, it is possible to cause the INR 16b to interfere with the CARS signal 16a as the local oscillator (LO) on the detector 50. Furthermore, the interference signal depends on the phase difference Δφ between the LO 16b and the CARS signal 16a, which includes a resonant component (res) and a non-resonant component (NR).

[0035] As shown in FIG. 6(a), when a hybrid probe light pulse 13 including two time-delayed probe pulses 13a and 13b is sent to a sample 5 (shown in FIG. 6(b)) simultaneously with a Stokes light pulse 11 and a pump light pulse 12, interference signals (target CARS signals) 15x and 15y shown in FIG. 7 are generated simultaneously at a focused point 5a. In the hybrid probe light pulse 13, the first probe light pulse 13a includes a time difference with the second probe light pulse 13b, and partially overlaps with the second probe light pulse 13b in time. In addition, the Stokes light pulse 11, the pump light pulse 12, and the hybrid probe light pulse 13 are emitted so that an overlapping portion 13m of the first probe light pulse 13a and the second probe light pulse 13b overlaps with the Stokes light pulse 11 and the pump light pulse 12. The probe pulses 13a and 13b of the hybrid probe light pulse 13 generate a scattering signal (CARS) 16a and a LO (local oscillator (internal reference)) 16b, respectively. The two generated signals 16a and 16b are interfered on a detector 50 to produce interfered or combined signals 15x and 15y, which form a set of CARS spectra (target CARS spectra) 15.

[0036] In this system 1, a plurality of hybrid probe light pulses 13 including two types 13x and 13y are provided by changing (altering) the phase φ2 of the second probe light pulse 13b for LO to plus or minus Pi / 2 ((+ / -)π / 2). One of the two types is a hybrid probe light pulse 13x including a first probe light pulse 13a and a second probe light pulse 13b with a phase difference Δφ of minus Pi / 2. The other of the two types is a hybrid probe light pulse 13y including a first probe light pulse 13a and a second probe light pulse 13b with a phase difference Δφ of plus Pi / 2. By changing the phase difference Δφ of the hybrid probe light pulse 13, the interference and signal shape of the target CARS spectrum 15 can be controlled. Therefore, by varying the phase difference Δφ using the hybrid probe light pulse 13, a set of target CARS spectra 15 including a target CARS spectrum 15x having a shape obtained by subtracting CARS 16a from LO 16b and a target CARS spectrum 15y having a shape obtained by adding CARS 16a to LO 16b can be generated and acquired. Therefore, the phase difference Δφ can be used to calculate or extract the resonance component 17 from the set of CARS spectra 15.

[0037] The resonant and non-resonant contributions exhibit different phase (and polarization) characteristics. Thus, the resonant and non-resonant fields have different phases, but the phase information is lost during detection. By using heterodyne detection, which overlaps the target signal with a coherent reference beam (local oscillator, LO) for interferometric detection, the different phase relationships can be exploited to measure the signal amplitude and phase, or to measure the chi-squared frequency. (3) Heterodyne detection involves overlapping a signal with a coherent, phase-stable LO that exhibits a constant phase relationship with the signal, and scanning the phase difference between the LO and the signal.

[0038] As shown in Fig. 6, both CARS 16a including NRB and resonance features and LO 16b only NRB are simultaneously acquired from the same spot 5a using the same optical path by the hybrid probe pulse 13. Therefore, both signals 16a and 16b have the same absorption, signal path and other experimental conditions. Moreover, this internal heterodyne method is applicable to forward scattering CARS and backward scattering CARS.

[0039] The local oscillator source can be generated from a second focus (non-resonant sample), an external laser, a laser generated by a coherent process (NOPA / OPA), or a broadband laser blue wing (SB-CARS). However, in order to obtain the LO signal, it is necessary to measure a reference for it, and it takes time to switch the measurement between the LO light source and the sample (target). In addition, due to fluctuations caused by switching the optical system, such as laser draft, the measurement conditions differ between the non-resonant and resonant components, making accurate measurement difficult. Therefore, it is desirable to obtain the local oscillator (LO) using only the sample to be measured (target) rather than using an object (volume) for local oscillation.

[0040] The advantages of this system 1 using the interheterodyne technique are as follows: (i) No external LO is required, and the LO is generated simultaneously from the same pulse at the same position, so it is inherently stable and phase-locked (allowing very long averaging). (ii) No reference water measurement is required, and no switching is required for non-resonant samples, so all signals, including the LO, can be obtained from the measurement target (sample). (iii) By using an etalon, detector interference can be eliminated and differences can be cancelled out. (iv) χ can be calculated without using the Maximum Entropy Method (MEM). (3) (v) System 1 can operate with scattering samples such as tissues (cells), i.e., the LOs are generated simultaneously via the exact same optical path, so phase shifts due to the sample are not an issue.

[0041] Figure 8 shows some examples of generating CARS signals using time-frequency maps. Figure 8(a) shows a typical measurement example using a first probe light pulse 13a without a probe delay. The first probe light pulse 13a overlaps with the excitation of the Stokes light pulse 11 and the pump light pulse 12, and a CARS signal 16a with resonance and NR is generated. The generated CARS signal 16a has a typical distributed CARS line shape due to the interference between the resonant and non-resonant electric fields (r-nr phase shift locking).

[0042] Figure 8(b) shows a measurement example using a TD probe light pulse 13c with a time delay (positive delay). The Stokes light pulse 11 and the pump light pulse arrive first, and the TD probe light pulse 13c arrives later. The NR signal decays faster than the vibration resonance signal, so the r / nr ratio increases but the signal strength decreases. Figure 8(c) shows a measurement example using a probe pulse for internal reference (a second probe light pulse with a negative delay) 13b. The probe light pulse 13b with a negative probe delay arrives first. Since the NR signal is generated electronically immediately, there is no time for coherent oscillations to build up and no resonance signal is generated. The generated signal 16b contains almost only the NR component (internal reference (INR)).

[0043] FIG. 9 shows an embodiment of a probe pulse generator (probe pulse generating device) 70. FIG. 10 shows a plurality of signals in the internal heterodyne method using a time-frequency map. The probe pulse generator (probe pulse conditioner) 70 includes a first probe pulse conditioner (first probe pulse unit) 71, a second probe pulse conditioner (second probe pulse unit) 72, an input interface 73, and an output interface 74. The input interface 73 includes a wave plate 76 for converting the polarization of the input probe pulse 30b, and a PBS (polarizing beam splitter) 75 for separating the first probe pulse (e.g., P-polarized light) 13a and the second probe pulse (e.g., S-polarized light) 13b from the input probe pulse 30b. The first probe pulse unit 71 includes a first probe path 77 for adjusting the first probe pulse 13a, which includes a wave plate 71a, a mirror 71b for reflecting the first probe pulse 13a to the PBS 75, and an actuator, such as a piezo actuator 71c, for controlling the difference in distance Δs between the wave plate 71a, the mirror 71b for reflecting the first probe pulse 13a to the PBS 75, and a second optical path 78 in the second probe pulse unit 72 (affecting the time difference Δt and the phase difference Δφ). The second probe pulse unit 72 includes a second probe optical path 78 for adjusting the second probe pulse 13b, which includes a wave plate 72a, a mirror 72b for reflecting the second probe pulse 13b to the PBS 75, and an actuator, such as a piezo actuator 72c, for controlling the difference in distance Δs. The output interface 74 includes an output path 79 including mirrors 74a, 74b and other optical elements for directing the first probe light pulse 13a and the second probe light pulse 13b overlapped by the PBS 75 as a hybrid (pair or composite) probe pulse 13.

[0044] The first probe path 77 and / or the second probe path 78 include a path that controls the distance difference Δs relative to another path, and the mirrors 71b and / or 72b are controlled by actuators 71c and / or 72c under the control of the phase controller 56t to set the time difference (delay time) Δt (relative to the Stokes pulse 11 and the pump pulse 12) for the first probe pulse 13a and to vary the phase difference Δφ between the first probe pulse 13a and the second probe pulse 13b. The generator 70 can perform fast and repeatable modulation by using polarization optics. The generator 70 includes two paths 77 and 78 to provide an adjustable relative delay selected by polarization. The waveplate 76 can be a rotating stage or an electro-optical modulator (EOM) for high speed (>kHz) modulation. High speed modulation can reduce noise, eliminate drifts in power, alignment, potential etaloning, etc., and improve the scanning speed for generating CARS images.

[0045] In this probe pulse generator 70, the wave plate 76 for controlling the polarization of the input probe light pulse 30b may have the function of a unit that controls the ratio of the first probe light pulse 13a to the second probe light pulse 13b. That is, the wave plate 76 may be set anywhere between 0 and 90°. This allows the intensity in the path 77 for the first probe pulse 13a and the intensity in the path 78 for the second probe pulse 13b to be controlled (and therefore the intensity of the LO signal (nr signal) and the CARS signal can be controlled). In the probe pulse generator (conditioner) 70, the polarizer is set to transmit only S-polarized light, so that the pulses after leaving this setup are S-polarized (same as the pump and Stokes) and have a well-ordered polarization. In this embodiment, in order to make the polarization of the Stokes light pulse 11 and the pump light pulse 12 the same, the input probe pulse 30b is P-polarized and the outputs of the first and second probe pulses 13a and 13b are S-polarized. However, such a configuration is one of the embodiments of the present invention. For example, the waveplate 76 may be set to (say) 22.5°. If the polarization before the PBS 75 is linear at 45°, then 50% of the pulse will go through one path (arm) 77 and the remaining 50% will go through the other path (arm) 78. After the PBS 75, 50% S and P polarizations remain. Since there is no way to simultaneously rotate them back to the same polarization, the polarizer may just cut off the parts that are not useful. In the example of a 22.5° waveplate (WP) setting, the S polarization will be linearly polarized at +45° and the P polarization will be polarized at -45°. The polarizer may then cut off all the P polarization components, producing two neat S-polarized pulses. The setting of the waveplate (WP) 76 allows adjustment of the percentage cut off and the amount cut off.

[0046] 10, a CARS signal (normal CARS signal) 16a having a resonant component and NR, and an LO signal (INR signal) 16b having only NR and a non-resonant component are generated synchronously by a hybrid probe light pulse 13 in which a first probe light pulse 13a and a second probe light pulse 13b are partially overlapped, as shown in FIG. 10. Therefore, in the present system 1, a target CARS spectrum 15 generated by the interference between the normal CARS signal 16a and the LO signal 16b can be obtained.

[0047] FIG. 11 shows a process in which the first probe light pulse 13a is generated in the probe pulse generator 70. FIG. 12 shows a number of signals for generating CARS by the first probe light pulse 13a using the time-frequency map. FIG. 13 shows a process in which the second probe light pulse 13b is generated in the probe pulse generator 70. FIG. 14 shows a number of signals for generating CARS by the second probe light pulse 13b using the time-frequency map. The original probe light pulse 30b supplied from the laser light source 30 is supplied to select one of two optical paths 77 and 78 depending on the polarization direction. The difference in optical path length between the optical paths 77 and 78 is controlled to change the probe delay Δt and the phase difference Δφ.

[0048] In one embodiment, the phase of the first probe pulse 13a may be modulated so that the phase Δφ between the first probe pulse 13a and the second probe pulse 13b is shifted by (−π / 2) and (+π / 2). In signal detection (heterodyne signal detection) in the present system 1, it is most important to periodically change the phase difference Δφ. Therefore, the probe pulse generator 70 may change the phase of either the first probe pulse 13a or the second probe pulse 13b, or both. As shown in the equation described later, it does not matter which phase is changed, but the second probe light pulse 13b having a negative delay time Δtn for generating LO(nr) is more suitable as the one to change the phase, since there is no resonance (resonance) and therefore the signal changes less even if the time delay is changed. Note that the phase change is performed using a small time delay, so it should be small enough that it does not matter which of the above is selected. In principle, for better control and simplification, the phase change may be applied to one of the paths 77 or 78, and the other path may be fixed.

[0049] In general, the modulation frequency should be as high as possible. However, the modulation frequency can be selected taking into account the following conditions: Since the modulation of the probe pulse generator 70 can be performed using a piezo (there may be other or better ways to achieve high-speed phase modulation), the piezo actuator needs some time to react and move, for example, on the order of milliseconds. When switching between two home positions, it is necessary to wait until the piezo reaches the home position. Therefore, the maximum usable frequency is limited to about 100 Hz. However, it can be improved.

[0050] Another point is the signal strength. In order to operate the system 1 even at very low concentrations, it is necessary to keep the detector noise low. To do this, it is necessary to adjust the integration time so that the detector 50 is fully filled to about 80% (in that case, measures against shot noise are mostly important, not readout noise, etc.). That is, the integration time can be adjusted depending on the signal strength, for example, 50 μs in one embodiment of this system. The fastest possible switching frequency may be selected, and if such high-speed modulation is possible, only one spectrum may be acquired per phase. It is also possible to use another detector, but in that case, the frequency is limited by the speed of phase modulation. In summary, there are ways to modulate the phase faster, for example, using an electro-optical modulator, and in principle the modulation frequency may be MHz by using several high-speed, highly sensitive photodetectors instead of a CCD camera. It should be noted that the advantages of this detection method can be achieved even if the modulation frequency is between 1 Hz-1 kHz.

[0051] FIG. 15 shows a process of extracting a resonance component 17 from a set of CARS spectra 15 including two CARS spectra 15x and 15y generated by hybrid probe light pulses 13x and 13y, respectively. FIG. 16 shows example waveforms of the two CARS spectra 15x and 15y and the resonance component 17 obtained by simulation. As shown in FIG. 15(a), a plurality of sets of hybrid probe light pulses 13x and 13y with different phase differences Δφ, such as plus and minus Pi / 2 ((+ / -)π / 2), are generated and supplied by changing the phase of the second probe light pulse 13b to φ1 and φ2 by controlling the voltage of the piezo actuator 72c of the second probe light path 78. As shown in FIG. 15(b), a set of these hybrid probe light pulses 13x and 13y generates a set of CARS spectra 15 including a CARS spectrum 15x (phase 1 (first phase), the sum of LO 16b and normal CARS signal 16a) and a CARS spectrum 15y (phase 2 (second phase), LO 16b minus normal CARS signal 16a). As shown in FIG. 15(c), the resonance components (resonance features) 17 are extracted by calculating the difference in these sets of CARS spectra 15 with reference to the phase difference Δφ.

[0052] Instead of or together with the piezo actuator 72c of the second probe optical path 78, the piezo actuator 71c of the first probe optical path 77 may be controlled. By scanning the voltage of the piezo actuator 71, the path length of the path 77 is changed, and the phase of the first probe optical pulse 13a is changed. This changes the phase difference Δφ between the phase of the LO 13b, and finally the detected interference signal 15 can be obtained. The piezo modulation may synchronize the voltage change with the acquisition of the CARS spectra 15x and 15y by the camera or detector.

[0053] Figure 17 shows the theoretical background of this interheterodyne method. As shown in Figure 17(a), the resonant and non-resonant contributions have different phase (and polarization) characteristics. Therefore, the resonant and non-resonant electric fields have different phases, but this phase information is lost during detection. By using heterodyne detection, which overlaps the signal with a coherent reference beam (reference beam, local oscillator, LO) during interferometric detection, the amplitude and phase of the signal can be measured by taking advantage of the different phase relationships, and the χ (3) It is possible to separate the real and imaginary parts of the detector signal, S Het In (ω), as shown in Figure 17(b), the phase difference Δφ of plus or minus Pi / 2 is measured and subtracted to remove the constant term and cosine term, and the χ (3) The imaginary part of can be extracted.

[0054] Figure 18 shows the relationship between the phase Δφ of the scanning probe (Figure 18(a)) and the intensity of a set of CARS spectra 15x and 15y (Figures 18(b)-(f)) obtained using hybrid probe pulses 13 with different phases Δφ. According to the signal intensities shown in Figures 18(b)-(f), as expected, the signal with the maximum intensity can be extracted by subtracting the spectrum with phase difference ±Pi / 2 (Figure 18(d)).

[0055] 19 shows another embodiment of a probe pulse generator (probe pulse generating device, probe pulse generating device) 70. The generator 70 includes a wave plate 65 for converting the polarization of the input probe light pulse 30b, at least one birefringent crystal 66 for separating the first probe light pulse 13a and the second probe light pulse 13b from the input probe light pulse 30b and providing a time difference Δφ between the first probe light pulse 13a and the second probe light pulse 13b, an EOM (electro-optical modulator) 67 for modulating at least one of the first probe light pulse 13a and the second probe light pulse 13b to control the phase difference Δφ between the first probe light pulse 13a and the second probe light pulse 13b, and an output path 79 for leading the first probe light pulse 13a and the second probe light pulse 13b after overlapping them by a polarization element such as a wave plate, a polarizing plate 68, or a PBS. In this generator 70, a single input pulse 30b is converted into two pulses 13a and 13b on each axis of a birefringent crystal 66 with a delay of about 2.5-3 ps between them. This delay can be adjusted by adding a birefringent wedge pair, which is suitable for fine tuning the delay. The phase modulation Δφ between the two pulses 13a and 13b is electrically controlled by an EOM (electro-optical modulator) 67.

[0056] FIG. 20 is a flow chart showing an overview of the process of the interheterodyne detection method. The method includes acquiring a set of CARS spectra (step 81) and extracting a resonance component (resonance feature) by comparing the acquired sets of CARS spectra (step 82). In step 81, a plurality of Stokes light pulses 11, a plurality of pump light pulses 12, and a plurality of hybrid probe light pulses 13 are irradiated onto a part 5a of a target 5 while changing the phase of the hybrid probe light pulses 13, thereby acquiring a plurality of sets of CARS spectra 15. These hybrid probe light pulses 13 include a plurality of first probe light pulses 13a and a plurality of second probe light pulses 13b. The plurality of first probe light pulses 13a and the plurality of second probe light pulses 13b are each partially overlapped in time, and the first probe light pulses 13a and the second probe light pulses 13b are partially overlapped with the Stokes light pulses 11 and the pump light pulses 12 while changing the phase of at least one of the first probe light pulses 13a and the second probe light pulses 13b.

[0057] In step 81, the second probe light pulse 13b is selected to generate a local oscillator (LO) signal 16b together with the Stokes light pulse 11 and the pump light pulse 12, and the first probe light pulse 13a is selected to generate a signal 16a including a resonance component together with the Stokes light pulse 11 and the pump light pulse 12. The pulse width PW2 of the first probe light pulse 13a and the second probe light pulse 13b may be larger than the pulse width PW1 of the Stokes light pulse 11 and the pump light pulse 12, and in step 81, the hybrid probe light pulse 13 may be emitted such that the first probe light pulse 13a has a first relative temporal relationship Δt1 to the Stokes light pulse 11 and the pump light pulse 12 and partially overlaps within the pulse width PW2 of the first probe light pulse 13a, and the second probe light pulse 13b has a second relative temporal relationship Δt2, which is a negative delay Δtn with respect to the first relative temporal relationship Δt1, to the Stokes light pulse 11 and the pump light pulse 12 and partially overlaps within the pulse width PW2 of the second probe pulse 13b.

[0058] In step 81, the hybrid probe light pulse 13 may be emitted such that the second probe light pulse 13b is emitted earlier than the pulses of the Stokes light 11 and the pump light 12. In step 81, the hybrid probe light pulse 13 may be emitted such that the Stokes light pulse 11, the pump light pulse 12 and the first probe light pulse 13 are substantially simultaneously, and the Stokes light pulse 11 and the pump light pulse 12 are substantially at the end of the pulse width PW2 of the second probe light pulse 13b.

[0059] 9, step 81 may include (i) converting the polarization of the input probe light pulse 30b using a wave plate 76, (ii) separating the first probe light pulse 13a and the second probe light pulse 13b from the input probe light pulse 30b using a PBS 75, (iii) conditioning the first probe light pulse 13a and the second probe light pulse 13b by a first probe path 77 and a second probe path 78, respectively, and (iv) directing the first probe light pulse 13a and the second probe light pulse 13b superimposed by the PBS 75 as a hybrid probe pulse 13. At least one of the first probe path 77 and the second probe path 78 may include a path for including a time difference with the other path and an actuator for moving a mirror for modulation.

[0060] When using the probe pulse generator 70 shown in FIG. 19 , step 81 may include (i) converting the polarization of the input probe light pulse 30 b using a wave plate 65; (ii) separating the first probe light pulse 13 a and the second probe light pulse 13 b from the input probe light pulse 30 b using at least one birefringent crystal 66 and including a time difference between the first probe light pulse 13 a and the second probe light pulse 13 b; (iii) modulating at least one of the first probe light pulse 13 a and the second probe light pulse 13 b using an EOM 67; and (iv) guiding the overlapped first probe light pulse 13 a and the second probe light pulse 13 b by a polarizing element 68 as a hybrid probe light pulse 13.

[0061] The method may further include a step of scanning the target 5 (step 83). In step 83, the target 5 is scanned with a plurality of Stokes optical pulses 11, a plurality of pump optical pulses 12, and a plurality of hybrid probe optical pulses 13 to obtain a set of CARS spectra 15 at each pixel to generate an image of the target 5. Step 83 may be a 3D scan, where a set of CARS spectra 15 is obtained at each voxel to generate a 3D image of the target 5. In step 84, the above steps may be repeated until all pixel or voxel information is obtained.

[0062] By focusing a laser light on a sample, e.g. a solution in a cuvette, a CARS spectrum is generated that can be analyzed to identify different molecules and even to quantitatively determine the concentration of solutes. In contrast to other methods such as fluorescence or Raman spectroscopy, in CARS and other nonlinear techniques, a signal is generated only at the focus. By increasing the focusing, an intrinsic spatial resolution of 1 μm is obtained. 3 It is possible to obtain signals originating only from a very small volume of the order of 100 nm. Instead of measuring a solution in a cuvette, it is also possible to apply CARS directly to structured materials such as tissues. System 1 can scan over the sample with beams 11, 12, and 13, providing spectra at different positions, so that images can be generated. System 1 can be applied not only as a CARS spectroscopy, but also as a CARS microscope, scanning and acquiring spectra at each pixel to form an image. For a thin sample (target) 5, the signal can be recorded in the forward scattering direction through the sample, and for a thick sample, the signal can be recorded in the backscattering direction. For example, imaging of locally high concentrations, such as lipids in fat cells, is desired. For locally high concentrations, focusing on a small volume allows the peak to stand out from the background.

[0063] As explained above, a method and system for using heterodyne detection is provided. The disclosed heterodyne detection (interheterodyne method, heterodyne internal reference method, heterodyne internal reference technique) is applicable to all kinds of CARS measurements, including forward scattering CARS and back scattering CARS. The disclosed heterodyne detection can use a multiple beam system to obtain LO signals from the same sample (target) 5 that obtains the resonant components 17.

[0064] The methods and systems described herein are applicable to biochemical and structural characterization of biological objects of interest, particularly invasive and non-invasive evaluation of the biochemical composition of biological objects of interest and their applications. The methods and systems described herein are applicable to all kinds of samples, even simple samples such as solutions that are unrelated to biochemistry.

[0065] This specification discloses a system 1 for detecting a CARS signal. The system includes: (i) a first optical path configured to provide at least one type of first optical pulse to a target to generate scattered light including a resonant component; (ii) a probe pulse conditioner (probe pulse generator) configured to provide a first probe optical pulse 13a and a second probe optical pulse 13b to a target 5 to generate scattered light (CARS signal); and (iii) an acquisition unit (analyzer) 56 that generates a difference between the scattered light acquired in synchronization with the modulation to extract the resonant component. The first probe optical pulse 13a includes a time difference between the first probe optical pulse 13a and the second probe optical pulse 13b, and they partially overlap in time. At least one of the first probe optical pulse 13a and the second probe optical pulse 13b is modulated and then optically overlapped to be emitted as a hybrid probe pulse (paired probe optical pulse or composite probe optical pulse) 13. A first light (beam) pulse including a Stokes light pulse 11 and a pump light pulse 12 is emitted so as to overlap the first probe light pulse 13a and the second probe light pulse 13b in time, thereby generating (generating) scattered light including a non-resonant component and a modulated resonant component. Therefore, the resonant component can be extracted by generating the difference between the acquired scattered light in synchronization with the modulation.

[0066] Another aspect disclosed above is a method for detecting a CARS signal. The method includes providing at least one type of first light pulse to a target to generate scattered light including a resonant component, providing a first probe light pulse and a second probe light pulse to the target to generate scattered light, and generating a difference of the scattered light acquired in synchronization with the modulation. The first probe light pulse includes a time difference between the first probe light pulse and the second probe light pulse, and they overlap in time. At least one of the first probe light pulse and the second probe light pulse is modulated and then optically overlapped. Then, the first light pulse is emitted such that the first probe light pulse and the second probe light pulse overlap in time.

[0067] The method may further include transmitting the first light pulse, the first probe light pulse, and the second probe light pulse to the target, and acquiring scattered light from the target. The method may further include scanning at least a portion of the target with the first light pulse, the first probe light pulse, and the second probe light pulse, and acquiring scattered light from the target. The step of providing the first light pulse may include the steps of providing a Stokes light pulse in a first wavelength range, providing a pump light pulse in a second wavelength range shorter than the first wavelength range so as to overlap in time with the Stokes light pulse, and providing the first probe light pulse and the second probe light pulse in a third wavelength range shorter than the second wavelength range. The method may further include acquiring a CARS signal from the target. The method may further include acquiring a decay over time of a resonant component of the CARS signal from the target.

[0068] Yet another aspect of the present invention is a computer program (computer program product) for a computer operating an apparatus. The target apparatus includes a first optical path configured to provide at least one type of first optical pulse to a target to generate scattered light including a resonant component, a probe pulse conditioner configured to provide a first probe optical pulse and a second probe optical pulse to the target to generate scattered light, and an acquisition unit to acquire scattered light from the target. The computer program includes executable code for executing the steps of: providing the first probe optical pulse and the second probe optical pulse such that the first probe optical pulse and the second probe optical pulse include a time difference in which the first probe optical pulse and the second probe optical pulse partially overlap in time, modulating at least one of the first probe optical pulse and the second probe optical pulse and then optically overlapping them, and generating a difference of the acquired scattered light in synchronization with the modulation to extract the resonant component.

[0069] The foregoing description of specific embodiments fully reveals the general nature of the embodiments herein, so that others can easily modify and / or adapt such specific embodiments for various applications by applying current knowledge without departing from the general concept. Therefore, such adaptations and modifications should be understood and are intended to be understood within the meaning and equivalents of the disclosed embodiments. It should be understood that the expressions or terms used herein are for the purpose of explanation and not for the purpose of limitation. Thus, although the embodiments herein are described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications within the spirit and scope of the appended claims.

Claims

1. acquiring a set of CARS spectra by irradiating a portion of a target with a Stokes light pulse, a pump light pulse, and a hybrid probe light pulse including a first probe light pulse and a second probe light pulse, the first probe light pulse and the second probe light pulse partially overlapping in time, the first probe light pulse and the second probe light pulse partially overlapping with the Stokes light pulse and the pump light pulse, and varying a phase of at least one of the first probe light pulse and the second probe light pulse to acquire the set of CARS spectra; and extracting resonance components by comparing the sets of acquired CARS spectra.

2. 10. The method of claim 1 , the second probe light pulse is selected to generate a local oscillator (LO) signal together with the Stokes light pulse and the pump light pulse, and the first probe light pulse is selected to generate a signal including a resonant component together with the Stokes light pulse and the pump light pulse.

3. 10. The method of claim 1 , the pulse widths of the first and second probe light pulses are greater than the pulse widths of the Stokes light pulse and the pump light pulse; 4. The method of claim 3, wherein acquiring the set of CARS spectra includes emitting the hybrid probe light pulse, wherein the first probe light pulse has a first relative temporal relationship with the Stokes light pulse and the pump light pulse that partially overlaps within the pulse widths of the Stokes light pulse and the pump light pulse and the first probe light pulse, and the second probe light pulse has a second relative temporal relationship that partially overlaps within the pulse widths of the Stokes light pulse and the pump light pulse and the second probe light pulse, the second relative temporal relationship including a negative delay with respect to the first relative temporal relationship.

4. 10. The method of claim 1 , The method of claim 1, wherein acquiring the set of CARS spectra includes launching the hybrid probe light pulse such that the second probe light pulse is launched earlier than the Stokes light and the pump light pulses.

5. 10. The method of claim 1 , acquiring the set of CARS spectra includes launching the hybrid probe light pulse such that the Stokes light pulse, the pump light pulse, and the first probe light pulse are launched simultaneously, and the Stokes light pulse and the pump light pulse are at edges of a pulse width of the second probe light pulse.

6. The method according to any one of claims 1 to 5, scanning the target with the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to obtain a set of CARS spectra at each pixel.

7. The method according to any one of claims 1 to 5, scanning the target with the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to obtain a set of CARS spectra for each voxel.

8. The method according to any one of claims 1 to 5, the Stokes light pulse comprises a first wavelength range, the pump light pulse comprises a second wavelength range shorter than the first wavelength range, and the hybrid probe light pulse comprises a third wavelength range shorter than the second wavelength range.

9. The method according to any one of claims 1 to 5, The method, wherein the Stokes optical pulse comprises a broadband Stokes beam.

10. The method according to any one of claims 1 to 5, Obtaining the set of CARS spectra comprises: converting the polarization of an input probe light pulse using a waveplate; separating the first probe light pulse and the second probe light pulse from the input probe light pulse using a PBS; modulating the first probe light pulse and the second probe light pulse through a first probe path and a second probe path, respectively, each including a waveplate, a mirror for reflecting light pulses to the PBS, at least one of the first probe path and the second probe path including a path including a time difference with another path, and an actuator for moving the mirror for modulation; guiding the first probe light pulse and the second probe light pulse so as to be overlapped by the PBS.

11. The method according to any one of claims 1 to 5, Obtaining the set of CARS spectra comprises: converting the polarization of an input probe light pulse using a waveplate; using at least one birefringent crystal to separate the first and second probe light pulses from the input probe light pulse and provide a time difference between the first and second probe light pulses; The first probe light pulse and the second probe light pulse are modulated by an electro-optic modulator (EOM). modulating at least one of the lobe light pulses; and guiding the overlapped first and second probe light pulses by a polarizing element.

12. an optical path configured to irradiate a portion of a target with a Stokes light pulse, a pump light pulse, and a hybrid probe light pulse including a first probe light pulse and a second probe light pulse, with the first probe light pulse and the second probe light pulse partially overlapping in time, with the first probe light pulse and the second probe light pulse partially overlapping with the Stokes light pulse and the pump light pulse, and with a change in phase of at least one of the first probe light pulse and the second probe light pulse; a detector configured to detect CARS spectra generated by the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to obtain a set of CARS spectra associated with a phase of the hybrid probe light pulse.

13. 13. The system of claim 12, a probe pulse generator configured to generate the hybrid probe light pulse comprising the first probe light pulse and the second probe light pulse comprising a negative delay with respect to the first probe light pulse.

14. 13. The system of claim 12, Further, a probe pulse generator is provided, the probe pulse generator includes a first probe pulse conditioner configured to generate the first probe light pulse having a pulse width greater than pulse widths of the Stokes light pulse and the pump light pulse, the first probe light pulse having a first relative temporal relationship of partial overlap within the pulse widths of the Stokes light pulse and the pump light pulse and the first probe light pulse; and a second probe pulse conditioner configured to generate the second probe light pulse having a pulse width greater than a pulse width of the Stokes light pulse and the pump light pulse, the second probe light pulse overlapping the Stokes light pulse and the pump light pulse within the pulse width of the second probe light pulse and having a second relative temporal relationship comprising a negative delay with respect to the first relative temporal relationship.

15. 15. The system according to claim 13 or 14, The probe pulse generator is configured to generate the hybrid probe light pulse such that the second probe light pulse is emitted earlier than the Stokes light and the pump light pulses.

16. 15. The system according to claim 13 or 14, the probe pulse generator is configured to generate the hybrid probe light pulse such that the first probe light pulse is emitted simultaneously with the Stokes light pulse and the pump light pulse, and the second probe light pulse is emitted at ends of the pulse width of the second probe light pulse.

17. 13. The system of claim 12, Further, a probe pulse generator is provided, The probe pulse generator includes: a waveplate for converting the polarization of the input probe light pulse; a PBS for separating the first probe light pulse and the second probe light pulse from the input probe light pulse; a first probe path and a second probe path for adjusting the first probe light pulse and the second probe light pulse, respectively, the first probe path and the second probe path each including a wave plate and a mirror for reflecting a light pulse to the PBS, at least one of the first probe path and the second probe path including a path for providing a time difference with the other path and an actuator for moving the mirror for modulation; The system, wherein the probe pulse generator further includes an output path for directing the first probe light pulse and the second probe light pulse superimposed by the PBS.

18. 13. The system of claim 12, Further, a probe pulse generator is provided, The probe pulse generator includes: a waveplate for converting the polarization of the input probe light pulse; at least one birefringent crystal for separating the first probe light pulse and the second probe light pulse from the input probe light pulse and for providing a time difference between the first probe light pulse and the second probe light pulse; an EOM for modulating at least one of the first probe light pulse and the second probe light pulse; an output path directing the first probe light pulse and the second probe light pulse superimposed by a polarizing element.

19. 14. The system according to claim 12 or 13, a first optical path configured to provide the Stokes light pulses in a first wavelength range; a second optical path configured to provide the pump light pulses in a second wavelength range that is shorter than the first wavelength range; and a third optical path configured to provide the hybrid probe light pulse in a third wavelength range that is shorter than the second wavelength range.

20. 20. The system of claim 19, The system, wherein the first optical path includes a first optical element for generating a broadband Stokes beam from the pump light pulse.

21. 14. The system according to claim 12 or 13, the system further comprising a scanner configured to scan the target with the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to acquire a set of CARS spectra at each pixel.

22. 14. The system according to claim 12 or 13, The system further comprises a scanner configured to scan the target with the Stokes light pulse, the pump light pulse, and the hybrid probe light pulse to acquire a set of CARS spectra for each voxel.

23. 14. The system according to claim 12 or 13, The system further comprises an analyzer configured to extract resonant components by comparing the sets of acquired CARS spectra.

24. 14. A computer program for causing a computer to operate as the system of claim 12 or 13, comprising instructions for controlling the system to acquire a set of CARS spectra and extract resonant components by comparing the sets of acquired CARS spectra.

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