CN114994017A - Coherent anti-Stokes Raman scattering microscopic imaging device and method - Google Patents
Coherent anti-Stokes Raman scattering microscopic imaging device and method Download PDFInfo
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Abstract
The invention discloses a coherent anti-Stokes Raman scattering microscopic imaging device and a coherent anti-Stokes Raman scattering microscopic imaging method, wherein the device comprises a laser pulse oscillator; the first reflector is arranged on a light path of the pulse laser sent by the laser pulse oscillator and forms a first reflected light beam; the galvanometer scanner is arranged on the light path of the first reflected light beam; the second reflector is loaded on the galvanometer scanner; the first reflected light beam forms a modulated light beam after passing through the second reflector, the first lens, the second lens, the fourth reflector, the excitation notch filter and the third reflector; the modulated light beam forms a second reflected light beam after passing through a fifth reflector; the sixth reflector, the objective lens, the object translation stage, the condensing lens, the short-pass filter and the detection trap filter are sequentially arranged on the light path of the second reflected light beam; the photomultiplier is arranged on a reflected light path of the detection trap filter; the output end of the photomultiplier is connected with the input end of the phase-locked amplifier; the invention does not need to specially customize a galvanometer scanner, and has lower cost and higher imaging efficiency.
Description
Technical Field
The invention belongs to the technical field of light scattering spectrum and microscopic imaging in nonlinear optics, and particularly relates to a coherent anti-Stokes Raman scattering microscopic imaging device and method.
Background
The raman scattering technology, one of the most effective optical technologies for detecting vibration modes and molecular structures, has been widely used in the fields of material science, biomedicine, and the like; however, spontaneous raman scattering is greatly limited in biomedical rapid imaging due to the problem of weak self-signals; the signal intensity of coherent anti-Stokes Raman scattering (CARS) is 5-6 orders of magnitude higher than that of spontaneous Raman scattering, and the method is widely applied to the field of biomedicine, particularly in the aspect of living cell imaging; CARS is a four-wave mixing optical process of interaction of pump light, Stokes light and anti-Stokes light with sample molecules, and belongs to a three-order nonlinear optical process; wherein the frequency is ω p Pump photon and frequency of omega S OfThe Kess photon excites the sample molecule to a vibration energy level, and the process needs to satisfy phase matching, namely the natural vibration frequency omega-omega of the sample molecule p -ω S (ii) a At this point, the energy of the sample molecules increases, with a reuse frequency of ω pr The probe light (probe) excites the sample molecule to a virtual energy level with higher energy, the energy of the sample molecule is unstable and returns to the ground state, and the release frequency is omega aS The process also satisfies a certain condition omega-omega aS -ω pr (ii) a CARS is typically implemented using a multi-beam or multi-source approach (to meet the frequency component requirements of pump photons, stokes photons, and probe photons), requiring that all excitation beams must be spatially coincident.
At present, in order to simplify the CARS system, a single-beam CARS method can be realized by using a notch filter; by producing notch characteristics (omega) on the laser spectrum pr ) And produce similar features (ω) on the CARS spectrum aS ) (ii) a The trap wave filter is small and simple, and is easy to be arranged on a galvanometer scanner and used for modulating laser at high frequency; since the position of the resonance signal is only related to the position omega of the trap frequency pr (ii) related; therefore, the non-resonance signal can be used as a local oscillator by utilizing phase-locked amplification, and the weak resonance signal can be amplified and extracted while the non-resonance signal is removed; compared with the common multi-beam CARS and the single-beam CARS scheme realized based on the pulse shaper, the single-beam CARS scheme realized based on the notch filter is much simpler and more compact; however, the notch filter is mounted on the galvanometer scanner, so that the modulation frequency generally does not reach the maximum frequency of the conventional galvanometer scanner; the scanning frequency of a commercial galvanometer can reach 3-4 kHz, but the scanning frequency is limited by the size and quality of a customized installation notch filter plate, and is only about 1kHz, so that the single-pixel residence time is limited to more than 1 millisecond, and the application of the technology in the aspect of biomedical rapid imaging is limited.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a coherent anti-Stokes Raman scattering microscopic imaging device and a coherent anti-Stokes Raman scattering microscopic imaging method, which are used for solving the technical problem that in the prior art, a notch filter is arranged on a galvanometric scanner, so that the modulation frequency cannot reach the maximum frequency of a conventional galvanometric scanner, and the imaging speed is slow.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a coherent anti-Stokes Raman scattering microscopic imaging device which comprises a laser pulse oscillator, a first reflector, a galvanometer scanner, a fifth reflector, a sixth reflector, an objective lens, an object carrying translation stage, a condensing lens, a short-pass filter, a detection notch filter, a photomultiplier and a lock-in amplifier, wherein the laser pulse oscillator is connected with the first reflector;
the laser pulse oscillator is used for emitting pulse laser; the first reflecting mirror is arranged on the light path of the pulse laser, and the pulse laser forms a first reflecting beam after passing through the first reflecting mirror; the galvanometer scanner is arranged on a light path of the first reflected light beam, and the first reflected light beam forms a modulated light beam after passing through the galvanometer scanner; the fifth reflector is arranged on a light path of the modulated light beam, and the modulated light beam forms a second reflected light beam after passing through the fifth reflector;
the galvanometer scanner is loaded with a second reflecting mirror, a first lens, a second lens, a third reflecting mirror, an excitation trapped wave filter and a fourth reflecting mirror;
the second reflecting mirror is arranged on the light path of the first reflecting light beam, and the first reflecting light beam forms a first light path after passing through the second reflecting mirror; the first light path forms a second light path after passing through the first lens, and the second light path forms a third light path after passing through the second lens; the third light path forms a fourth light path after passing through a fourth reflector, and the fourth light path forms a fifth light path after passing through an excitation notch filter; the fifth light path forms a sixth light path after passing through a third reflector, and the sixth light path forms a seventh light path after passing through a second lens; the seventh light path forms an eighth light path after passing through the first lens, and the eighth light path forms the modulated light beam after passing through the second reflector;
the sixth reflector, the objective lens, the object carrying translation table, the condensing lens, the short-pass filter and the detection trap filter are sequentially arranged on a light path of the second reflected light beam; the photomultiplier is arranged on a reflected light path of the detection notch filter and is used for converting the reflected light of the detection notch filter into an electric signal; the output end of the photomultiplier is connected with the input end of the phase-locked amplifier.
Furthermore, the device also comprises a pulse compressor and a long-pass filter; the pulse compressor and the long-pass filter are sequentially arranged on a light path of the laser pulse and are sequentially arranged between the laser pulse oscillator and the first reflector.
Further, the first reflector is arranged at a focal length of one time in front of the first lens, the second lens is arranged at a focal length of two times behind the first lens, and the excitation notch filter is arranged at a focal length of one time behind the second lens; wherein the focal length of the first lens is equal to that of the second lens.
Further, the device also comprises a beam splitter and a first spectrometer; the beam splitter is arranged on a light path of the second reflected light beam and is arranged between the fifth reflector and the sixth reflector; the second reflected light beam passes through the beam splitter to form a reflection light path and a transmission light path; and the reflected light path enters the first spectrometer, and the transmitted light enters the sixth reflecting mirror.
Further, the device also comprises a third lens and a second spectrometer; the third lens is arranged on a transmission light path of the detection notch filter; the transmission light path of the detection notch filter plate enters the second spectrometer after passing through the third lens; the second spectrometer is used for CARS spectral measurement.
Further, the laser pulse oscillator is a femtosecond laser pulse oscillator.
Further, the pulse laser is an ultrashort pulse laser; wherein the center wavelength of the ultrashort pulse laser is 793-808nm, the bandwidth is 10-100nm, the repetition frequency is 10-100MHz, and the pulse width is 5-120 fs.
The invention also provides a coherent anti-stokes Raman scattering microscopic imaging method, which utilizes the coherent anti-stokes Raman scattering microscopic imaging device;
the imaging method comprises the following steps:
placing a sample to be imaged on the object translation stage;
selecting the reflection wavelength of the detection notch filter, and determining the characteristic vibration mode of the sample to be imaged; simultaneously, moving the position of the photomultiplier to maximize the current value or the voltage value of the electric signal output by the photomultiplier;
observing the amplitude and phase change of the CARS microscopic imaging result output by the phase-locked amplifier by changing the parameters of a galvanometer scanner, and recording the electric signal output by the photomultiplier;
and carrying out data processing by utilizing Matlab software or Python software, and carrying out data processing on the electric signal output by the photomultiplier to obtain a coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be imaged.
Further, by changing parameters of a galvanometer scanner, the amplitude and phase changes of the CARS microscopic imaging result output by the phase-locked amplifier are observed, and the process of recording the electric signals output by the photomultiplier tube is as follows:
when the relative distance between the objective lens and the sample to be imaged is unchanged, establishing an xoy coordinate system by taking the horizontal plane of the sample to be imaged as a coordinate plane;
determining coordinates (x, y) of the position of the light spot on the sample to be imaged;
when the y value of the position coordinate of the sample to be imaged, where the light spot is located, is a constant value, increasing the voltage value applied to the x-axis direction of the object stage translation stage, where the sample to be imaged is located;
translating the x value of the position coordinate of the sample to be imaged, where the light spot is located, by the step length of 1 micrometer, and recording the electric signals of the photomultiplier at N different x values;
when the y value of the position coordinate of the sample to be imaged where the light spot is located is y +1 micrometer, simultaneously recording the electric signals of the photomultiplier tube when the N different x values are obtained; when the y value of the position coordinate of the sample to be imaged, where the light spot is located, is y + N micrometers, simultaneously recording the electric signals of the photomultiplier at N different x values again;
scanning an N micron multiplied by N micron area on a sample to be imaged to obtain an N multiplied by N two-dimensional matrix; and the electrical signals of all the photomultiplier tubes on the grid points of the N × N two-dimensional matrix are recorded.
Further, the dwell time of the light spot in a single pixel during the measurement is set to 400 microseconds.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a coherent anti-Stokes Raman scattering microscopic imaging device and a coherent anti-Stokes Raman scattering microscopic imaging method.A second reflecting mirror is adopted to replace an excitation notch filter in a galvanometer scanner customized with the excitation notch filter by utilizing the relation between an object and an image in optics, the second reflecting mirror is placed at the position of the object, and the excitation notch filter is placed at the position of the image; special customization on the galvanometer scanner is not needed, so that the cost is low; meanwhile, the scanning speed of a single pixel can be effectively improved, and the imaging efficiency is higher; and can meet the application in the field of tumor diagnosis.
Furthermore, a pulse compressor and a long-pass filter are sequentially arranged between the laser pulse oscillator and the first reflector, so that dispersion compensation of the pulse laser is realized.
Drawings
FIG. 1 is a schematic structural diagram of a coherent anti-Stokes Raman scattering microscopic imaging device according to the present invention;
FIG. 2 is CARS original spectrum of lung cancer tissue of white mouse in the invention; wherein, the graph (a) is a CARS original spectrogram when the pulse laser is 10mW and the excitation notch filter is at 779.6nm and 779.9 nm; panel (b) is a raman spectrogram of lung cancer tissue extracted using the original CARS spectrum;
FIG. 3 shows the lung cancer tissue of the mouse at a vibration frequency of 216cm -1 CARS micrographs at time.
The device comprises a laser pulse oscillator 1, a pulse compressor 2, a long-pass filter 3, a first reflector 4, a second reflector 5, a first lens 6, a second lens 7, a third reflector 8, an excitation notch filter 9, a fourth reflector 10, a fifth reflector 11, a beam splitter 12, a first spectrometer 13, a sixth reflector 14, an objective lens 15, an objective translation table 16 and a condenser lens 17, wherein the laser pulse oscillator is connected with the laser pulse oscillator 1; 18 short-pass filter, 19 detection notch filter, 20 photomultiplier, 21 phase-locked amplifier, 22 third lens and 23 second spectrometer.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
As shown in the attached figure 1, the invention provides a coherent anti-Stokes Raman scattering microscopic imaging device, which comprises a laser pulse oscillator 1, a pulse compressor 2, a long-pass filter 3, a first reflector 4, a galvanometer scanner, a fifth reflector 11, a beam splitter 12, a first spectrometer 13, a sixth reflector 14, an objective lens 15, an object translation stage 16, a condenser lens 17, a short-pass filter 18, a detection notch filter 19, a photomultiplier 20, a lock-in amplifier 21, a third lens 22 and a second spectrometer 23.
The laser pulse oscillator 1 is used for emitting pulse laser; wherein, the laser pulse oscillator 1 adopts a femtosecond laser pulse oscillator; the pulse laser is ultrashort pulse laser; preferably, the central wavelength of the ultrashort pulse is 793-808nm, the bandwidth is 10-100nm, the repetition frequency is 10-100MHz, and the pulse width is 5-120 fs.
The pulse compressor 2 and the long-pass filter 3 are sequentially arranged on a light path of the laser pulse, and the pulse compressor 2 is used for performing dispersion compensation on the laser pulse to form a compensated laser pulse; and the long-pass filter wave plate 3 is used for filtering the compensated laser pulse to form a filtered laser pulse.
The first reflector 4 is arranged on a light path of the laser pulse after the filtering processing, and the laser pulse after the filtering processing forms a first reflected light beam after passing through the first reflector 4; the galvanometer scanner is arranged on a light path of the first reflected light beam, and the first reflected light beam forms a modulated light beam after passing through the galvanometer scanner.
The galvanometer scanner is loaded with a second reflecting mirror 5, a first lens 6, a second lens 7, a third reflecting mirror 8, an excitation trap filter 9 and a fourth reflecting mirror 10; wherein the first reflector 5 is arranged in front of the first lens 6 at a focal length of one time, the second lens 7 is arranged behind the first lens 6 at a focal length of two times, and the excitation notch filter 9 is arranged behind the second lens 7 at a focal length of one time; wherein the focal length of the first lens 6 is the same as that of the second lens 7; the focal length of the first lens 6 and the focal length of the second lens 7 are both 30mm-500 mm.
According to the method, the relation between an object and an image in optics is utilized, an excitation notch filter in a galvanometer scanner with the excitation notch filter customized is replaced by a second reflecting mirror, the second reflecting mirror is placed at the position of the object, and the excitation notch filter is placed at the position of the image; the galvanometer scanner can adopt the existing common commercial galvanometer scanner, does not need to be specially customized, and has lower cost.
The second reflecting mirror 5 is arranged on the light path of the first reflected light beam, and the first reflected light beam forms a first light path after passing through the second reflecting mirror 5; the first light path forms a second light path after passing through the first lens 6, and the second light path forms a third light path after passing through the second lens 7; the third light path forms a fourth light path after passing through the fourth reflector 10, and the fourth light path forms a fifth light path after passing through the excitation notch filter 9; the fifth light path forms a sixth light path after passing through a third reflector 8, and the sixth light path forms a seventh light path after passing through the second lens 7; the seventh light path forms an eighth light path after passing through the first lens 6, and the eighth light path forms a modulated light beam after passing through the second reflecting mirror 5.
The fifth reflecting mirror 11 is arranged on the light path of the modulated light beam, and the modulated light beam forms a second reflected light beam after passing through the fifth reflecting mirror 11; the beam splitter 12 is arranged on the light path of the second reflected light beam, and the second reflected light beam forms a reflection light path and a transmission light path after passing through the beam splitter 12; the first spectrometer 13 is disposed on a reflection light path of the second reflected light beam, and is configured to observe a wavelength position of a notch on the laser spectrum.
The sixth reflector 14, the objective lens 15, the object translation stage 16, the condenser lens 17, the short pass filter 18 and the detection notch filter 19 are sequentially arranged on the transmission light path of the second reflected light beam; wherein, the objective lens 15 is installed on the objective lens adjusting bracket, and the object translation stage 16 is a two-dimensional translation stage; the photomultiplier 20 is arranged on a reflection light path of the detection notch filter 19 and is used for converting the reflection light of the detection notch filter 19 into an electric signal; the output end of the photomultiplier 20 is connected with the input end of the phase-locked amplifier 21, and the phase-locked amplifier 21 is used for obtaining a CARS microscopic imaging result according to the electric signal; the third lens 22 is arranged in the transmission light path of the detection notch filter 19; the transmission light path of the detection notch filter 19 passes through the third lens 22 and then enters the second spectrometer 23; the second spectrometer 23 is used for CARS spectral measurement.
Working principle and imaging method
When the coherent anti-stokes raman scattering microscopic imaging device is used for imaging a sample to be imaged, the method specifically comprises the following steps:
s1, roughly placing all components in the device in sequence; the second reflector 5 loaded on the galvanometer scanner is placed at a focal length one time in front of the first lens 6, the second lens 7 with the same focal length as that of the first lens 6 is selected, the second lens 7 is placed at a focal length two times behind the second lens 6, and the excitation notch filter 9 is placed at a focal length one time behind the second lens 7; in step S1, a needle is placed at the position of the second reflector 5, a camera is placed at the position of the excitation notch filter 9, and the relative distances between the first lens 6, the second lens 7, the third reflector 8 and the excitation notch filter 9 are adjusted for multiple times by adjusting the second reflector 5 until the image of the needle in the camera cannot be clear.
S2, starting a laser pulse oscillator, roughly adjusting the focus of the objective lens 15 to enable the laser to be focused on the sample to be imaged until an original CARS signal is seen from the second spectrometer 23 or the photomultiplier 20; meanwhile, the positions of the sixth reflector and the condensing lens 17 are adjusted to maximize the CARS spectral intensity measured by the second spectrometer 23; in step S2, the laser focus is positioned on the sample to be imaged, and the coupling efficiency between the detecting element and the collected CARS signal is high; during specific operation, the position of a sample to be imaged is observed by adjusting the object translation stage, the focus position of the objective lens is adjusted by adjusting a support knob of the objective lens, and the pitching angle and the collimation degree of laser are adjusted by adjusting the fifth reflector 11 and the sixth reflector 14.
S3, optimizing the relative position of a prism pair in the pulse compressor, so that the dispersion of the laser on the sample to be imaged is compensated until the original CARS signal cannot be increased; in step S3, by compensating for group velocity dispersion of the laser light due to passing through the objective lens; during specific operation, a one-dimensional precision translation stage in the pulse compressor is adjusted to change the position of the prism pair, and meanwhile, the change of the original CARS spectral intensity is observed by using the second spectrometer until the CARS spectral intensity is not increased any more.
S4, observing the CARS spectrum intensity measured by the second spectrometer 23, and adjusting the beam splitter 12 to ensure that the laser and the objective lens 15 are not increased until the observed original CARS signal is not increased any more, so that the incident laser penetrates through the objective lens 15 as far as possible; in specific operation, the CARS spectrum intensity measured by the second spectrometer is observed until the observed original CARS signal is not increased any more, and at the moment, the laser is matched with the objective lens to the optimal position.
S5, adjusting the angle of the optical fiber of the second spectrometer 23 until the original CARS signal observed by the second spectrometer 23 is not increased any more, so that the optical coupling efficiency reaches the highest value; in step S5, two sets of raw CARS spectra are measured and subjected to data processing by changing the position of the super-steep filter in the second spectrometer 23 to obtain the raman spectrum of the sample to be imaged.
The specific operation process of step S5 includes:
s501, adjusting the angle of the excitation notch filter, and utilizingThe first spectrometer observes the wavelength position of the notch on the laser spectrum, and when the notch wavelength is 779.6nm, the second spectrometer is used for recording original CARS spectrum data I 1 ；
S502, adjusting the angle of the excitation notch filter, and recording a second group of original CARS spectral data I by using a second spectrometer when the notch wavelength is 779.9nm 2 ；
S503, differentiating the two groups of original CARS spectra to obtain a Raman spectrum of the sample to be detected, drawing by utilizing Matlab software to obtain intensity changes of different vibration frequencies by taking the horizontal axis as vibration frequency and the vertical axis as CARS spectrum intensity; in step S503, the influence of background noise is removed and the resonance raman signal to be extracted is amplified through differentiation and normalization processing;
wherein, in step S503, the Raman spectrum I of the sample to be measured Raman The following were used:
wherein, I 1 And I 2 The primary CARS spectrograms of the excitation notch filter at 779.6nm and 779.9nm respectively;
is to use Matlab to I 1 And I 2 And smoothing and filtering for 50 times.
S6, preparing a section of animal tissue, placing the section on a glass slide, and placing the section on the object translation stage 16
S7, determining a characteristic vibration mode of the sample to be imaged by using the notch filter; specifically, by observing the original CARS spectrum signal measured by the second spectrometer, the reflected wavelength is selected by using the detection notch filter 19, and the position of the photomultiplier tube 20 is moved at the same time, so that the current value or the voltage value of the electrical signal output by the photomultiplier tube 20 is maximized;
s8, observing the amplitude and phase change of the CARS microscopic imaging result output by the lock-in amplifier 21 by changing the parameters of the galvanometer scanner, and recording the electric signal output by the photomultiplier 20; and carrying out data processing by utilizing Matlab software or Python software, and carrying out data processing on the electric signal output by the photomultiplier tube 20 to obtain a coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be imaged.
In the invention, the amplitude and phase change of the CARS microscopic imaging result output by the lock-in amplifier 21 is observed by changing the parameters of the galvanometer scanner, and the process of recording the electric signal output by the photomultiplier 20 is as follows:
when the relative distance between the objective lens and the sample to be imaged is unchanged, establishing an xoy coordinate system by taking the horizontal plane of the sample to be imaged as a coordinate plane;
determining coordinates (x, y) of the position of the spot on the sample;
when the y value of the position coordinate of the sample to be imaged, where the light spot is located, is a constant value, increasing a voltage value applied to the x-axis direction of an object carrying translation stage, where the sample to be imaged, where the light spot is located, translating the x value of the position coordinate of the sample to be imaged, where the light spot is located, by taking the step length as 1 micrometer, and simultaneously recording electric signals of a photomultiplier tube at 130 different x values;
when the y value of the position coordinate of the sample to be imaged where the light spot is located is y +1 micrometer, simultaneously recording the electric signals of the photomultiplier tube 20 at 130 different x values again;
when the y value of the position coordinate of the sample to be imaged where the light spot is located is y +130 micrometers, simultaneously recording the electric signals of the photomultiplier 20 at 130 different x values again, and scanning a 130-micrometer square area on the sample to be imaged to obtain a 130-130 two-dimensional matrix; recording all values on the lattice points of the 130 x 130 two-dimensional matrix; wherein, the dwell time of the light spot in a single pixel during the measurement is set to 400 microseconds.
According to the coherent anti-Stokes Raman scattering microscopic imaging device and method, an excitation notch filter in a galvanometer scanner customized with an excitation notch filter is replaced by a second reflecting mirror by utilizing the relation between an object and an image in optics, the second reflecting mirror is placed at the position of the object, and the excitation notch filter is placed at the position of the image; the single-pixel speed is improved by 2.5 times, the time is saved by 10.14 seconds under the condition of the same scanning area of 130 micrometers and 130 micrometers, and the method has important significance in the application aspect of in-vivo imaging; in the invention, the purpose of setting the bandwidth to be 10-100nm is as follows: the combination of photons required by CARS excitation is selected by wide-bandwidth incident laser, so that the signal sensitivity is high; the pulse repetition frequency is 10-100MHz, and the pulse width is 120 fs: the CARS excitation with low average power is realized by utilizing the high peak power of the ultra-steep pulse, and the phototoxicity on biological samples is low.
In the invention, when the focal length of the third lens 22 is 50mm, the coupling efficiency with the optical fiber connected with the second spectrometer 23 is high; meanwhile, the intensity of the generated CARS signal can be gradually and orderly increased, and the position of each element for CARS spectrum detection can be optimized; has the advantage of high imaging speed and has important significance in the aspect of biomedical living body imaging.
Aiming at the problem that the scanning speed of a customized galvanometer scanner used in the prior art is limited, the imaging device and the imaging method separate the notch filter from the galvanometer scanner by using the 4F system consisting of the pair of lenses with the same focal length, so that the scanning speed of the galvanometer scanner is not limited by the notch filter any more, the scanning speed can be improved by 3-5 times, and the cost is reduced.
In the invention, the galvanometer and the excitation notch filter plate can be used without being detached at any time, and the notch generated by the notch filter plate can not be synchronized when the galvanometer scanner rotates, so that the notch filter plate can not play a role in modulating laser; the 4F system solves the problem by utilizing the relation between an object and an image between the vibrating mirror and the excitation notch filter, so that the rotation of the vibrating mirror can synchronize the notches generated by the excitation notch filter on the laser spectrum, and the function of modulating laser is played as a key role for demodulating signals at the back.
The test process comprises the following steps:
take the imaging process of lung cancer tissue of a white mouse as an example
In the invention, a laser pulse with the pulse width of 20fs is dispersed and compensated by a pulse compensator which is arranged on a one-dimensional precision translation stage after the central wavelength of the laser pulse is 795nm and the bandwidth of the laser pulse is 45nm, and then the laser pulse passes through a long-pass filter 3; the model of the long-pass filter plate is Semrock LP01-785 RU; then passes through a first reflector 4, a second reflector 5 loaded on a galvanometer scanner, a first lens 6, a second lens 7, a fourth reflector 10 and an excitation notch filter 9; wherein the type of the excitation notch filter 9 is optigram, BNF 785; the light beam returns to the second reflecting mirror 5 along the original path through the third reflecting mirror 10, is reflected by the beam splitter 12 after passing through the fifth reflecting mirror 11, and enters the first spectrometer 13; wherein the first spectrometer is of the type: thorlabs, CCS 175; the first spectrometer 13 is used for detecting the wavelength of a notch generated by the excitation notch filter 9, and transmitted light is focused on a sample to be imaged on an object translation stage 16 by an objective lens 15 which is arranged on a two-dimensional precision translation stage through a sixth reflecting mirror 14; the type of the objective lens 15 is: newport, 20x, 0.4 NA; a sample to be imaged is placed on a sample groove of a two-dimensional precision translation stage; the transmission signals are converged by a condenser lens 17 arranged on a second two-dimensional precision translation stage; the type of the condensing lens 17 is: edmund Optics, 0.5 NA; the transmission part filters the incident laser through a short pass filter 18, wherein the model of the short pass filter 18 is: semrock SP01-785 RU; then the signal is reflected by a detection notch filter plate 19 and guided into a photomultiplier 20, and then is converted into an electric signal to be input into a phase-locked amplifier 21 for CARS microscopic imaging; the transmission signal is then guided into the fiber connected to a second spectrometer 23 through a third lens 22 with a focal length of 50mm for measuring the original CARS spectrum; wherein the third lens 22 is of the type Thorlabs, AC 254-030-AB; the second spectrometer 23 has the following model: jobin Yvon Triax 320.
As shown in figure 2, the CARS original spectrogram of lung cancer tissue of white mouse is shown in figure 2; wherein in figure (a), the pulse laser is 10mW, and the excitation notch isCARS original spectrogram of the filter at 779.6nm and 779.9 nm; FIG. (b) is a Raman spectrogram of lung cancer tissue extracted using the original CARS spectrum; starting laser, respectively recording CARS original spectrograms of the excitation notch filter at 779.6nm and 779.9nm when the laser power is measured to be 10mW by using a power meter, and respectively using I to obtain two spectrums 1 And I 2 As shown in figure (a); using a formula
Calculating the Raman spectrum of the lung tumor is shown in figure (b), wherein,
is to use Matlab to I 1 And I 2 Filtering for 50 times to obtain; it can be seen from fig. 2 that the low frequency raman spectrum of the lung tumor can be clearly shown by processing the original CARS spectrum.
As shown in FIG. 3, the vibration frequency of lung cancer tissue of a white mouse is 216cm in FIG. 3 -1 CARS microimaging graph; the laser scanning system can perform high-frequency modulation on incident laser by using a galvanometer scanner so as to perform high-frequency modulation on a resonance item in a CARS signal; the resonance CARS in the generated CARS signal is only related to the notch generated by the notch filter, and the non-resonance CARS is not related to the notch; the angle of the super steep long-pass filter plate is adjusted in a micro-scale mode, two groups of original CARS spectrums are measured, and the two groups of original CARS spectrums are differentiated and normalized to obtain a low-frequency vibration spectrum or a Raman spectrum. The long-pass filter and the short-pass filter ensure that the incident laser infinitely approaches to the detected CARS signal, so that the detection of the terahertz waveband vibration spectrum can be realized. Removing non-resonance signals in the CARS signal through phase-locked amplification, and extracting weaker resonance signals in the CARS signal; selecting the vibration frequency of 216 in the figure (b), calculating to obtain the wavelength for detecting the notch of the notch filter, then obtaining 130 x 130 points by moving the objective table, and outputting the voltage value or the current value of each point, as shown in fig. 3; the spatial distribution of the tumors in the lung tissue of the white mouse can be seen in FIG. 3.
According to the invention, the 4F system is adopted to separate the excitation notch filter from the galvanometer scanner, so that the scanning speed of the galvanometer is not influenced by the notch filter, the cost is reduced, and the imaging speed is improved; replacing an excitation notch filter in a galvanometer scanner customized with the excitation notch filter by a second reflecting mirror by utilizing the relation between an object and an image in optics, placing the second reflecting mirror at the position of the object, and placing the excitation notch filter at the position of the image; the single-pixel scanning speed can be increased by 2-4 times, and the time is saved by 10.14 seconds under the conditions that the scanning area is 130 micrometers and 130 micrometers; because the galvanometer scanner provided with the excitation notch filter needs to be specially customized and produced, the galvanometer scanner provided with the reflector can adopt a common commercial galvanometer scanner without professional customization, so that the cost can be greatly reduced; the method can be applied to lung tumor diagnosis and has important significance in the application of biomedical rapid imaging.
The above-described embodiment is only one of the embodiments that can implement the technical solution of the present invention, and the scope of the present invention is not limited by the embodiment, but includes any variations, substitutions and other embodiments that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed.
Claims (10)
1. A coherent anti-Stokes Raman scattering microscopic imaging device is characterized by comprising a laser pulse oscillator (1), a first reflector (4), a galvanometer scanner, a fifth reflector (11), a sixth reflector (14), an objective (15), an object carrying translation table (16), a condensing lens (17), a short-pass filter (18), a detection notch filter (19), a photomultiplier (20) and a phase-locked amplifier (21);
the laser pulse oscillator (1) is used for emitting pulse laser; the first reflector (4) is arranged on the light path of the pulse laser, and the pulse laser forms a first reflected light beam after passing through the first reflector (4); the galvanometer scanner is arranged on a light path of the first reflected light beam, and the first reflected light beam forms a modulated light beam after passing through the galvanometer scanner; the fifth reflector (11) is arranged on the light path of the modulated light beam, and the modulated light beam forms a second reflected light beam after passing through the fifth reflector (11);
the galvanometer scanner is provided with a second reflecting mirror (5), a first lens (6), a second lens (7), a third reflecting mirror (8), an excitation notch filter (9) and a fourth reflecting mirror (10);
the second reflecting mirror (5) is arranged on the light path of the first reflected light beam, and the first reflected light beam forms a first light path after passing through the second reflecting mirror (5); the first light path forms a second light path after passing through the first lens (6), and the second light path forms a third light path after passing through the second lens (7); the third light path forms a fourth light path after passing through a fourth reflector (10), and the fourth light path forms a fifth light path after passing through an excitation notch filter (9); the fifth light path forms a sixth light path after passing through a third reflector (8), and the sixth light path forms a seventh light path after passing through a second lens (7); the seventh light path forms an eighth light path after passing through a first lens (6), and the eighth light path forms the modulated light beam after passing through a second reflector (5);
the sixth reflector (14), the objective lens (15), the object translation stage (16), the condensing lens (17), the short-pass filter (18) and the detection notch filter (19) are sequentially arranged on the light path of the second reflected light beam; the photomultiplier (20) is arranged on a reflected light path of the detection notch filter (19) and is used for converting the reflected light of the detection notch filter (19) into an electric signal; the output end of the photomultiplier (20) is connected with the input end of the phase-locked amplifier (21).
2. A coherent anti-stokes raman scattering microscopic imaging device according to claim 1, further comprising a pulse compressor (2) and a long pass filter (3); the pulse compressor (2) and the long-pass filter (3) are sequentially arranged on a light path of the laser pulse and are sequentially arranged between the laser pulse oscillator (1) and the first reflector (4).
3. A coherent anti-stokes raman scattering microimaging device according to claim 1, wherein the first mirror (5) is arranged at a focal length one times in front of the first lens (6), the second lens (7) is arranged at a focal length two times behind the first lens (6), and the excitation notch filter (9) is arranged at a focal length one times behind the second lens (7); wherein the focal length of the first lens (6) is equal to the focal length of the second lens (7).
4. A coherent anti-stokes raman scattering microscopy imaging device according to claim 1, further comprising a beam splitter (12) and a first spectrometer (13); the beam splitter (12) is arranged on the light path of the second reflected light beam and is arranged between the fifth reflector (11) and the sixth reflector (14); the second reflected light beam passes through the beam splitter (12) to form a reflection light path and a transmission light path; wherein the reflected light path enters a first spectrometer (13) and the transmitted light is incident on a sixth mirror (14).
5. A coherent anti-stokes raman scattering microscopic imaging device according to claim 1, further comprising a third lens (22) and a second spectrometer (23); the third lens (22) is arranged on a transmission light path of the detection notch filter (19); the transmission light path of the detection notch filter (19) enters the second spectrometer (23) after passing through the third lens (22); the second spectrometer (23) is used for CARS spectral measurement.
6. A coherent anti-stokes raman scattering microscopic imaging device according to claim 1, wherein the laser pulse oscillator (1) is a femtosecond laser pulse oscillator.
7. The coherent anti-stokes raman scattering microscopic imaging device according to claim 1, wherein the pulsed laser is an ultra-short pulsed laser; wherein the center wavelength of the ultrashort pulse laser is 793-808nm, the bandwidth is 10-100nm, the repetition frequency is 10-100MHz, and the pulse width is 5-120 fs.
8. A coherent anti-stokes raman scattering microscopic imaging method, characterized by using a coherent anti-stokes raman scattering microscopic imaging device according to any one of claims 1 to 7;
the imaging method comprises the following steps:
-placing a sample to be imaged on the object translation stage (16);
selecting the reflection wavelength of the detection notch filter (19) and determining the characteristic vibration mode of the sample to be imaged; simultaneously, moving the position of the photomultiplier (20) to maximize the current value or the voltage value of the electrical signal output by the photomultiplier (20);
observing the amplitude and phase change of the CARS microscopic imaging result output by the phase-locked amplifier (21) by changing the parameters of a galvanometer scanner, and recording the electric signal output by the photomultiplier (20);
and carrying out data processing by utilizing Matlab software or Python software, and carrying out data processing on the electric signal output by the photomultiplier (20) to obtain a coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be imaged.
9. The coherent anti-stokes raman scattering microscopic imaging method according to claim 8, wherein the amplitude and phase changes of the CARS microscopic imaging result output by the lock-in amplifier (21) are observed by changing the parameters of the galvanometer scanner, and the process of recording the electric signal output by the photomultiplier tube (20) is specifically as follows:
when the relative distance between the objective lens (15) and the sample to be imaged is unchanged, establishing an xoy coordinate system by taking the horizontal plane of the sample to be imaged as a coordinate plane;
determining coordinates (x, y) of the position of the light spot on the sample to be imaged;
when the y value of the position coordinate of the sample to be imaged where the light spot is located is a constant value, increasing a voltage value applied to the x-axis direction of an object translation stage where the sample to be imaged is located;
translating the x value of the position coordinate of the sample to be imaged where the light spot is located by taking the step length as 1 micron, and recording the electric signals of the photomultiplier at N different x values;
when the y value of the position coordinate of the sample to be imaged where the light spot is located is y +1 micrometer, simultaneously recording the electric signals of the photomultiplier (20) at N different x values; when the y value of the position coordinate of the sample to be imaged where the light spot is located is y + N micrometers, simultaneously recording the electric signals of the photomultiplier (20) at N different x values again;
scanning an N micron multiplied by N micron area on a sample to be imaged to obtain an N multiplied by N two-dimensional matrix; and the electrical signals of all the photomultiplier tubes (20) at the grid points of the N x N two-dimensional matrix are recorded.
10. A coherent anti-Stokes Raman scattering microscopic imaging method according to claim 9, wherein the dwell time of the light spot in a single pixel during the measurement is set to 400 μ s.
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