CN114994017B - 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 method, comprising a laser pulse oscillator; the first reflecting mirror is arranged on the 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 optical path of the first reflected light beam; the vibrating mirror scanner is provided with a second reflecting mirror; the first reflected light beam passes through the second reflector, the first lens, the second lens, the fourth reflector, the excitation notch filter and the third reflector to form a modulated light beam; the modulated light beam passes through a fifth reflecting mirror to form a second reflected light beam; the sixth reflecting mirror, the objective lens, the object carrying translation stage, the condensing lens, the short-pass filter and the detection notch filter are sequentially arranged on the light path of the second reflected light beam; the photomultiplier is arranged on a reflection light path of the detection notch filter; the output end of the photomultiplier is connected with the input end of the lock-in amplifier; the invention does not need to specially customize the 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 is one of the most effective optical technologies for detecting vibration modes and molecular structures, and has been widely used in the fields of material science, biological medicine 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 has wide application in the biomedical field, in particular in the field of living cell imaging; CARS is an optical process of four-wave mixing by interaction of pump light, stokes light, and anti-stokes light with sample molecules, belonging to a third-order nonlinear optical process; wherein the pump photon with the frequency of omega p and the stokes photon with the frequency of omega S excite the sample molecule to a vibration level, and the process needs to meet the phase matching, namely the natural vibration frequency omega=ω p-ωS of the sample molecule; at this time, the energy of the sample molecule is increased, then the sample molecule is excited to a virtual energy level with higher energy by using the probe light (probe) with the frequency of omega pr, the energy of the sample molecule is unstable and returns to the ground state, and the anti-stokes photon with the frequency of omega aS is released, and the process also meets a certain condition omega=omega aS-ωpr; CARS are typically implemented using a multi-beam or multi-source scheme (to meet the frequency component requirements of pump photons, stokes photons, and probe photons), requiring that all excitation beams must coincide spatially.
Currently, in order to simplify the CARS system, a single-beam CARS method can be implemented using notch filters; by producing notch features (ω pr) on the laser spectrum and similar features (ω aS) on the CARS spectrum; because the notch filter is small and simple, the notch filter is easy to install on a galvanometer scanner and modulates laser at high frequency; since the resonance signal position is only related to notch frequency position omega pr; therefore, the non-resonance signal can be used as a local oscillator by using 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 single-beam CARS scheme realized based on a 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 such that the modulation frequency generally does not reach the maximum frequency of a conventional galvanometer scanner; the scanning frequency of the commercial galvanometer can reach 3-4 kHz, but the scanning frequency is limited by the customized installation notch filter due to the problems of volume and quality, and the scanning frequency is only about 1kHz, so that the stay time of a single pixel is limited to be more than 1 millisecond, and the application of the technology in the aspect of quick imaging in biomedicine is restricted.
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 method, which are used for solving the technical problems that a notch filter is arranged on a galvanometer scanner in the prior art, so that the modulation frequency can not reach the maximum frequency of a conventional galvanometer scanner, and the imaging speed is slow.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The invention provides a coherent anti-Stokes Raman scattering microscopic imaging device, which comprises a laser pulse oscillator, a first reflecting mirror, a galvanometer scanner, a fifth reflecting mirror, a sixth reflecting mirror, an objective lens, a carrying translation stage, a condensing lens, a short-pass filter, a detection notch filter, a photomultiplier and a phase-locked amplifier, wherein the first reflecting mirror is arranged on the first reflecting mirror;
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 reflected light beam after passing through the first reflecting mirror; the galvanometer scanner is arranged on the light path of the first reflected light beam, and the first reflected light beam passes through the galvanometer scanner to form a modulated light beam; the fifth reflecting mirror 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;
The vibrating mirror scanner is provided with a second reflecting mirror, a first lens, a second lens, a third reflecting mirror, an excitation notch filter and a fourth reflecting mirror;
The second reflecting mirror 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; 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 reflecting mirror, 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 reflecting mirror, the objective lens, the object carrying translation stage, the condensing lens, the short-pass filter and the detection notch filter are sequentially arranged on the light path of the second reflected light beam; the photomultiplier is arranged on a reflection light path of the detection notch filter and is used for converting the reflection light of the detection notch filter into an electric signal; the output end of the photomultiplier is connected with the input end of the lock-in amplifier.
Further, the device also comprises a pulse compressor and a long-pass filter; the pulse compressor and the long-pass filter are sequentially arranged on the optical path of the laser pulse and are sequentially arranged between the laser pulse oscillator and the first reflecting mirror.
Further, the first reflecting mirror 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 first lens is equal with the focal length of second lens.
Further, the device also comprises a beam splitter and a first spectrometer; the beam splitter is arranged on the light path of the second reflected light beam and is arranged between the fifth reflecting mirror and the sixth reflecting mirror; the second reflected light beam passes through the beam splitter to form a reflected light path and a transmitted light path; 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 passes through the third lens and then enters the second spectrometer; the second spectrometer is used for CARS spectrum measurement.
Further, the laser pulse oscillator adopts a femtosecond laser pulse oscillator.
Further, the pulse laser is ultra-short pulse laser; wherein the center wavelength of the ultra-short pulse laser is 793-808nm, the bandwidth is 10-100nm, the repetition frequency is 10-100MHz, and the pulse width is 5-120fs.
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 carrying translation stage;
Selecting the reflection wavelength of the detection notch filter, and determining the characteristic vibration mode of the sample to be imaged; simultaneously, the position of the photomultiplier tube is moved so as to maximize the current value or the voltage value of the electric signal output by the photomultiplier tube;
By changing parameters of a galvanometer scanner, observing amplitude and phase changes of CARS microscopic imaging results output by the lock-in amplifier, and recording electric signals output by the photomultiplier;
And carrying out data processing by utilizing Matlab software or Python software, and carrying out data processing on the electric signals 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 the galvanometer scanner, the amplitude and phase change of the CARS microscopic imaging result output by the lock-in amplifier is observed, and the electric signal output by the photomultiplier is recorded, which 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 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 the object carrying 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, with the step length of 1 micron, and recording the electric signals of the photomultiplier when 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=y+1 micrometers, simultaneously recording the electric signals of the photomultiplier when N different x values are recorded again; recording the electric signals of the photomultiplier when the light spot is positioned at the position coordinate of the sample to be imaged, wherein the y value of the position coordinate of the sample to be imaged is y=y+N micrometers, and the N different x values are recorded at the same time;
scanning an N micron-by-N micron area on a sample to be imaged to obtain an N two-dimensional matrix; and the electrical signals of all photomultiplier tubes at grid points of the N x N two-dimensional matrix are recorded.
Further, the dwell time of the spot at a single pixel during 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 method, which utilize the relation between an object and an image in optics, wherein an excitation notch filter in a galvanometer scanner customized with the excitation notch filter is replaced by a second reflector, the second reflector is placed at the position of the object, and the excitation notch filter is placed at the position of the image; the vibrating mirror scanner does not need to be specially customized, and the cost is low; meanwhile, the scanning speed of single pixels can be effectively improved, and the imaging efficiency is high; and satisfies the application to the field of tumor diagnosis.
Furthermore, the pulse compressor and the long-pass filter are sequentially arranged between the laser pulse oscillator and the first reflecting mirror, so that dispersion compensation of pulse laser is realized.
Drawings
FIG. 1 is a schematic diagram of a coherent anti-Stokes Raman scattering microscopic imaging device according to the present invention;
FIG. 2 is a CARS original spectrum of a small white mouse lung cancer tissue; wherein, the graph (a) is a CARS original spectrum graph when the pulse laser is 10mW and the excitation notch filter is 779.6nm and 779.9 nm; FIG. (b) is a Raman spectrum of lung cancer tissue extracted using the original CARS spectrum;
FIG. 3 is a CARS microscopic image of lung cancer tissue of mice in the present invention at a vibration frequency of 216cm -1.
The laser pulse oscillator comprises a laser pulse oscillator 1, a pulse compressor 2, a long-pass filter plate 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 plate 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, a 16-carrier translation stage and a condensing lens 17; the device comprises a short-pass filter 18, a detection notch filter 19, a photomultiplier 20, a phase-locked amplifier 21, a third lens 22 and a second spectrometer 23.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the following specific embodiments are used for further describing the invention in detail. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
As shown in fig. 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 reflecting mirror 4, a galvanometer scanner, a fifth reflecting mirror 11, a beam splitter 12, a first spectrometer 13, a sixth reflecting mirror 14, an objective lens 15, a carrier translation stage 16, a condensing 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 ultra-short pulse laser; preferably, the center wavelength of the ultrashort pulse is 793-808 nm, the bandwidth is 10-100 nm, the repetition frequency is 10-100 MHz, and the pulse width is 5-120 fs.
The pulse compressor 2 and the long-pass filter 3 are sequentially arranged on the optical 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; the long-pass filter wave plate 3 is used for performing filter processing on the compensated laser pulse to form a laser pulse after filter processing.
The first reflecting mirror 4 is arranged on the optical path of the filtered laser pulse, and the filtered laser pulse passes through the first reflecting mirror 4 to form a first reflected light beam; the galvanometer scanner is arranged on the light path of the first reflected light beam, and the first reflected light beam passes through the galvanometer scanner to form a modulated light beam.
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; wherein the first reflecting mirror 5 is arranged at a focal length of one time in front of the first lens 6, the second lens 7 is arranged at a focal length of two times behind the first lens 6, and the excitation notch filter 9 is arranged at a focal length of one time behind the second lens 7; wherein the focal length of the first lens 6 and the second lens 7 is the same; the focal length of the first lens 6 and the second lens 7 is 30mm-500mm.
In the invention, by utilizing the relation between an 'object' and an 'image' in optics, an excitation notch filter in a vibrating mirror scanner customized with the excitation notch filter is replaced by a second reflecting mirror, the second reflecting mirror is placed at the 'object' position, and the excitation notch filter is placed at the 'image' position; the galvanometer scanner can adopt the existing general commercial galvanometer scanner, special customization of the galvanometer scanner is not needed, and the cost is low.
The second reflecting mirror 5 is arranged on the optical path of the first reflected light beam, and the first reflected light beam forms a first optical 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 reflecting mirror 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 optical path forms an eighth optical path after passing through the first lens 6, and the eighth optical path forms a modulated light beam after passing through the second reflecting mirror 5.
The fifth reflecting mirror 11 is arranged on the optical 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 disposed on the optical path of the second reflected light beam, and the second reflected light beam forms a reflected optical path and a transmitted optical path after passing through the beam splitter 12; the first spectrometer 13 is disposed on the reflected light path of the second reflected light beam, and is used for observing the wavelength position of the notch on the laser spectrum.
The sixth reflecting mirror 14, the objective lens 15, the object carrying translation stage 16, the condensing lens 17, the short-pass filter 18 and the detection notch filter 19 are sequentially disposed on the transmission optical path of the second reflected light beam; wherein, the objective lens 15 is mounted on the objective lens adjusting bracket, and the object carrying translation stage 16 is a two-dimensional translation stage; the photomultiplier 20 is disposed on the reflected light path of the detection notch filter 19, and is configured to convert the reflected light of the detection notch filter 19 into an electrical signal; the output end of the photomultiplier 20 is connected with the input end of the lock-in amplifier 21, and the lock-in amplifier 21 is used for obtaining CARS microscopic imaging results according to the electric signals; the third lens 22 is arranged on 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 of the embodiment is used for imaging a sample to be imaged, the method specifically comprises the following steps of:
S1, roughly placing all components in the device in sequence; wherein, the second reflecting mirror 5 loaded on the galvanometer scanner is placed at the position of one focal length in front of the first lens 6, the second lens 7 with the same focal length as the first lens 6 is selected, the second lens 7 is placed at the position of two focal lengths behind the second lens 6, and the excitation notch filter 9 is placed at the position of one focal length behind the second lens 7; in step S1, a needle is placed at the position of the second reflecting mirror 5, a camera is placed at the position of the excitation notch filter 9, and the relative distances among the second reflecting mirror 5, the first lens 6, the second lens 7, the third reflecting mirror 8 and the excitation notch filter 9 are adjusted multiple times until the image of the needle in the camera can not be clear any more.
S2, starting a laser pulse oscillator, and roughly adjusting the focal point of the objective lens 15 to enable laser to be focused on a 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 reflecting mirror and the condensing lens 17 are adjusted to maximize the CARS spectrum 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 of the detection element and the collected CARS signals is high; in specific operation, the position of the sample to be imaged is observed by adjusting the object carrying translation stage, the focal position of the objective is adjusted by adjusting the bracket knob of the objective, and the pitching angle and the collimation degree of the laser are adjusted by adjusting the fifth reflecting mirror 11 and the sixth reflecting mirror 14.
S3, optimizing the relative positions of the prism pairs in the pulse compressor, so that the dispersion of laser on the sample to be imaged is compensated until the original CARS signal cannot be increased; in step S3, the group velocity dispersion of the laser light due to passing through the objective lens is compensated; during specific operation, the one-dimensional precise translation stage in the pulse compressor is adjusted to change the position of the prism pair, and meanwhile, the second spectrometer is utilized to observe the change of the original CARS spectrum intensity until the CARS spectrum intensity is not increased any more.
S4, observing the CARS spectrum intensity measured by the second spectrometer 23, and adjusting the beam splitter 12 until the laser and the objective lens 15 are not increased any more until the observed original CARS signal is transmitted through the objective lens 15 as much as possible; in a specific operation, the intensity of the CARS spectrum measured by the second spectrometer is observed, so that the observed original CARS signal is not increased any more, and the laser is matched with the objective lens to an optimal position.
S5, adjusting the optical fiber angle 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, by changing the position of the ultra-steep filter in the second spectrometer 23, two sets of raw CARS spectra are measured and data processing is performed to obtain a raman spectrum of the sample to be imaged.
The specific operation procedure in step S5 includes:
S501, adjusting the angle of an excitation notch filter, observing the wavelength position of a notch on a laser spectrum by using a first spectrometer, and recording original CARS spectrum data I 1 by using a second spectrometer when the wavelength of the notch is 779.6 nm;
S502, adjusting the angle of the excitation notch filter, and recording second group of original CARS spectrum data I 2 by using a second spectrometer when the notch wavelength is 779.9 nm;
S503, differentiating the two groups of original CARS spectrums to obtain a Raman spectrum of a sample to be tested, and drawing by Matlab software to obtain intensity changes of different vibration frequencies by taking a horizontal axis as vibration frequency and a vertical axis as CARS spectrum intensity; in step S503, the influence of background noise is removed by differentiation and normalization processing, and the resonance raman signal to be extracted is amplified at the same time;
In step S503, the raman spectrum I Raman of the sample to be measured is as follows:
Wherein, I 1 and I 2 are CARS original spectrograms when the excitation notch filter is 779.6nm and 779.9nm respectively; Is obtained by smooth filtering I 1 and I 2 for 50 times by Matlab.
S6, preparing animal tissue slices, placing the slices on a glass slide and placing the slices on the carrier translation stage 16
S7, determining a characteristic vibration mode of a sample to be imaged by using the notch filter; specifically, by observing the original CARS spectrum signal measured by the second spectrometer, the wavelength reflected by the detection notch filter 19 is selected, and the position of the photomultiplier 20 is moved at the same time, so that the current value or the voltage value of the electric signal output by the photomultiplier 20 is maximized;
S8, observing amplitude and phase changes of CARS microscopic imaging results output by the lock-in amplifier 21 by changing parameters of a galvanometer scanner, and recording electric signals output by the photomultiplier 20; and carrying out data processing by Matlab software or Python software, and carrying out data processing on the electric signals 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 present invention, by changing the parameters of the galvanometer scanner, the amplitude and phase change of the CARS microscopic imaging result output by the lock-in amplifier 21 is observed, and the electric signal output by the photomultiplier 20 is recorded, which is specifically 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 location 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 a carrying 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 using a step length of 1 micrometer, and simultaneously recording the electric signals of the photomultiplier when 130 different x values are recorded;
When the y value of the position coordinate of the sample to be imaged where the light spot is located is y=y+1 micrometers, simultaneously recording the electric signals of the photomultiplier 20 when 130 different x values are recorded again;
When the y value of the position coordinate of the sample to be imaged where the light spot is located is y=y+130 micrometers, simultaneously recording the electric signals of the photomultiplier 20 when 130 different x values again, and scanning the area of 130 square micrometers on the sample to be imaged to obtain a 130×130 two-dimensional matrix; and all values on the 130×130 two-dimensional matrix lattice points are recorded; wherein the dwell time of the spot at a single pixel during measurement is set to 400 microseconds.
The coherent anti-Stokes Raman scattering microscopic imaging device and method utilize the relation between an object and an image in optics, an excitation notch filter in a galvanometer scanner customized with the excitation notch filter is replaced by a second reflector, the second reflector 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 living imaging; in the invention, the purpose of setting the bandwidth to be 10-100 nm is that: the combination of photons required by CARS excitation is selected more by the incident laser with wide bandwidth, and the signal sensitivity is high; the pulse repetition frequency is selected to be 10-100 MHz and the pulse width is selected to be 120fs for the purpose of: the CARS excitation with low average power is realized by using the high peak power of the ultra-steep pulse, and the phototoxicity to biological samples is small.
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 positions of each element detected by CARS spectrum can be optimized; has the advantage of high imaging speed and has important significance in biomedical living imaging.
According to the imaging device and the imaging method, aiming at the problem that the scanning speed of the customized galvanometer scanner used in the prior art is limited, the notch filter is separated from the galvanometer scanner by using the 4F system consisting of the lenses with the same focusing distance, so that the scanning speed of the galvanometer scanner is not limited by the notch filter, the scanning speed can be increased by 3-5 times, and meanwhile, the cost is reduced.
In the invention, the vibrating mirror and the excitation notch filter can be used without being detached at random, and the notch generated by the notch filter can not be synchronous when the vibrating mirror scanner rotates, so that the function of modulating laser can not be realized; the adopted 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 notch generated by the excitation notch filter on the laser spectrum can be synchronized when the vibrating mirror rotates, thereby playing a key role in modulating the laser and demodulating signals later.
The test process comprises the following steps:
Taking the imaging process of a lung cancer tissue of a certain mouse as an example for explanation
In the invention, a beam of laser pulse with the pulse width of 20fs is subjected to dispersion compensation by a pair of pulse compensators placed on a one-dimensional precise translation stage, and then passes through a long-pass filter 3, wherein the central wavelength of the laser pulse is 795nm, and the bandwidth of the laser pulse is 45 nm; the model of the long-pass filter is Semrock LP01-785RU; then passes through the first reflecting mirror 4, then passes through the second reflecting mirror 5 loaded on the galvanometer scanner, respectively passes through the first lens 6 and the second lens 7, then passes through the fourth reflecting mirror 10, and then passes through the excitation notch filter 9; the model number of the excitation notch filter 9 is Optigrate, and BNF785; the light returns to the second reflector 5 along the original path through the third reflector 10, is reflected by the beam splitter 12 after passing through the fifth reflector 11, and enters the first spectrometer 13; the model of the first spectrometer is as follows: thorlabs, CCS175; the first spectrometer 13 is used for detecting the wavelength of the notch generated by exciting the notch filter 9, and the transmitted light is focused on the sample to be imaged on the carrying translation stage 16 by the objective lens 15 arranged on the two-dimensional precise translation stage through the sixth reflecting mirror 14; the model of the objective lens 15 is as follows: newport,20x,0.4na; the sample to be imaged is placed on a sample groove of a two-dimensional precise translation table; the transmitted signals are converged by a condensing lens 17 placed on a second two-dimensional precision translation stage; wherein, the model of the condensing lens 17 is: edmund Optics,0.5NA; the transmission part filters out the incident laser light through a short-pass filter 18, wherein the type of the short-pass filter 18 is as follows: semrock SP01-785RU; then reflected by the detection notch filter 19 and led into the photomultiplier 20, and converted into an electric signal which is input into the lock-in amplifier 21 for CARS microscopic imaging; the transmitted signal is then directed through a third lens 22 of 50mm focal length into an optical fiber connected to a second spectrometer 23 for measuring the original CARS spectrum; wherein the model of the third lens 22 is Thorlabs, AC254-030-AB; the model of the second spectrometer 23 is: jobin Yvon Triax 320.
As shown in fig. 2, fig. 2 shows a CARS original spectrum of a lung cancer tissue of a white mouse; the drawing (a) is a CARS original spectrogram of the pulse laser with the power of 10mW when the excitation notch filter is between 779.6nm and 779.9 nm; drawing (b) is a raman spectrum of lung cancer tissue extracted using the original CARS spectrum; starting laser, respectively recording CARS original spectrograms of an 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 representing two spectrums by using I 1 and I 2, wherein the figure (a) shows; using the formulaThe Raman spectrum of the lung tumor is calculated as shown in figure (b), wherein/>Is obtained by filtering I 1 and I 2 for 50 times by Matlab; from fig. 2 it can be seen that by processing the original CARS spectrum, the low frequency raman spectrum of the lung tumor can be clearly presented.
As shown in fig. 3, a CARS microscopic imaging image of a lung cancer tissue of a white mouse with a vibration frequency of 216cm -1 is shown; the incident laser can be modulated at high frequency by using a galvanometer scanner, so that resonance items in CARS signals are modulated at high frequency; because the resonance CARS in the generated CARS signal is only related to the notch generated by the notch filter, the non-resonance CARS is not related to the notch; by slightly adjusting the angle of the ultra-steep long-pass filter, two groups of original CARS spectrums are measured, and the low-frequency vibration spectrum or Raman spectrum can be obtained by differentiating and normalizing the two groups of original CARS spectrums. The long-pass filter and the short-pass filter ensure that the incident laser approaches the detected CARS signal infinitely, so that the detection of the terahertz wave band vibration spectrum can be realized. Removing non-resonance signals in the CARS signals through phase-locking amplification, and extracting weaker resonance signals in the CARS signals; obtaining the wavelength of detecting notch of notch filter by selecting 216 vibration frequency in the drawing (b) and calculating, then obtaining 130×130 points by moving the stage, and outputting voltage value or current value of each point as shown in fig. 3; the spatial distribution of tumors in the lung tissue of mice 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; using the relation between an 'object' and an 'image' in optics, replacing an excitation notch filter in a vibrating mirror scanner customized with the excitation notch filter by a second reflecting mirror, placing the second reflecting mirror at the 'object' position, and placing the excitation notch filter at the 'image' position; the single-pixel scanning speed can be improved by 2-4 times, and the time is saved by 10.14 seconds under the conditions of the same scanning area of 130 micrometers and 130 micrometers; because the vibrating mirror scanner provided with the excitation notch filter needs to be specially customized and produced, the invention can adopt a common commercial vibrating mirror scanner by adopting the vibrating mirror scanner provided with the reflecting mirror, and the cost can be greatly reduced without professional customization; the method has important significance in the aspect of application in lung tumor diagnosis and in the aspect of application of biomedical rapid imaging.
The above embodiment is only one of the implementation manners capable of implementing the technical solution of the present invention, and the scope of the claimed invention is not limited to the embodiment, but also includes any changes, substitutions and other implementation manners easily recognized by those skilled in the art within the technical scope of the present invention.
Claims (10)
1. The coherent anti-Stokes Raman scattering microscopic imaging device is characterized by comprising a laser pulse oscillator (1), a first reflecting mirror (4), a galvanometer scanner, a fifth reflecting mirror (11), a sixth reflecting mirror (14), an objective lens (15), a carrier 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 reflecting mirror (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 reflecting mirror (4); the galvanometer scanner is arranged on the light path of the first reflected light beam, and the first reflected light beam passes through the galvanometer scanner to form a modulated light beam; 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 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 optical path of the first reflected light beam, and the first reflected light beam forms a first optical 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 reflecting mirror (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 the first lens (6), and the eighth light path forms the modulated light beam after passing through the second reflecting mirror (5);
the sixth reflecting mirror (14), the objective lens (15), the object carrying 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 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 lock-in amplifier (21).
2. A coherent anti-stokes raman scattering microscopy 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 the optical path of the laser pulse and are sequentially arranged between the laser pulse oscillator (1) and the first reflecting mirror (4).
3. A coherent anti-stokes raman scattering microscopy imaging device according to claim 1, characterized in that the first mirror (5) is arranged at a focal length in front of the first lens (6), the second lens (7) is arranged at a focal length twice behind the first lens (6), and the excitation notch filter (9) is arranged at a focal length twice behind the second lens (7); wherein the focal length of the first lens (6) and the second lens (7) is equal.
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 optical path of the second reflected light beam and is arranged between the fifth reflecting mirror (11) and the sixth reflecting mirror (14); the second reflected light beam forms a reflected light path and a transmitted light path after passing through the beam splitter (12); wherein the reflected light path enters a first spectrometer (13), and the transmitted light path enters a sixth reflecting mirror (14).
5. A coherent anti-stokes raman scattering microscopy 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 spectrum measurement.
6. A coherent anti-stokes raman scattering microscopy imaging device according to claim 1, characterized in that the laser pulse oscillator (1) is a femtosecond laser pulse oscillator.
7. A coherent anti-stokes raman scattering microscopy imaging device according to claim 1, wherein the pulsed laser is an ultra-short pulsed laser; wherein the center wavelength of the ultra-short pulse laser is 793-808nm, the bandwidth is 10-100nm, the repetition frequency is 10-100MHz, and the pulse width is 5-120fs.
8. A method of coherent anti-stokes raman scattering microscopy imaging, characterized by using a coherent anti-stokes raman scattering microscopy imaging device according to any one of claims 1-7;
The imaging method comprises the following steps:
placing a sample to be imaged on the carrier 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) so as to maximize the current value or the voltage value of the electric signal output by the photomultiplier (20);
by changing parameters of a galvanometer scanner, observing amplitude and phase changes of CARS microscopic imaging results output by the lock-in amplifier (21), and recording electric signals 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 signals output by the photomultiplier tube (20) to obtain a coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be imaged.
9. A method of coherent anti-stokes raman scattering microscopy imaging according to claim 8, characterized by the process of observing the amplitude and phase variations of the CARS microscopy imaging result output by the lock-in amplifier (21) and recording the electrical signal output by the photomultiplier tube (20) by varying the parameters of a galvanometer scanner, in particular 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 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 the object carrying 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, with the step length of 1 micron, and recording the electric signals of the photomultiplier when 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=y+1 micrometers, simultaneously recording the electric signals of the photomultiplier (20) when N different x values are recorded again; when the y value of the position coordinate of the sample to be imaged where the light spot is located is y=y+N micrometers, simultaneously recording the electric signals of the photomultiplier (20) when N different x values are recorded again;
scanning an N micron-by-N micron area on a sample to be imaged to obtain an N two-dimensional matrix; and recording the electrical signals of all photomultiplier tubes (20) on grid points of the N two-dimensional matrix.
10. A method of coherent anti-stokes raman scattering microscopy as defined in claim 9 wherein the dwell time of the spot in a single pixel during measurement is set to 400 microseconds.
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